Ex Parte Winquist et al

Patent Trial and Appeal Board

May 3, 2018

12528849 (P.T.A.B. May. 3, 2018)

UNITED STA TES p A TENT AND TRADEMARK OFFICE
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR
12/528,849 12/15/2009
23570 7590 05/07/2018
PORTER WRIGHT MORRIS & ARTHUR, LLP
INTELLECTUAL PROPERTY GROUP
41 SOUTH HIGH STREET
29THFLOOR
COLUMBUS, OH 43215
Ola Winquist
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www .uspto.gov
ATTORNEY DOCKET NO. CONFIRMATION NO.
4007636-185958 7461
EXAMINER
HIBBERT, CATHERINE S
ART UNIT PAPER NUMBER
1636
NOTIFICATION DATE DELIVERY MODE
05/07/2018 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address( es):
ipdocket@porterwright.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte OLA WINQUIST and MAGNUS THORN
Appeal2017-004106
Application 12/528,849
Technology Center 1600
Before DEMETRA J. MILLS, ERIC B. GRIMES, and
ELIZABETH A. LA VIER, Administrative Patent Judges.
MILLS, Administrative Patent Judge.
DECISION ON APPEAL
This is an appeal 1 under 35 U.S.C. § 134. The Examiner has rejected
claims 114, 115, 117-121, 123, 126, and 128-130 for obviousness. We have
jurisdiction under 35 U.S.C. § 6(b ).
We REVERSE and enter a NEW GROUND OF REJECTION
pursuant to our authority under 37 C.F.R. § 41.50(b).
1 The Appeal Brief states "[ t ]he real party in interest for the present
application is the Assignee, Sentoclone International AB." App. Br. 1.
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Application 12/528,849
STATEMENT OF CASE
The following claim is representative.
114. A method for detecting a cancer cell in a biological
sample comprising a single-cell suspension of sentinel or
metinel lymph node cells, wherein the biological sample
comprising a single-cell suspension of sentinel or metinel
lymph node cells is a sample in which the presence of a cancer
cell is unknown, comprising:
i) adding to said biological sample comprising a single-
cell suspension of sentinel or metinel lymph node cells a panel
of agents which bind to two or more different extracellular
markers,
wherein at least one of the agents binds to an
extracellular marker that is specific for cancer and is selected
from the group consisting of:
markers for colon cancer cells selected from the group
consisting of carcino embryonic antigen (CEA), carbohydrate
antigen 15-3 (CA15-3), carbohydrate antigen 125 (CA125),
carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 242
(CA242), Ep-CAM, G250, RCASl, STN, and TA-90;
markers for urinary bladder cancer cells selected from the
group consisting ofBC-10, CA19-9, carcino embryonic antigen
(CEA)1 and NY-ES0-1;
markers for malignant melanoma cells selected from the
group consisting ofMSH-R, NY-ES0-1, and TA-90;
markers for breast cancer cells selected from the group
consisting of CA15-3, CA125, carcino embryonic antigen
(CEA), Ep-CAM, NY-ES0-1, PRL-R, RCASl, STN, and TA-
90·
'
markers for prostate cancer cells selected from the group
consisting ofEp-CAM1 NY-ES0-1, PRL-R, PSMA1RCAS1,
andB2M;
markers for ovarian cancer cells selected from the group
consisting ofCA15-3, CA125, G250, RCASl, STN and B2M;
and
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Appeal2017-004106
Application 12/528,849
markers for lung cancer cells selected from the group
consisting of CA 15-3, CA 125, carcino embryonic antigen
(CEA), Ep-CAM, RCASl, and TA-90;
ii) subjecting the biological sample to flow cytometry;
and
iii) detecting any binding of one or more of said agents to
an extracellular marker, wherein binding of an agent that is
specific for cancer to an extracellular marker is indicative of a
cancer cell in the biological sample_,_
wherein steps (i)-(iii) are performed in 25 minutes or
less.
Cited References
L.G. Durrant et al., Enhanced recognition of human colorectal
tumour cells using combinations of monoclonal antibodies, 60
BR. J. CANCER 855-860 (1989) ("Durrant")
Wilma E. Mesker, Automated Analysis of Multiple Sections for
the Detection of Occult Cells in Lymph Nodes, 9 CLINICAL
CANCER RESEARCH 4826-4834 (2003) ("Mesker")
BECTON DICKINSON BIOSCIENCES, INTRODUCTION TO FLOW
CYTOMETRY: A LEARNING GUIDE (2002) ("Guide")
UT Health Science Center, FAQ/or Flow Cytometry (updated
Feb. 26, 2015), http://research.uthscsa.edu/facs/faq.asp (last
visited Oct. 17, 2016) ("Flow Cytometry")
Ground of Rejection
Claims 114, 115, 117-121, 123, 126, and 128-130 are rejected under
35 U.S.C. § 103(a) as being unpatentable over Durrant in view of Mesker.
FINDINGS OF FACT
The Examiner's findings of fact are set forth in the Final Office
Action at pages 2-17.
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Application 12/528,849
PRINCIPLES OF LAW
In making our determination, we apply the preponderance of the
evidence standard. See, e.g., Ethicon, Inc. v. Quigg, 849 F.2d 1422, 1427
(Fed. Cir. 1988) (explaining the general evidentiary standard for proceedings
before the Office).
"The combination of familiar elements according to known methods
is likely to be obvious when it does no more than yield predictable results."
KSR Int'! Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
The Examiner must provide specific factual findings predicated on
sound technical and scientific reasoning to support his or her conclusion of
common knowledge. See, In re Soli, 317 F .2d 941, 946 ( CCP A 1963 ); In re
Chevenard, 139 F.2d 711, 713 (CCPA 1943).
Pending Obviousness Rejection
The Examiner finds that Durrant teaches each element claimed except
using a biological sample comprising sentinel or metinel lymph node cells,
and that the procedure is capable of being performed in 25 minutes or less.
Ans. 3. The Examiner cites Mesker as disclosing a method for detecting a
cancer cell in a biological sample comprising sentinel or metinel lymph node
cells and CEA antibodies. Ans. 3. The Examiner cites "Guide" and Flow
Cytometry as evidence that the flow cytometry procedure described in
Durrant provides a result in less than 25 minutes. Ans. 8-9. The Examiner
concludes that one of ordinary skill in the art
would have been motivated to combine the elements of the
cited references, specifically using sentinel lymph node samples
such used by Mesker et al for the flow cytometry technique of
Dur[ r ]ant et al to arrive at the instantly claimed invention for
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Appeal2017-004106
Application 12/528,849
the rationale of improving correct negative detection probability
combined with improved correct positive detection, improving
speed and efficiency, and for being able to determine the origin
of metastasis. Durant et al disclose that although expression of
CEA on colorectal tumours have previously been shown [by]
immunoperoxidase staining, they recite that "there are
advantages in analyzing tumour cell suspensions by indirect
immunofluorescence and flow cytometric analysis" (page 857,
lines 1-5).
Final Act. 5. The Examiner further concludes that
In view of the high level of skill in the art, one of ordinary skill
in the art would have had a reasonable expectation of success to
use sentinel lymph node samples as shown by Mesker et al in
combination with the flow cytometry methods of Dur[ r ]ant et al
because both references are in the same field of study and seek
to solve the same problem and have successfully used the CEA
marker in combination with a general epithelial marker to
detect cancer cells with high accuracy.
Final Act. 6.
Appellants contend that
At page 8, the Examiner's Answer newly cites "Introduction to
Flow Cytometry: A Leaming Guide" (December 2002) (no
publisher listed) and at page 9, the Examiner's Answer newly
cites "UT Health Science Center FAQ for Flow Cytometry"
(updated February 28, 2015 and printed October 17, 2016).
Neither of these references were previously cited in the
prosecution of this application.
Reply Br. 1.
Appellants contend that "[ n ]either Durrant nor Mesker provide one of
ordinary skill in the art with any apparent reason to use Mesker's lymph
node cells in Dur[r]ant's study of antibodies and antibody combinations"
and "neither of these references provide one of ordinary skill in the art with
a reasonable expectation of success." App. Br. 7. Appellants further argue
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Appeal2017-004106
Application 12/528,849
that, "Durrant does not teach or suggest a method for detecting a cancer cell
in a biological sample or, more specifically, a biological sample in which the
presence of cancer cells is unknown," and that "Durrant provides no
suggestion or recognition if the presence of cancer cells could be detected in
a single-cell suspension of sentinel or metinel lymph node cells using flow
cytometry." App. Br. 9.
ANALYSIS
Claims 114, 115, 117-121, 123, 126, and 128-130 are on appeal.
We acknowledge Appellants' argument that the "Introduction to Flow
Cytometry: A Leaming Guide," published December 2002, and
UT Health Science Center "FAQ for Flow Cytometry" updated 02/26/2015,
references were not properly cited in the rejection. In view of the
Examiner's error in failing to properly cite these references, we reverse the
pending rejection and enter a new ground of rejection, by adding "Guide"
and "Flow Cytometry" to the original rejection, as well as an additional
reference, U.S. Patent 6,558,916, issued May 6, 2003 to Veerapandian.
NEW GROUND OF REJECTION
Claims 114, 115, 117-121, 123, 126, and 128-130 are rejected under
35 U.S.C. § 103(a) as being unpatentable over Durrant in view of Mesker,
Veerapandian, "Introduction to Flow Cytometry: A Leaming Guide,"
published December 2002, and UT Health Science Center "FAQ for Flow
Cytometry" updated 02/26/2015.
Durrant and Mesker are discussed above in the Examiner's original
rejection. Durrant does not specifically disclose that the flow cytometry
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Appeal2017-004106
Application 12/528,849
process including adding agents that bind to biological markers to a single
cell suspension, subjecting the sample to flow cytometry, and detecting the
binding of the agents, takes 25 minutes or less.
Veerapandian2 discloses a flow cytometry method for real-time
measurement of patient cellular responses. Veerapandian, Title, Fig. 19.
The method may involve contacting and processing a single cell suspension.
Id. at col. 24, 11. 45---60. Multiwell plate panels may be used. Id. at col. 65,
1. 20. Individual cells may be tagged with an antibody specific for a cell
type. Id. at col. 59, 11. 49--50; col. 68, 11. 28-31. Antibody labeling and
staining of cells "is within 15 minutes on ice." Id. at col. 60, 11. 25-51.
Cells are then analyzed by the flow cytometer (PCM). Id. at col. 60, 11. 59--
63. The PCM flow rate was was chosen so that the reaction time from
mixing to detection was forty seconds; and the time to complete an
additional reagent concentration gradient run was 5 minutes. Id. at col. 48,
11. 40-44, col. 49, 11. 5-6. The cells may be cancer cells. Id. at col. 21, 11.
25-27. Thus, in Veerapandian, the total run time for one cell is on the order
of about 16 minutes (including binding of antibody label marker and staining
incubation time of 15 minutes, plus flow cytometry rate of 40 seconds for
detection of markers for one cell; Example 8A), which is within the claimed
time of 25 minutes or less.
2 Veerapandian et al., US 6,558,916 B2, issued May 6, 2003 (filed
Feb. 7, 2001) ("Veerapandian").
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Appeal2017-004106
Application 12/528,849
As further evidenced by page 39 of "Guide", one of ordinary skill in
the art of flow cytometry would have understood that about 300 cells per
second could be detected (i.e., 18,000 cells per minute), as early as 2002.
Therefore, it would have been obvious to one of ordinary skill in the
art at the time of the invention to use Mesker's CEA reactive lymph node
cells in Durrant's flow cytometry assay using anti-CEA antibodies, because
Durrant suggests that although expression of CEA on colorectal tumors has
previously been shown by immunoperoxidase staining (the method of
Mesker), Durrant recites that "there are advantages in analyzing tumour cell
suspensions by indirect immunofluorescence and flow cytometric analysis"
(Durrant 857 (lines 1-5)). There would have been a reasonable expectation
of success because Mesker discloses that sentinel lymph nodes reacted with
CEA antibody can detect tumor node metastasis (Mesker 4829), and Durrant
discloses that multiple antibodies, including CEA antibody, can be used to
detect cancer in a cell flow cytometry assay. Veerapandian evidences that it
was known in the art at the time of the invention that a single cell suspension
of cells, which may be cancer cells, can be reacted with first or second
antibodies and detected in a flow cytometer in 25 minutes or less.
As to Appellants' arguments of record, we are not persuaded by
Appellants' argument that, "Durrant does not teach or suggest a method for
detecting a cancer cell in a biological sample or, more specifically, a
biological sample in which the presence of cancer cells is unknown." App.
Br. 7. One of ordinary skill in the art would readily recognize that, in the
assay of Durrant, whether an antibody set reacts or fails to react with a
sample is indicative of a positive or negative result, whether the nature of
cancer cells in a sample is known or unknown. Durrant and Veerapandian
8
Appeal2017-004106
Application 12/528,849
provide a suggestion or recognition that the presence of cancer cells could be
detected in a single-cell suspension of cancer cells using flow cytometry.
CONCLUSION OF LAW
The Examiner's§ 103 rejection is reversed, and a New Ground of
Rejection is added.
Pursuant to 37 C.F.R. § 41.50(b), we enter a NEW GROUND OF
REJECTION of claims 114, 115, 117-121, 123, 126, and 128-130 under 35
U.S.C. § 103 as being unpatentable over Durrant in view of Mesker,
Veerapandian, and "Guide."
This decision contains a new ground of rejection pursuant to 37
C.F.R. § 41.50(b). 37 C.F.R. § 41.50(b) provides "[a] new ground of
rejection pursuant to this paragraph shall not be considered final for judicial
review." 37 C.F.R. § 41.50(b) also provides:
When the Board enters such a non-final decision, the Appellant,
within two months from the date of the decision, must exercise one of the
following two options with respect to the new ground of rejection to avoid
termination of the appeal as to the rejected claims:
( 1) Reopen prosecution. Submit an appropriate
amendment of the claims so rejected or new Evidence relating
to the claims so rejected, or both, and have the matter
reconsidered by the examiner, in which event the prosecution
will be remanded to the examiner. The new ground of rejection
is binding upon the examiner unless an amendment or new
Evidence not previously of Record is made which, in the
opinion of the examiner, overcomes the new ground of rejection
designated in the decision. Should the examiner reject the
claims, appellant may again appeal to the Board pursuant to this
subpart.
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Appeal2017-004106
Application 12/528,849
(2) Request rehearing. Request that the proceeding be
reheard under§ 41.52 by the Board upon the same Record. The
request for rehearing must address any new ground of rejection
and state with particularity the points believed to have been
misapprehended or overlooked in entering the new ground of
rejection and also state all other grounds upon which rehearing
is sought. Further guidance on responding to a new ground of
rejection can be found in the Manual of Patent Examining
Procedure§ 1214.01.
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a). See 37 C.F.R.
§ 1.136(a)(l )(iv).
REVERSED; 37 C.F.R. § 41.50(b)
10
Application/Control No. Applicant(s)/Patent Under Patent
12/528,849 Appeal No. 2017-004106
Notice of References Cited
Examiner Art Unit
1636 Page 1 of 1
U.S. PATENT DOCUMENTS
*
Document Number Date
Country Code-Number-Kind Code MM-YYYY Name Classification
A US- 6,558,916 Veerapandian et al. , May 6, 2003
B US-
c US-
D US-
E US-
F US-
G US-
H US-
I US-
J US-
K US-
L US-
M US-
FOREIGN PATENT DOCUMENTS
*
Document Number Date
Country Code-Number-Kind Code MM-YYYY Country Name Classification
N
0
p
Q
R
s
T
NON-PATENT DOCUMENTS
* Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages)
u
v
w
x
*A copy of this reference is not being furnished with this Office action. (See MPEP § 707.05(a).)
Dates in MM-YYYY format are publication dates. Classifications may be US or foreign.
U.S. Patent and Trademark Office
PT0-892 (Rev. 01-2001) Notice of References Cited Part of Paper No.
c12) United States Patent
Veerapandian et al.
(54) CELL FLOW APPARATUS AND METHOD
FOR REAL-TIME MEASUREMENTS OF
PATIENT CELLULAR RESPONSES
(75) Inventors: Pandi Veerapandian, San Diego, CA
(US); Gregory Kaler, San Diego, CA
(US)
(73) Assignee: Axiom Biotechnologies, Inc., San
Diego, CA (US)
( *) Notice: Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 0 days.
(21)
(22)
(65)
(63)
(51)
(52)
(58)
This patent is subject to a terminal dis-
claimer.
Appl. No.: 09/779,690
Filed: Feb.7,2001
Prior Publication Data
US 2002/0086340 Al Jul. 4, 2002
Related U.S. Application Data
Continuation-in-part of application No. 09/568,778, filed on
May 10, 2000, now Pat. No. 6,242,209, which is a continu-
ation of application No. 09/370,786, filed on Aug. 5, 1999,
now Pat. No. 6,280,967, which is a continuation-in-part of
application No. 09/317,793, filed on May 24, 1999, now Pat.
No. 6,096,509, which is a continuation of application No.
08/904,904, filed on Aug. 1, 1997, now Pat. No. 5,919,646,
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121 ~119 I
~
I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
US006558916B2
(10) Patent No.: US 6,558,916 B2
*May 6, 2003 (45) Date of Patent:
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_ci!J
~CELL~
SUSP£tvSION
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l/7
US 6,558,916 B2
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Dunne, Time Window Analysis and Sorting, (1991), Cytom-
etry 12:597-601.
* cited by examiner
U.S. Patent May 6, 2003 Sheet 1of65 US 6,558,916 B2
JOI !OJ
TEST COMPOUNDS ~--~------.-i STANDARDS 125
SAMPLER SAMPLER
!05 109
JO? SOFTWARE
GRADIENT
DEVICE
!lJ
121
CONTROLLER
GRADIENT
DEVICE
117
~1---1 CAL/BRA TION
REACTION
DEVELOPING
LINES
115
119
UNIT
123
COMPUTER
DRAIN ~---l DETECTOR i-----------__J
FIG. 1A
U.S. Patent May 6, 2003 Sheet 2 of 65 US 6,558,916 B2
101 !OJ
125 TEST COMPOUNDS ~---.----------i STANDARDS
SAMPLER SAMPLER
!05 109 107
SOFTWARE
GRADIEN.T
DEVICE
llJ
CELL
SUSPENSION
!21
CONTROLLER
GRADIENT
DEVICE
117
i...-1-----i CALI BR A Tl ON
REACTION
DEVELOPING
LINES
COUPLING
SYSTEM
UNIT
115
120
119
123
COMPUTER
DRAIN }-olll----; DETECTOR ~--------------'
FIG. 1B
U.S. Patent May 6, 2003 Sheet 3 of 65 US 6,558,916 B2
SAMPLE
LASER(S) SAMPLE/LASER -
INTERCEPT
PARAMETER 1
PARAMETER 2
PARAMETER 3 -
PARAMETER 4 -
PARAMETER 5 -
- PARAMETER 6
PARAMETER 7
AND HIGHER
SIGNAL
PROCESSING
• I DATA OUTPUT I
~
DATA MANIPULATION
t
I DATA STORAGE I
FIG. 2
FIG. 3A 20!
AUTOSAMPLER
COMPOUNDS
207
2!!
24J.
GRADIENT
PUMP
24!
PUMP
-205
215
213
AUTOSAMPLER
STANDARDS I ,2/7
M L BUFFER
'209 22!
-227
(
MIXING
CHAMBER
GRADIENT
PUMP
23!
235
PUMP
245
REACTION
DEVELOPING
LINES
247-
DETECTOR I .., r
249
·223
225
219
233
CAL) BR.
MAX
CAL) BR.
MIN
237-
~ •
rJ'l •
~
~
"""'" ~=
"""'"
~
= '"<
!?'
N c
6
Cf'l ::r
(!;)
(!;)
""" ...
~ ......
0-,
Ul
e
00
"" 111
(Ii
CIJ
't.c
~
"" t:=
N
U.S. Patent May 6, 2003 Sheet 5 of 65
20/
COMPOUNDS
207
2//
243
GRADIENT
PUMP
241
PUMP
213
:205
AUTOSAMPLER
2/5 STANDARDS
M
22/
227
MIXING
CHAMBER
REACTION
DEVELOPING
LINES
247
251
L
GRADIENT
PUMP
PUMP
249
DETECTOR 1-------l._... DRAIN
FIG. 3B
US 6,558,916 B2
2/9
223
225
233
CALJBR.
MAX
CALIBR.
MIN
237
U.S. Patent May 6, 2003 Sheet 6 of 65
JOJ JO!
AUTOSAMPLER
COMPOUNDS
Ni
307
PROPORTIONING
VALVE
J49
347
305
BUFFER
359
DRAIN
315
305 AUTOSAMPLER
STANDARDS
Jll
ANTAGONISTS
M· J
317
331
MIXING
CHAMBER
3JJ
J42
348
PRIMING
VALVE
351 345
357
PER/STAL TIC
PUMP
FIG. 4A
AGONISTS
Lk
PROPORTIONING
VALVE
JJ5
PRIMING
VALVE
REACTION
DEVELOPING
LINES
US 6,558,916 B2
319
J05
325
337
CALIBRATION
MAX
339
341
353
355
U.S. Patent May 6,2003 Sheet 7 of 65 US 6,558,916 B2
JOJ, JO!
AUTOSAMPLER
COMPOUNDS
Ni
307
PROPORTIONING
VALVE
312
349
347
305
BUFFER
359
DRAIN
315
305 AUTOSAMPLER
STANDARDS
JI/
ANTAGONISTS
Mj
317 323
331
MIXING
CHAMBER
332
333
342
348
PRIMING
VALVE
351 345
-357
PER/STAL TIC
PUMP
FIG. 4B
AGONISTS
Lk
PROPORTIONING
VALVE
335
J40
PRIMING
VALVE
REACTION
DEVELOPING
LINES
COUPLING
SYSTEM
319
J05
325
337
CALIBRATION
MAX
J.39
CALIBRATION
MIN
34!
353
36!
J55
U.S. Patent May 6, 2003 Sheet 8 of 65 US 6,558,916 B2
NO
405
401
PROVIDE CELt-/COMPOUND
MIXTURE TO DETECTOR
YES~---< NO
ADD STANDARD
AGONIST
ADD STANDARD
ANTAGONIST
TO RECEPTOR Ri TO RECEPTOR R.
I
YES
413
THE COMPOUND IS
AGONIST TO THE
RECEPTOR Ri
YES
THE COMPOUND IS
ANTAGONIST TO THE
RECEPTOR Ri
417
GO TO THE
POTENCY MODE
FIG. 5
415
407
NO
FIG. 6
AGONIST
REGISTER
CONCENTRATION
DEPENDENCE OF CELL
RESPONSE ACTIVATION
CALCULATE
EC50
505
503
ANTAGONIST
507
REGISTER CONCENTRATION
DEPENDENCE OF INHIBITION
OF CELL RESPONSE IN THE
PRESENCE OF A STANDARD
509
AGONIST
CALCULATE
IC50
513
51!
REGISTER CONCENTRATION
DEPENDENCIES OF CELL
RESPONSE ACTIVATION WITH
STANDARD AGONIST IN THE
PRESENCE OF SEVERAL
CONCENTRATIONS OF THE
COMPOUND
CALCULATE pA2;
DETERMINE THE NATURE OF
INHIBITION:
COMPETITIVE/NONCOMPETITIVE
~ •
rJ'l •
~
~
"""'" ~=
"""'"
~
= '"<
"'"" N c
6
Cf'l ::r
(!;)
(!;)
"""
\C:)
~ ......
"" Ul
e
00
"" 111
(Ii
CIJ
~
~
"" t:=
N
U.S. Patent May 6, 2003 Sheet 10 of 65 US 6,558,916 B2
FIG. 7A
FIG. 7B
FIG. 7C
FIG. 70
FIG. 7
U.S. Patent May 6,2003 Sheet 11 of 65 US 6,558,916 B2
FIG. 7A
614
TURN OFF
PROPORTIONING
VALVE 327
START
600
602
SET INITIAL
PARAMETERS
SET AUTOSAMPLERS
AND VALVES INTO
ORIGINAL POSITION
TURN PUMP ON
604
606
608
CLOSE PRIMING
VALVES 345 & 313
610
TURN ON
PROPORTIONING
VALVE 327
DELAY
616
OPEN PRIMING
VALVE 313
TURN ON
PROPORTIONING
VALVE 311
612
620
618
CLOSE PRIMING
VALVE 329
U.S. Patent May 6, 2003 Sheet 12 of 65
626
TURN ON DIVERTING
VALVES 333 & 335
621
TURN OFF
PROPORTIONING
VALVE 311
622
627
DELAY
TURN OFF
DIVERTING VALVE
335
629
DELAY
FIG 7B
628
US 6,558,916 B2
624
OPEN PRIMING
VALVE 329
U.S. Patent May 6,2003 Sheet 13 of 65
/632
OPEN PRIMING
VALVE 345
640
TURN OFF
DIVERTER VALVE
347
630
TURN OFF
DIVERTING VALVE
333
635
DELAY
TURN ON
DIVERTER VALVE
347
636
637
TURN OFF
PUMP
642
643
FIG. 7C
US 6,558,916 B2
634
CLOSE PRIMING
VALVE 341
638
OPEN PRIMING
VALVE 341
FIG. 7D
PRIMING
VALVES
STEP 345 341 313 329
START - - - -
608 + +
612
614
616 -
618 +
620
622
624 -
626
628
630
632 - +
636
638 -
STOP - - - -
DIVERTING
VALVES
335 333 347
- - -
+ +
-
-
+
-
- - -
PROPORTIONING
VALVES
327 311
- -
+
-
+
-
- -
~ •
rJ'l •
~
~
"""'" ~=
"""'"
~
= '"<
!?'
N c
6
Cf'l ::r
(!;)
(!;)
"""
"""'
""" ~
"""=
~
VI
e
00
"" 111
(Ii
CIJ
~
~
"" t:=
N
U.S. Patent May 6, 2003 Sheet 15 of 65 US 6,558,916 B2
FIG. BA
FIG. BB
FIG. BC
FIG. 80
FIG. BE
FIG. BF
FIG. BC
FIG. 8
U.S. Patent May 6,2003 Sheet 16 of 65 US 6,558,916 B2
FIG. BA
TURN OFF VALVES:
327 & 311;
700
SET INITIAL
PARAMETERS
706
702
704
335, 333 & 34 7
345, 341, 313 & 329
SET AUTOSAMPLERS
INTO ORIGINAL
POSITION
708
TURN. PUMP
ON
REGISTER
BASAL ·SIGNAL
"BS"
709
SAVE BASAL SIGNAL
INCREMENT COUNTER
N
N. =N. 1 +1 I 1-
7/J
710
712
714
STR
~__.__, M
FROM FfG BEi
U.S. Patent May 6, 2003 Sheet 17 of 65 US 6,558,916 B2
717
SET COUNTER N
TO N=O
719
725\
FLAG
COMPOUND Ni
"POSITIVE"
K
FROM FIG. Be
7/J
MOVE AUTOSAMPLER 301
TO POSITION Ni
TURN PROPORTIONING
VALVE 311 ON
718
720
722
REGISTER COMPOUND
INDUCED SIGNAL
SNi
72.J
SAVE SNi
NO
FIG. BB
726
FLAG
COMPOUND Ni
"NEGATIVE"
L
TO FIG. Bf
U.S. Patent May 6, 2003 Sheet 18 of 65
FROM FIG. Bb
K
730
INCREMENT
01-----....i COUNTER M: ~-----{ p
US 6,558,916 B2
FROM FIG. Bd Mj =Mj-1+ 1 FROM FIG. Be
YES
N
TO FIG. Be
734
736
738
740
MOVE AUTOSAMPLER
315 TO M j POSITION
TURN PROPORTIONING
VALVE 327 ON
REGISTER & SAVE
SIGNAL SN i M j
TURN PROPORTIONING
VALVES 311 & 327 OFF
742
MOVE AUTOSAMPLER
315 TO POSITION M=O
FIG. BC
U.S. Patent
75!
May 6, 2003 Sheet 19 of 65
FLAG
744
TURN PROPORTIONATING
VALVE 327 ON
746
748
TURN PROPORTIONATING
VALVE 327 OFF
752
US 6,558,916 B2
FLAG
COMPOUND Nr - "POSITIVE"
ANTAGONIST Mj - "POSITIVE"
COMPOUND Nr- "POSITIVE"
ANTAGONIST Mj - "NEGATIVE"
753
SAVE DATA
IN DATABASE
0
TO FIG. Be
FIG BD
U.S. Patent May 6, 2003 Sheet 20 of 65 US 6,558,916 B2
Bf
SET
N --- COUNTER M TO i-----R
FROM FIG. Be ZERO FROM FIG. Bf
MOVE AUTOSAMPLERS
301 & 315 TO ZERO
POSITION
TURN
PROPORTION A TING
VALVES 311 & 327 ON
WASH·DELAY
TURN PROPORTIONATING
VALVES 311 & 327 OFF
M
TO FIG. Ba
FIG. BE
U.S. Patent May 6, 2003 Sheet 21 of 65
INCREMENTAL
S 1---..-i COUNTER: i-.----1 Q
US 6,558,916 B2
FROM FIG. Bg .____L_K ____,LK.---_1+_1 ----' FROM FIG. Be
796
YES
)-------.-! R
768
MOVE AUTOSAMPLER 315
TO POSITION LK
770
TURN ON 327
TURN OFF 311
REGISTER & SAVE
SIGNAL SLK
776
780
REGISTER SIGNAL
SL~Ni
TURN OFF 327
TURN OFF 311
FIG. BF
U.S. Patent May 6, 2003 Sheet 22 of 65
FLAG
MOVE AUTOSAMPLER 315
TO POSITION "o"
TURN PROPORTIONATING
VALVE 327 ON
786
TURN PROPORTIONING
VALVE 327 OFF
790
US 6,558,916 B2
784
785
787
792
FLAG
STANDARD L~- "POSITIVE" STANDARD Lk·_ "NEGATIVE"
SAVE DATA IN
DATABASE
s
TO FIG. Bf
794
FIG. BC
U.S. Patent May 6, 2003 Sheet 23 of 65 US 6,558,916 B2
FIG. 9A
FIG. 9B
FIG. 9C
FIG. 90
FIG. 9£
FIG. 9
U.S. Patent
808
May 6, 2003 Sheet 24 of 65 US 6,558,916 B2
800
802
SET INITIAL PARAMETERS AND
INCREMENTAL COUNTERS L, M AND N
TO ZERO
804
INCREMENT. COUNTER N ~------{ X
Ni =Ni-1+1
-----.------~FROM FIGS. 9c & 9e
SET INCREMENTAL
COUNTER N TO ZERO
FIC. 9A
YES
TURN PUMP ON
TURN DIVERTING
VALVE 333 ON
REGISTER MAX
CALIBRATION SIGNAL
TURN Dl_VERTING
VALVE 335 ON
REGISTER MIN
CALIBRATION SIGNAL
816
820
824
832
836
U.S. Patent
YES
846'
TO FIG. 9c
May 6, 2003 Sheet 25 of 65
TURN DIVERTING
VALVES 335 & 333 OFF:
FIG. 9B
US 6,558,916 B2
844
NO
848
TO FIG. 9e
U.S. Patent May 6, 2003 Sheet 26 of 65 US 6,558,916 B2
FROM FIG. 9b
850
MOVE AUTOSAMPLER 301 TO
POSITiON N 1
860
TURN GRADIENT MODE OF
PROPORTIONING VALVE 311
ON
REGISTER DOSE RESPONSE
OF A COMPOUND N1
STIMULATED SIGNAL
INCREMENT COUNTER M
.---~~ M·=M.
1
+1
J 1-
NO
YES
v
FROM FIG. 9d
-------852
854
856
CALCULATE ACTIVATION
PARAMETERS FOR THE
COMPOUND Ni
858
SAVE DATA IN A DATABASE
9a
FIG. 9C
U.S. Patent May 6, 2003 Sheet 27 of 65
-----------...----------
MOVE AUTOSAMPLER 315 /864
TO POSITION M j
TURN PROPORTIONATING ~866
VALVE 311 ON
TURN GRADIENT MODE OF r 868
PROPORTIONING VALVE 327
ON
I /870
REGISTER DOSE RESPONSE INHIBITION
BY Mj STANDARD OF A COMPOUND N 1
STIMULATED SIGNAL
,, /871
US 6,558,916 B2
/872
TURN PROPORTIONATING
V VALVES
TO FIG. 9c 311 AND 327 OFF
CALCULATE INHIBITION
PARAMETERS FOR
THE STANDARD Mj
FIG. 90
/874
SAVE DATA IN A
DATABASE
U.S. Patent May 6,2003 Sheet 28 of 65 US 6,558,916 B2
876
INCREMENT
FROM FIG. 9b COUNTER L~-----~
Lk =Lk-1+1
FIG. 9£
"\------.rx
TO FIG. 9o
NO
MOVE AUTOSAMPLER MOVE AUTOSAMPLER
301 TO POSITION N; 315 TO POSITION Lk
882
892
CALCULATE ACTIVATION
PARAMETERS FOR THE
STANDARD Lk
894
SAVE DATA IN A
DATABASE
896
CALCULATE INHIBITION
PARAMETERS FOR THE
COMPOUND Ni
TURN GRADIENT MODE OF
PROPORTIONING VALVE 327 ON
884
886
REGISTER DOSE RESPONSE
OF Lk STANDARD
STIMULATED SIGNAL
TURN GRADIENT MODE
OF PROPORTIONING
VALVE 311 ON
888
REGISTER Ni COMPOUND DOSE
DEPENDENT INHIBITION OF A
STANDARD Lk STIMULATED SIGNAL
890
TURN OFF
PROPORTIONATING
VALVES 327 AND 311
U.S. Patent
750
~ 500
c
,=.,
+
N
0
()
L__J
250
0
750
~ 500
c
,=.,
+
N
0
()
L__J 250
0
10-1
May 6, 2003
0 25
I I 11111
1 o0
Sheet 29 of 65
FIG. 10
50 75
ET-1 , pM
FIG. 11
I I I I 11111 I I I
10
1
Log [ET-1, pM]
US 6,558,916 B2
BQ-123, nM
0
10
40
100
BQ-123, nM
0
10
40
125
I 11111 I I 111 ii
10
2
10
U.S. Patent
500
2
c
~
c
,:.=,
+ 250
N
0
u
L__J
0
-0.2
500
~
c
c
,:.=,
+ 300 N
0
u
L__J
100
May 6,2003 Sheet 30 of 65 US 6,558,916 B2
FIC.12
' BQ-123
ET-1
t
0.0 0.2 0.4
0 25
ET-1
'
pM
BQ-123, »M
FIC. 13
100 300
BQ-123, nM
F/C, 14
IJ/2
/JOI IJOfJ
SUPPLY CELLS
FROM· NEW
CELL LINE
1302
SUPPLY CELLS
ADD STANDARD AGONIST H FROM ORIGINAL
TO CELLS CELL LINE
SUPPLY MIXTURE TO DETECTOR
1304
INCREMENT.STANDARD
AGONIST COUNTER
/309-...
REGISTER I
!JO? _/I POTENCY PROFILE
I INCREMENT CELL I
COUNTER
' I RECORD /308/I POTENCY. PROFILE
I ~'' .. II .. -· ,... ....... ,.... I I II.·--"' It:.~
/J!!-- ............_r ,,/" ( END)
RESET STANDARD I· I ..)/..) -
AGONIST COUNTER
~
•
rJ'l
•
~
~
"""'" ~=
"""'"
~
= '"<
!?'
N c
6
C/'.J ::r
(!;)
(!;)
"""
~
~
~
"""=
~
VI
e
00
"" 111
(Ii
CIJ
"'
""' ~
"" t:=
N
U.S. Patent May 6, 2003
1407
..-----I
SUPPLY HOST
CELLS
1408
COMBINE HOST CELLS
WITH AGONIST
1409
SUPPL y_ MIXTURE
TO DETECTOR
Sheet 32 of 65 US 6,558,916 B2
1401
SUPPLY TRANFECTED CELLS
1402
COMBINE TRANSFECTED CELLS
WITH STANDARD AGONIST
1403
SUPPLY MIXTURE TO DETECTOR
1404
1405
~Y_E_S~~~~~~~ INCREMENT.STANDARD
AGONIST COUNTER
NO
NO
1412 YES
END
FIG. 15
U.S. Patent May 6, 2003 Sheet 33 of 65 US 6,558,916 B2
FIG. 16A
FIG. 16B
FIG. 16C
FIG. 160
FIG. 16£
FIG. 16F
FIG. 16
U.S. Patent May 6,2003 Sheet 34 of 65 US 6,558,916 B2
1500
1502
1503
1506
TURN OFF VALVES
SET INITIAL
PARAMETERS
.----------------.
ENTER:
LMAX
CMAX
311 & 3~27, 333 ..,.... __ ___.__ _ ~
335 & 347, 313
329, 341 & 345
1508
TURN. PUMP
357 ON
1504
MOVE AUTOSAMPLERS
TO ZERO. POSITION
1-------~p
1510 .___-.--______. FROM FIG. 16f
1512
REGISTER
BASAL .SIGNAL
SAVE BASAL SIGNAL
"Bs. "
I
FIG. 16A
U.S. Patent May 6, 2003 Sheet 35 of 65 US 6,558,916 B2
1514
INCREMENT COUNTER L
0 t--~ · ~----1 M
Lk =Lk-1+1
FROM FIG. 16e ....___ _______ _____, FROM FIG. 16c
1516
N~-----{
TO FIG. 16f
1518
MOVE AUTOSAMPLER 315
AT POS.ITION Lk
TURN PROPORTIONING
VALVE 327 ON
1521
REGISTER AGONIST INDUCED
SIGNAL SLk
SAVE AGONIST INDUCED
SIGNAL Slk
FIG. 16B
1520
1522
1523
U.S. Patent
1540
FLAG
AGONIST Lk AS
"POSI Tl VE"
K
TO FIG. 16d
May 6, 2003 Sheet 36 of 65
TURN PROPORTIONING
VALVE 327 OFF
MOVE AUTOSAMPLER 315
TO ZERO POSITION
TURN PROPORTIONING
VALVE 327 ON
US 6,558,916 B2
1530
1532
1534
1536
WASH DELAY
TURN PROPORTIONING
VALVE 327 OFF
1538
1542
YES FLAG
r----~ AGONIST Lk AS
"NEGATIVE"
M
TO FIG. 16b
FIG. 16C
U.S. Patent May 6, 2003 Sheet 37 of 65 US 6,558,916 B2
16c
!550
MOVE
AUTOSAMPLER 315
AT Lk POSITION
TURN GRADIENT MODE OF
PROPORTIONING VALVE 327
ON
REGISTER DOSE RESPONSE OF
A STANDARD AGONIST ~
STIMULATED SIGNAL
CALCULATE POTENCY
PARAMETERS FOR THE
STANDARD AGONIST Lk
SAVE DATA IN THE
DATABASE
FIG. 160
1552
1554
1556
1558
U.S. Patent May 6, 2003 Sheet 38 of 65
TURN PROPORTIONING
VALVE 327 OFF
MOVE
AUTOSAMPLER 315
AT ZERO POSITIOB
TURN PROPORTIONING
VALVE 327 ON
WASHING DELAY
TURN PROPORTIONING
VALVE 327 OFF
0
TO FIG. 16b
FIG. 16£
US 6,558,916 B2
U.S. Patent May 6, 2003
TO FIG. 16a
p
SWITCH MUL TYCHANNEL
VALVE 347 TO NEXT
POSITION
SET COUNTER L TO
L = 0
Sheet 39 of 65 US 6,558,916 B2
FROM FIG. 16b
N
INCREMEN.TAL CELL
COUNTER Ci =Ci +1
NO
MOVE AUTO$AMPLER 315
TO ZERO POSITION
TURN PROPORTIONING
VALVE 327 ON
WASH ·DELAY
TURN PROPORTIONING
VALVE 327 OFF
TURN PUMP 357 OFF
STOP
FIG. 16F
U.S. Patent May 6, 2003 Sheet 40 of 65 US 6,558,916 B2
FIG. 17A
FIG. 17B
FIG. 17C
FIG. 170
FIG. 17£
FIG. 17F
FIG. 17C
FIG. 17
U.S. Patent May 6,2003
FIG. 17A
_:_606' ~ ·-·--
33, 335, & 34 7
Sheet 41 of 65 US 6,558,916 B2
START
SET INITIAL
PARAMETERS
ENTER
LMAX
1600
1602
1603
1604
SET AUTOSAMPLERS
AT ZERO. POSITION
RN OFF VALVE~J):
311 & 327
. 329, 341 & 345
[ ____ , __ -,---___ _____I
SET VALVE 347 TO
POSITION 1
TURN PUMP 357
ON
1612
REGISTER HOST CELL
BASAL. SIGNAL
SAVE BASAL SIGNAL
FOR HOST CELLS
"(BS)Hc"
-···---- - ----
1608
1610
1614
1616
U.S. Patent
FIG. 17B
May 6, 2003 Sheet 42 of 65
SET VALVE 347 TO
POSITION 2
1622
DELAY
US 6,558,916 B2
1620
1624
REGISTER
TRANSFECTED CELL
BASAL SIGNAL
SAVE BASAL SIGNAL FOR
TRANSFECTED CELLS
,, (BS) TC"
TURN PUMP 357 OFF
1626
1628
U.S. Patent May 6, 2003 Sheet 43 of 65 US 6,558,916 B2
1630
INCREMENT
0 r----~ COUNTER L ~-------\ M
FROM FIG. 17e Lk =~+1+ 1 FROM FIG. 17d
!632
YES
N~-----<
TO FIG. 179
MOVE AUTOSAMPLER 315
AT POSiTION Lk
TURN PUMP 357 ON
TURN PROPORTIONING
VALVE 327 ON
!634
1636
1638
!640
DELAY
REGISTER AGONIST INDUCED
SIGNAL IN TRANSFECTED CELLS
(SLK )re
1642
1643
SAVE SIGN.AL (SLK)Tc
IN A DATABASE
FIG. 17C
U.S. Patent May 6, 2003 Sheet 44 of 65
TURN PROPORTIONING
VALVE 327 OFF
MOVE AUTOSAMPLER
315 TO ZERO
POSITION
TURN PROPORTIONING
VALVE 327 ON
US 6,558,916 B2
1644
1646
1648
1650
1656
WASH-DELAY
TURN PROPORTIONING
VALVE ·327 OFF
TURN PUMP
FLAG
AGON I ST L AS ..,.Y._E_S--< NO
"POSITIVE TC,,
K
TO FIG. 17e
FIG 170
165.2
1654
1660
FLAG
AGONIST Lk AS
"NEGATIVE"
M
TO FIG. 17c
U.S. Patent
!676
May 6,2003 Sheet 45 of 65
FROM FIG. 17d
MOVE
AUTOSAMPLER 315
AT POSITION LK
SET VALVE 347 TO
POSITION 1
TURN VALVE 327 ON
TURN PUMP 357 ON
!662
!664
!666
1668
1670
DELAY
US 6,558,916 B2
1672
REGISTER AGONIST INDUCED
SIGNAL IN HOST CELLS,
(SLk ~c
!678
FLAG AS
"POSITIVErc ~-Y--=E=-=S-< NO
FLAG AS
"POSITIVE TC
NEGATIVEHc" NEGA Tl VE He"
SAVE DATA IN
A DATABASE
FIG 17£
U.S. Patent May 6, 2003 Sheet 46 of 65
SET VALVE 347 TO
POSITION 2
TURN VALVE 327 OFF
MOVE AUTOSAMPLER
315 AT ZERO POSITION
TURN VALVE 327 ON
WASH DELAY
TURN VALVE 327 OFF
TURN PUMP 357 OFF
0
TO FIG. 17c
FIG. 17F
US 6,558,916 B2
U.S. Patent May 6, 2003 Sheet 47 of 65
FROM FIG. 1 7c
1700
SET COUNTER L
TO L=O
1702
MOVE AUTOSAMPLERE
315 AT ZERO POSITION
TURN VALVE 327 ON
!704
!706
WASH DELAY
!708
TURN VALVE 327 OFF
!710
TURN PUMP 357 OFF
1712
FIG. 17C
US 6,558,916 B2
U.S. Patent May 6, 2003 Sheet 48 of 65 US 6,558,916 B2
UNLABELED CELLS
HARVEST CELLS
LOAD CELLS WITH
BIORESPONSE INDICATORS
WASH AND _RESUSPEND
CELLS FOR ANALYSIS
I'
EXPOSE CELLS TO TEST MIXTURES
COMPRISING TES_T COMPOUND(S)
OR TEST COMPOUND(S) + STANDARD(S)
IN MIXING APPARATUS
DELIVER SAMPLE FROM MIXING
APPARATUS TO DETECTOR
SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
I DATA ANALYSIS
I'
DATA OU_TPUT TO
FIG 18 DATABASE
U.S. Patent May 6,2003 Sheet 49 of 65 US 6,558,916 B2
LABEL CELLS WITH I UNLABELED CELLS I
CELL TYPE INDICATOR
I I
I HARVEST CELLS I
LABEL CELLS WITH
~------1cELL TYPE SPECIFIC
ANTIBODIES
LOAD CELLS WI TH
RESPONSE INDICATOR
WASH AND RESUSPEND
CELLS FOR ANALYSIS
EXPOSE CELLS TO TEST MIXTURES
COMPRISING TEST COMPOUND(S)
OR TEST COMPOUND(S) + STANDARD(S)
IN MIXING APPARATUS
DELIVER SAMPLE FROM
MIXING APPARATUS
TO DETECTOR
SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
' "
I DATA ANALYSIS I
FIG. 19 DATA OUTPUT
TO DATABASE
U.S. Patent May 6, 2003 Sheet 50 of 65 US 6,558,916 B2
UNLABELED CELLS
I HARVEST CELLS I
LOAD CELLS WITH SEVERAL
RESPONSE iNDICATORS
WASH AND RESUSPEND
CELLS FOR ANALYSIS
EXPOSE CELLS TO TEST MIXTURES
COMPRISING TEST COMPOUND(S)
OR TEST COMPOUND.(S) + STANDARD(S)
IN MIXING APPARATUS
'
DELIVER SAMPLE FROM
MIXING APPARATUS
TO DETECTOR
,
SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
'
I DATA ANALYSIS I
DATA OUTPUT TO
DATABASE
FIG. 20
U.S. Patent May 6, 2003 Sheet 51 of 65
LABEL CELLS WITH
CELL TYPE
INDICATORS
UNLABELED CELLS
l.___ __ --11-i: HARVEST CELLS I
US 6,558,916 B2
LABEL CELLS WITH
CELL TYPE SPECIFIC
ANTIBODIES
LOAD CELLS WITH RECEPTOR
ACTIVITY IN°DICATOR(S)
WASH AND RESUSPEND
CELLS FOR AN AL YSI S
EXPOSE CELLS TO TEST MIXTURES
COMPRISING TEST COMPOUND(S) OR
TEST COMPOUND(S) + STANDARD(S)
IN MIXING APPARATUS
DELIVER SAMPLE FROM MIXING
APPARATUS TO DETECTOR
SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
I DATA ANALYSIS I
DA TA OUTPUT TO
DATABASE
FIG. 21
U.S. Patent May 6, 2003 Sheet 52 of 65 US 6,558,916 B2
LABEL CE.LLS WITH LABEL CELLS WI TH
CELL TYPE INDICATORS CELL TYPE SPECIFIC
ANTIBODIES
LABEL CELLS WI TH
CELL TYPE SPECIFIC I UNLABELED CELLS I
ANTIBODIES i
I L-1 HARVEST CELLS:
I
t UNLOADED CELL SET
LOAD CELLS WITH ca2+, IF NECESSARY
OTHER ION. OR OTHER
BIORESPONSE INDICATORS
' WASH AND_RESUSPEND CELLS FOR ANAL YSlS
• EXPOSE CELLS TO TEST MIXTURES EXPOSE CELLS TO TEST MIXTURES
COMPRISING TEST COMPOUND(S) OR COMPRISING TEST COMPOUND(S) OR
TEST COMPOUND(S) + STANDARD(S) TEST COMPOUND(S) + STANDARD(S)
AND TO CATIONS IN AND TO BIOSTAINS IN
MIXING APPARATUS MIXING APPARATUS
I
• DELIVER SAMPLE FROM
MIXING APPARATUS
TO DETECTOR
SINGLE PARTICLE AND/OR
MUL Tl PARAMETRIC ANALYSIS
BY DETECTOR
• I DATA ANALYSIS I
DATA OUTPUT
TO DATABASE
FIG. 22
U.S. Patent May 6, 2003 Sheet 53 of 65 US 6,558,916 B2
LABEL CELLS WITH I UNLABELED CELLS I
CELL TYPE INDICATORS
I
FIG. 23
I
I HARVEST CELLS I
LABEL CELLS WI TH
~---~ CELL TYPE SPECIFIC
ANTIBODIES
''
LOAD CELLS WITH RESPONSE
INDICATOR(S) INDICATIVE OF
THE ACTIVITY OF SECOND
MESSENGERS
WASH AND RESUSPEND
CELLS FOR ANALYSIS
EXPOSE CELLS TO TEST MIXTURES
COMPRISING TEST COMPOUND(S)
OR TEST COMPOUND(S) + STANDARD(S)
IN MIXING APPARATUS
DELIVER SAMPLE FROM
MIXING APPARATUS
TO DETECTOR
SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BT DETECTOR
I DATA ANALYSIS I
DATA OUTPUT
TO DATABASE
U.S. Patent May 6, 2003 Sheet 54 of 65 US 6,558,916 B2
LABEL CELLS WITH CELL I UNLABELED CELLS I
TYPE INDICATORS • I : HARVEST CELLS I
• LABEL CELLS WITH CELL
TYPE SPECIFIC ANTIBODIES
• LOAD CELLS WITH BIORESPONSE
INDICATOR(S) INDICATIVE OF APOPTOSIS
OR CELLULAR TOXI Cl TY
t
WASH AND RESUSPEND
CELLS FOR. ANALYSIS
' EXPOSE CELLS TO TEST MIXTURES COMPRISING TEST· COMPOUND(S) OR
TEST COMPOUND(S) + STANDARD(S)
' INCUBATE (,:ELLS WITH
TEST MIXTURES
• RAPIDLY INPUT ~XPOSED CELLS
TO MIXING APPARATUS
' EXPOSE CELLS TO APOPTOSIS SENSING
COMPOUND(S) OR APOPTOSIS
DETECTING COMPOUND(S)
IN MIXING APPARATUS
• DELIVER SAMPLE FROM
APPARATUS TO DETECTOR
t
SINGLE PARTICLE AND /OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
• I DATA ANALYSIS I
IC.24 t
DATA OUTPUT
TO DATABASE
U.S. Patent May 6, 2003 Sheet 55 of 65 US 6,558,916 B2
LABEL CELL$ WITH CELL I UNLABELED CELLS I
TYPE INDICATORS
' I - HARVEST CELLS I ' LABEL CELLS WI TH CELL TYPE SPECIFIC
ANTIBODIES
+
LOAD CELLS WITH BIORESPONSE
INDICATOR(S) INDICATIVE
OF NECROSIS
+
WASH AND RESUSPENDI
CELLS FOR ANALYSIS
' EXPOSE CELLS TO TEST MIXTURES COMPRISING TEST ·COMPOUND(S) OR
TEST COMPOUND(S) + STANDARD(S)
' I INCUBATE CELLS WITH I TEST MIXTURES
+
RAPIDLY INPUT EXPOSED
CELLS TO MIXING
APPARATUS
' EXPOSE CELLS TO NECROSIS SENSING COMPOUND(S) OR NECROSIS DETECTING
COMPOUNO(S) IN MIXING APPARATUS
• DELIVER SAMPLE FROM MIXING
APPARATUS TO DETECTOR
' SINGLE PARTICLE AND/OR MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
' FIG. . 25 I DATA ANALYSIS I ' DATA OUTPUT TO DATABASE
U.S. Patent May 6, 2003 Sheet 56 of 65 US 6,558,916 B2
LABEL CELLS WITH CELL
TYPE INDICATORS
UNLABELED CELLS
FIG. 26
'---------1-i HARVEST CELLS
LABEL CELL$ WI TH CELL
TYPE SPECIFIC ANTIBODIES
LOAD CELLS WITH BIORESPONSE
JN DI CA TOR(S) INDICATIVE
OF NECROSIS AND APOPTOSIS
WASH AND RESUSPEND
CELLS FOR ANALYSIS
EXPOSE CELLS TO TEST MIXTURES
COMPRISING TEST COMPOUND(S)
OR TEST COMPOUND(S) + STANDARD(S)
INCUBATE CELLS WITH
TEST MIXTURES
RAPIDLY INPUT
EXPOSED ·CELLS TO
MIXING APPARATUS
EXPOSE CELLS TO NECROSIS AND
APOPTOSIS SENSIN_G COMPOUNDS OR
NECROSIS AND APOPTOSIS DETECTING
COMPOUNDS IN MIXING APPARATUS
DELIVER SAMPLE FROM MIXING
APPARATUS TO DETECTOR
SINGLE PARTICLE AND/OR
MUL Tl PARAMETRIC ANALYSIS
BY DETECTOR
DATA ANALYSIS
DATA OUTPUT
TO DATABASE
U.S. Patent May 6, 2003 Sheet 57 of 65 US 6,558,916 B2
LABEL CELLS WITH
CELL .TYPE I UNLABELED CELLS I
INDICATORS
I : HARVEST CELLS I
LABEL . CELLS WI TH CELL
TYPE SPECIFIC ANTIBODIES
LOAD CELLS WITH BIORESPONSE
INDICA TOR(S)
WASH AND RESUSPEND
CELLS FOR ANALYSIS
EXPOSE CELLS TO TEST MIXTURES COMPRISING
TEST COMPOUND(S) OR TEST
COMPOUND(S) + STANDARD(S)
IN MIXING APPARATUS
DELIVER SAMPLE FROM
MIXING APPARATUS TO
DETECTOR
SINGLE PARTICLE AND /OR
MULTIPARAMETRIC ANALYSIS
BY DETECTOR
EXPAND AND
FLUORESCENCE-ACTIVATED CELL SORTING REANALYZE
DECISIONS BY DETECTOR TO SELECT i-.. SELECT!;D CELL
DESIRED SUBPOPULATION OF CELLS POPULATIONS TO
CONFIRM
HOMOGENEITY
I DATA ANALYSIS I
DATA OUTPUT
TO DATABASE
FIG. 27
U.S. Patent May 6,2003 Sheet 58 of 65 US 6,558,916 B2
EXPOSE CELLS TO
TEST COMPOUNDS
INCUBATE CELLS WITH
TEST COMPOUND(S)
l
FIG. 28
PREPARE ADDRESSABLE
ANTIBODY COATED
MICROSPHERES
LYSE CELLS AND ADD
ANTIBODY-COATED
MICROSPHERES
I INCUBATE I
t
INPUT BEAD SAMPLE
INTO MIXING APPARATUS
ADD SECONDARY
ANTIBODY· TO BEAD
SAMPLE
SINGLE PARTICLE AND/OR
MUL Tl PARAMETRIC ANALYSIS
BY DETECTOR
I DATA ANALYSIS I
t
DATA OUTPUT
TO DATABASE
U.S. Patent May 6, 2003 Sheet 59 of 65 US 6,558,916 B2
EXPOSE CELLS TO TEST
COMPOUND(S) PREPARE ADDRESSABLE
OLIGONU~LEOTIDE
PROBE-COATED
MICROSPHERES INCUBATE CELLS WITH
TEST COM.POUND(S)
FIG. 29
LYSE CELL AND ADD
OLIGONUCLEOTIDE
PROBE-COATED
MICROSPHERES AND
BIOTINYLATED
OLIGONUCLEOTIDE PROBES
I INCUBATE I
t
INPUT BEAD SAMPLE
INTO MIXING APPARATUS
ADD ANTI-BIOTIN
ANTIBODY· TO BEAD
SAMPLE
SINGLE PARTICLE AND/OR
MULTIPARAMETRIC ANALYSIS
BY DETECTOR
I DATA ANALYSIS I
+
DATA OUTPUT
TO DATABASE
U.S. Patent May 6, 2003 Sheet 60 of 65 US 6,558,916 B2
ISOLATE GENOMIC
DNA FROM CELLS
' I PCR AMPLIFY DNA I
• MINISEQUENCING
REACTION TO INSERT
FLUORESCENT BASE AT
SNP SITE
+
INPUT EXTENDED
FLUORESCENT PRIMERS FROM
WELL TO MIXING APPARATUS
t
ADD STREPTAVIDIN-COATED
BEADS TO EXTENDED PRIMERS
IN MIXING APPARATUS
t
PERMIT BINDING OF
PRIMERS TO BEADS
' SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
' I DATA ANALYSIS I
' DATA OUTPUT
TO DATABASE
FIG. 30
U.S. Patent May 6,2003 Sheet 61 of 65 US 6,558,916 B2
PREPARE UNIQUE ADDRESSABLE
POPULATIONS OF BEADS COUPLED
WITH DISTINCT, FLUORESCENT
ENZYME-SPECIFIC SUBSTRATES
!
INPUT BEAD POPULATIONS
INTO MIXING APPARATUS
1
MIX SUBSTRATE COATED BEADS WITH
ENZYME TARGET IN THE PRESENCE
OF TEST MIXTURES COMPRISING
TEST COMPOUND(S) OR TEST
COMPOUND(S) + STANDARD(S)
'
SET REACTION TIME IN MIXING
APPARATUS ACCORDING TO
DESIRED KINETIC PARAMETERS
SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
DA TA ANALYSIS
DATA OUTPUT
TO DATABASE
FIG. 31
U.S. Patent May 6,2003 Sheet 62 of 65 US 6,558,916 B2
PREPARE UNIQUE ADDRESSABLE
POPULATIONS OF-BEADS COUPLED
WITH DISTINCT BIOMOLECULES
r
INPUT BEAD POPULATIONS
INTO MIXING APPARATUS
'
MIX MOLECULAR TARGET-COATED
BEADS WITH COGNATE MOLECULAR
TARGET IN THE PRESENCE OF TEST
COMPOUND(S) IN MIXING APPARATUS
SET REACTION TIME IN MIXING
APPARATUS ACCORDING TO DESIRED
KINETIC PARAMETERS
SINGLE PARTICLE AND/OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
I DATA ANALYSIS I
DATA OUTPUT
TO DATABASE
FIG. 32
U.S. Patent May 6, 2003 Sheet 63 of 65 US 6,558,916 B2
PREPARE UNIQUE ADDRESSABLE
POPULATIONS OF BEADS COUPLED
~TH REC~PTOR TARGETS
--·-
INPUT BEAD POPULATIONS
INTO MIXING APPARATUS
!
t
MIX RECEPTOR TARGET-COATED
BEADS WITH FLUORESCENT LIGANDS
IN THE PRESENCE OF TEST MIXTURES
COMPRISING TEST COMPOUND(S) OR
TEST COMPOUND(S) + STANDARD(S)
, '
SET REACTION TIME IN MIXING
APPARATUS ACCORDING TO DESIRED
KINETIC PARAMETERS
t
~·~· L
SINGLE PARTICLE AND /OR
MUL TIPARAMETRIC ANALYSIS
BY DETECTOR
r
DATA ANALYSIS
DATA OUTPUT
TO DATABASE
r----- -----
Figure 34: Toxicity Screening by Flow Cytometry
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1-
u.
40
30
20
10
0' I "'"'' Y;
Calcium Glutathione Pl. Mcmb. 1 Pl. Mcrnb. 2 Side Sctr. Forw. Sctr.
Parameter
---- ------------
0 Oubain IZJ Etoposide El Acetaminophen 0 lbuprofcn G Control
---_-------=------
~ •
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~
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Plasma Membrane Plasma Membrane I
Probe 1 Probe 2
----
111 uM 40% 34%
DSOOnM 29% 28%
-·----·--··
111250 nM 25% 21%
D 125 nM 10% 11%
m63nM 11% 8%
&131 nM 7% 6%
-----~~----
£316 nM 7% 5%
l?JB nM 8% 6% L___ ____
ctmomycm
}::'.:
~ti~
t}
§~~
{Calcium]i I Light Scatter
54% 55%
60% 55%
~,
42% 50%
---------------~
26% 41%
--------- - ---- - --------
17% 33%
11% 30%
-----
12% 24%
11% 24%
~ •
rJ'l •
~
~
"""'" ~=
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US 6,558,916 B2
1
CELL FLOW APPARATUS AND METHOD
FOR REAL-TIME MEASUREMENTS OF
PATIENT CELLULAR RESPONSES
RELATED APPLICATIONS 5
2
surface molecules, the cell response would be abnormal
causing malfunctioning of a tissue or an organ.
In eukaryotic cells, receptor molecules determine the
selective response of the cell. Each type of receptor can
interact only with a specific set of ligand molecules. For
example, adrenergic receptors interact with adrenaline and
noradrenaline, cholinergic receptors interact with
acety lcholine, serotoninergic receptors interact with
5-hydroxytriptamine, dopamineergic with DOPA and so on.
The present application is a continuation-in-part of U.S.
patent application Ser. No. 09/568,778 filed May 10, 2000,
now U.S. Pat. No. 6,242,209 the disclosure of which is
incorporated herein by reference in its entirety, which is a
continuation of U.S. patent application Ser. No. 09/370, 786,
filed Aug. 5, 1999, now U.S. Pat. No. 6,280,967 which is a
continuation-in-part of U.S. patent application Ser. No.
09/317,793, filed May 24, 1999 now U.S. Pat. No. 6,096,509
which is a continuation of U.S. patent application Ser. No.
08/904,904, filed Aug. 1, 1997 (now U.S. Pat. No. 5,919,
646, issued Jul. 6, 1999), which was a continuation-in-part
of U.S. patent application Ser. No. 08/691,356 filed Aug. 2,
1996 (now U.S. Pat. No. 5,804,436, issued Sep. 8, 1998), the
disclosures of which are incorporated herein by reference in
their entireties.
rn The cells derived from the different tissues invariably
express specific sets of tissue receptors. Different types of
receptors are connected to different signal transduction
pathways. For example, nicotinic cholinergic receptor, upon
binding acetylcholine molecule, directly activates sodium
15 channel (Claudio et al., 1987, is incorporated herein by
reference). G-protein coupled receptors activate enzymes of
second messenger pathways, for example, adenylate cyclase
or phospholipase C with subsequent activation of cAMP or
phosphoinositide cascades (Divecha and Itvine, 1995, is
FIELD OF THE INVENTION
This invention relates generally to an apparatus for
screening and the pharmacological profiling of compounds
modulating a cellular physiological response. This invention
also relates to devices for rapid assessment of the properties
20 incorporated herein by reference). Receptor tyrosine kinases
activate cascade of MEK/MAPK kinases leading to cell
differentiation and proliferation (Marshall, 1995 and
Herskowitz, 1995, are incorporated herein by reference)].
Cytokine receptors activate JAK/STAT cascade which in
25 turn can regulate other pathways as well as activate gene
transcription (Hill & Treisman, 1995, is incorporated herein
by reference).
Together with the receptors, the cell surface meirane
carries ion pumps, ion transporters and ion channels. These
molecular assemblies work in concert to maintain intracel-
lular ion homeostasis. Any changes in the activity of these
systems would cause a shift in the intracellular concentra-
tions of ions and consequently to the cell metabolic
of compounds that modulate the activities of cells. The
compounds investigated may be involved in regulating the
activity of signal transduction pathways, cellular responses, 30
cell surface receptors, ion channels, non-selective pores,
second messenger pathways, dov-'llstream signal transduc-
tion pathways, apoptosis, cellular necrosis or any other
cellular responses. The devices and methods of the present
invention may also be used to perform biochemical
analyses, such as Western analyses, Northern analyses,
detection of single nucleotide polymorphisms (SNPs),
detection of enzymatic activities, or molecular assembly
35
response.
Ion pumps act to maintain transmembrane ion gradients
utilizing ATP as a source of energy. The examples of the ion
pumps are: Na+;K+-ATPase maintaining transmembrane
gradient of sodium and potassium ions, Ca2 + -ATPase main-
assays.
In some embodiments, this invention relates to methods
and apparatus for detecting, evaluating and characterizing
the ability and potency of substances to act as agonists or
antagonists against receptors and ion channels localized on
a cell surface membrane.
40 taining transmembrane gradient of calcium ions and
H+ -ATPase maintaining transmembrane gradient of protons.
BACKGROUND INFORMATION
Ion transporters use the electrochemical energy of trans-
membrane gradients of one ion species to maintain gradients
of other ion counterpart. For example, the Na+/Ca2 +-
45 exchanger uses the chemical potential of the sodium gradi-
ent directed inward to pump out calcium ions against their
chemical potential.
Biological cells contain receptor molecules located on
their external membrane. The function of these receptors is
to "sense" the cell environment and supply the cell with an 50
input signal about any changes in the environment. In
eukaryotic organisms such cell environment is comprised of
the neighboring cells and the function of the receptor is to
allow cells to communicate with each other directly (the
paracrine regulatory system) or indirectly (the endocrine 55
regulatory system) thus achieving harmonized response of a
tissue, organ or a whole organism. In prokaryotic cells, the
surface localized receptors provide a means for detecting
extracellular environment.
Ion channels, upon activation, allow for the ions to move
across the cell membrane in accordance with their electro-
chemical potential. There are two main types of ion chan-
nels: voltage operated and ligand-gated. Voltage operated
channels are activated to the open state upon changes in
transmembrane electric potential. Sodium channels in the
neuronal axon or L-type calcium channels in neuromuscular
junctions exemplify this kind of channel. Ligand-gated
channels are activated to the open state upon binding a
certain ligand with the chemoreceptor part of their mol-
ecules. The classical example of ligand-gated channels is
nicotinic cholinergic receptor which, at the same time, is the
60 sodium channel. Having received such a signal, neurotransmitters,
hormones, chemoattractant or chemorepellant substances
for example, the surface localized receptors transmit this
information about extracellular environment into the cell
through specific intracellular pathways in such a way that
the cell responds in the specific fashion to accommodate 65
these changes. When there is an altered supply of the
external signal molecules or an altered activity of the cell
There are numerous methods for detecting ligand/receptor
interaction. The most conventional are methods where the
affinity of a receptor to a substance of interest is measured
in radioligand binding assays. In these assays, one measures
specific binding of a reference radiolabeled ligand molecule
in the presence and in the absence of different concentrations
of the compound of interest. The characteristic inhibition
US 6,558,916 B2
3
parameter of the specific binding of the reference radiola-
beled ligand with the compound of interest, IC50 , is taken as
a measure of the affinity of the receptor to this compound
(Weiland & Molinoff, 1981 and Swillens et all., 1995, are
incorporated herein by reference). Recent advances in 5
microchip sensor technology made it possible to measure
direct interactions of a receptor molecule with a compound
of interest in real time. This method allows for determination
4
cell from extracellular environment through specialized
voltage-independent Ca2 +-selective channels triggered by
specific ligands. In nonexcitable cells, hyperpolarization of
the plasma cell membrane also enhances entry of calcium
ions through passive transmembrane diffusion along the
electric potential. For example, opening of potassium chan-
nels brings the membrane potential to more negative values
inside the cell, thus facilitating Ca2 + entry across the plasma
membrane. Excitable cells contain voltage-dependent Ca2 + of both association and dissociation rate constants with
subsequent calculation of the affinity parameter (F _gerstam
et al., 1992, is incorporated herein by reference). While
being very precise and convenient, these methods do not
allow to distinguish between agonist and antagonist activity
of the compound.
The type of biological activity of the compounds, agonist
or antagonist, may be determined in the cell based assays. In
the methods described in Harpold & Brust, 1995, which is
incorporated herein by reference, cells cotransfected with a
receptor gene and reporter gene construct, are used to
provide means for identification of agonist and antagonist
potential pharmaceutical compounds. These methods are
inconvenient because they require very laborious manipu-
lations with gene transfection procedures, are highly time
consuming and use artificially modified cells. Besides, to
prove that the agonistic effect of a particular compound is
connected to the stimulation of a transfected receptor, sev-
eral control experiments with a positive and negative control
cell lines should be performed as well.
10
channels on their plasma membrane, which, upon membrane
depolarization, open for a short period of time and allow
inflow of Ca2 + from external media into cytoplasm. The
endoplasmic reticulum membrane as well as plasma mem-
brane of the excitable cells contains InsP 3 receptors and
Ca2 +-sensitive ryanodine receptors (RyR) releasing Ca2 +
15 from intracellular stores upon membrane receptor triggered
phospholipase C activation or depolarization-induced short
burst of Ca2 + entry into cell cytoplasm from extracellular
media respectively.
20
It is well established that G-protein coupled serpentine
receptors initiate Ca2 + mobilization through the activation of
phospholipase Cf3 (Sternweis and Smrcka, 1992, is incor-
porated herein by reference) whereas tyrosine kinase recep-
tors activate phospholipase Cy with subsequent intracellular
Ca2 + mobilization (Berridge & Irvine, 1989, is incorporated
25
herein by reference).
Most closely related to the methods of this invention are
the methods described in Paree et al., 1994, which is
incorporated herein by reference. These prior art methods
use natural cells and are based on registering the natural cell
responses, such as the rate of metabolic acidification, to the
biologically active compounds. The disadvantage of the
prior art is low throughput speed, each measurement point 35
taking about three minutes. Another disadvantage of the
prior art is the use of cells immobilized on the internal
surface of the measuring microflow chamber. This leads to
the necessity of using separate silicon sensors, or cover slips,
with the cells adherent to them for each concentration point 40
of the agonist or antagonist, for the receptors that undergo
desensitization upon binding to the agonist molecule. This
results in high variability of the experimental results.
There are many plasma membrane G-protein coupled
serpentine receptors, tyrosine kinase growth factor receptors
and voltage- and ligand-regulated channels known to initiate
30
intracellular Ca2 + mobilization.
Ca2 + plays an essential role in many functional processes
of a cell. For example, Ca2 + affects the cell cycle (Means,
1994, is incorporated herein by reference) and activates
specific transcription factors (Sheng et al., 1991, is incor-
porated herein by reference). Scores of receptors and ion
channels use the Ca2 + signal to initiate events as basic as cell
Ionized calcium, unlike other intracellular ion events, e.g.
changes in the intracellular concentrations of protons, 45
sodium, magnesium, or potassium, serves as the most com-
mon element in different signal transduction pathways of the
cells ranging from bacteria to specialized neurons (Clapham,
1995, is incorporated herein by reference). There are two
major pools which supply signal transduction pathways in 50
the cell with the calcium ions, extracellular space and the
endoplasmic reticulum. There are several mechanisms to
introduce small bursts of calcium into cytosol for signal
transduction.
Both excitable and nonexcitable cells have on their 55
motility, contraction, secretion, division etc.
Increases in cytosolic and, consequently, in nuclear con-
centration of the Ca2 + can also be a cell death promoting
signal. For example, prolonged increase in free Ca2 + acti-
vates degradation processes in programmed cell death,
apoptosis, activates nucleases that cleave DNA and degrade
cell chromatin, promotes DNA digestion by direct stimula-
tion of endonucleases, or indirectly by activation of Ca2 + -
dependent proteases, phosphatases and phospholipases,
resulting in a loss of chromatin structural integrity (Nicotera
et all., 1994, is incorporated herein by reference).
A development of intracellular fluorescent calcium indi-
cators (Grynkiewicz et all., 1985, is incorporated herein by
reference) made it possible for intracellular concentration of
free calcium to be measured directly in the living cell. Thus
the ability to register changes in intracellular calcium con-
centration provide the means for monitoring effects of
different compounds useful in treating various diseases,
whose action is thought to be a result of an interaction with
membrane receptors and ion channels.
With the advent of combinatorial chemistry approaches to
identify pharmacologically useful compounds, it is increas-
ingly evident that there is a need for methods and appara-
plasma membrane predominantly two receptor classes,
G-protein coupled serpentine receptors (GPCSR) and the
receptor tyrosine kinases (RTK), that control calcium entry
into cell cytoplasm. Both GPCSR and RTK receptors acti-
vate phospholipase C to convert phosphatidylinositol into
inositol(l,4,5)-trisphosphate (InsP 3 ) and diacylglicerol.
InsP 3 acts as an intracellular second messenger and activates
specialized receptor that spans the endoplasmic reticular
membrane. The activation of this receptor triggers release of
calcium ions from the endoplasmic reticulum (Berridge,
1993, is incorporated herein by reference). The calcium ions
can also enter the cytoplasm of excitable and nonexcitable
60 tuses capable of performing automated characterization of
pharmacological profiles and corresponding potencies of the
compounds in synthesized combinatorial libraries. This
would enable the rapid screening of a large number of
compounds in the combinatorial library the identification of
65 those compounds which have biological activity, and the
characterization of those compounds in terms of potency,
affinity and selectivity.
US 6,558,916 B2
5
It is an object of this invention to provide methods for
screening and the quantitative characterization of potentially
pharmacologically effective compounds that specifically
interact with and modulate the activity of cell membrane
receptors, ion pumps and ion channels using living cells.
It is an additional object of this invention to provide
methods capable of characterizing an affinity of the active
compounds to the binding sites of the cell.
It is another additional object of this invention to provide
methods to distinguish between agonistic and antagonistic
activity of the compounds.
It is yet another additional object of this invention to
provide methods to determine the nature of the receptor, ion
channel or ion pump entity which is sensitive to the active
compounds discovered during the screening process.
It is yet another additional object of this invention to
provide methods to characterize cell receptor pattern for
particular cell source tissue.
5
6
cells is mixed with flows of the compound and a standard
substance. The effects of the compound alone and in mixture
with the standard substance are measured and provide the
means for pharmacological profiling of the compounds, drug
screening and cell receptor pattern characterizing.
In a preferred embodiment of the invention, the com-
pounds to be screened and standard agonist and antagonist
substances are organized in a 96-well plate format, or other
regular t\vo dimensional array, such as a 48-well and 24-well
rn plate format or an array of test tubes. In another preferred
embodiment of the invention, the non-adherent cells are
grown in a suspension of freely flowing cells by growing
them in an appropriate cell cultivating system.
In another preferred embodiment of the invention, the
15 naturally adherent cells which need attachment to a surface
for their growth, are grown in the appropriate cell cultivating
system containing commercially available micro spherical
beads to which the cells adhere during the growth.
It is yet another additional object of this invention to 20
perform each of the above methods on each member of a
series of cell types.
In yet another preferred embodiment of the invention, the
naturally adherent cells which need attachment to a surface
for their growth, are grown in the cell culture flasks with a
subsequent detachment of the cells from the flask bottom
with an appropriate detaching reagent. It is yet another additional object of the invention to
determine the pattern of cell surface receptors expressed in
one or more cell types.
It is yet another additional object of the invention to
confirm that a test compound influences the activity of a
particular receptor.
It is yet an additional object of the invention to determine
the activity of a given receptor in a variety of cell types in
which it is expressed.
It is a specific object of this invention to provide an
apparatus for fulfillment of the objectives above.
In accordance with the present invention, either eukary-
25 otic or prokaryotic cells can be used. The cells can be
transfected with a gene coding to express a receptor of
interest, for example, an orphaned receptor. In addition, the
variety of compounds having biologically relevant activity
may be used including but not limited to neurotransmitters,
30 hormones, toxins, receptor activators and inhibitors, ion
channel<> and ion pump modulators, irritants and/or drugs.
It is yet another additional object of this invention to 35
provide an apparatus for fulfillment of each of the objectives
above for each member of a series of cell types.
The cells grown in accordance with the preferred embodi-
ments described above, are mixed \Vith an appropriate
fluorescent dye, for example FURA-2AM for measurements
concentrations of intracellular calcium or BCECF-AM for
measurements of intracellular pH, and are incubated in the
appropriate conditions to allow the dye to penetrate into the
cell. The cells loaded with a dye are supplied to the appa-At least some of these and other objectives are addressed
by the various embodiments of the invention disclosed
herein.
SUMMARY OF THE INVENTION
The present invention addresses the above and other
needs by providing a method and corresponding apparatuses
which allows the automated characterization of pharmaco-
logical profiles and corresponding potencies of compounds
in synthesized combinatorial libraries. This enables the rapid
screening of a large number of compounds in the combina-
torial library, the identification of those compounds which
have biological activity, and the characterization of those
compounds in terms of potency, affinity and selectivity.
A variety of effects caused by the compounds to be
screened may be detected and quantitatively characterized
according to the present invention. Preferably, these effects
include but are not limited to changes in intracellular con-
centration of ionized calcium, cAMP or pH, transmembrane
potential and other physiological and biochemical charac-
teristics of living cell which can be measured by a variety of
conventional means, for example using specific fluorescent,
luminescent or color developing dyes.
40 ratus. In the apparatus, the cells are successively mixed with
a solutions of the compounds to be tested.
The methods of the present invention may be performed
automatically using the apparatti disclosed herein. In
particular, the cells or particles may be automatically mixed
45
with test compound(s) and or standard compound(s). The
cells or particles may be automatically delivered to the
detector. The detector may automatically analyze the cells
for particular characteristics, automatically identify test
compounds having the desired cellular effects, or automati-
50 cally identify the presence of a particular molecule in a
sample. In some embodiments, a coupling device may
automatically deliver the cells or particles to the detector.
Calibration solutions may be input automatically. In
addition, gradients of test compounds or standard com-
55 pounds may be automatically provided. The automation of
the present methods increases the number of samples which
can be evaluated over a given time period.
A first embodiment of the present invention is a method
for identifying compounds which produce a cellular
60 response comprising:
The present invention also includes methods of screening
for agonist or antagonist activity of drugs, methods of
characterizing their potency profiles, methods of identifying
the receptor expression pattern of cell membrane ("receptor 65
fingerprinting") and methods of determining toxicity pro-
files for the compounds. In these methods, a steady flow of
(a) using an apparatus to combine a suspension of cells
with one or more test compounds to form test mixtures;
(b) directing the test mixtures through a detection zone,
the detection zone being capable of detecting a plurality
of cellular responses simultaneously; and
(c) simultaneously measuring a plurality of cellular
responses to the one or more test compounds in the
US 6,558,916 B2
7
suspended cells as the test mixtures are flowing through
the detection zone.
In one aspect of the first embodiment, the plurality of
cellular responses includes a cellular response selected from
the group consisting of activation or inhibition of a receptor 5
mediated response, activation or inhibition of an ion
channel, activation or inhibition of a non-selective pore,
activation or inhibition of a second messenger pathway at a
point downstream of a receptor or channel, activation or
inhibition of apoptosis, and activation or inhibition of eel-
10
lular necrosis, and cellular toxicity.
In another aspect of the first embodiment, the plurality of
cellular responses are measured by contacting the cells with
one or more response indicating reagents which generate
signals indicative of particular cellular responses. For
example, the signal generated by the response indicating 15
reagent<> may be fluorescence.
In another aspect of the first embodiment, the step of
directing the test mixtures through a detection zone com-
prises directing the text mixtures through a detection zone
which is capable of detecting the cellular responses in 20
individual cells.
In another aspect of the first embodiment, the step of
directing the test mixtures through a detection zone com-
prises directing the test mixtures through a flow cytometer.
In another aspect of the first embodiment, the step of 25
simultaneously measuring a plurality of cellular responses
comprises measuring the plurality of cellular responses in
the suspended cells as a slug is flowing through the detection
zone.
In another aspect of the first embodiment, the suspension
30 of cells comprises more than one type of cell.
A second embodiment of the present invention is a
method for identifying compounds which produce a cellular
response comprising:
(a) using an apparatus to combine a suspension of cells
with one or more test compounds to form test mixtures; 35
(b) directing the test mixtures through a detection zone,
the detection zone being capable of detecting cellular
responses in individual cells; and
(c) measuring one or more cellular responses to the one or
more test compounds in individual suspended cells as 40
the test mixtures are flowing through the detection
zone.
8
A third embodiment of the present invention is a method
of determining whether a sample contains one or more
molecules comprising:
using an apparatus to combine one or more agents capable
of binding one or more molecules with the sample to
form test mixtures;
directing the test mixtures through a detection zone; and
determining whether the one or more agents are bound to
the one or more molecules as the one or more agents are
flowing through the detection zone.
In one aspect of the third embodiment, the one or more
agents are fixed to a solid support. For example, a plurality
of agents capable of binding a plurality of molecules may be
fixed to uniquely identifiable solid supports. The determin-
ing step may comprise detecting a signal from an individual
solid support wherein the signal indicates that the agent on
the solid support has bound to the molecule.
In another aspect of the third embodiment, the one or
more agents are selected from the group consisting of
antibodies, nucleic acids, polypeptides, enzymatic
substrates, and receptors.
In another aspect of the third embodiment, the one or
more molecules are selected from the group consisting of
polypeptides, nucleic acids, receptor ligands, enzymatic
agonists and enzymatic antagonists.
A fourth embodiment of the present invention is a method
of determining whether a sample contains one or more
polypeptides comprising:
attaching one or more antibodies which specifically bind
the one or more polypeptides to solid supports;
using an apparatus to combine the one or more antibodies
attached to solid supports with samples to form test
mixtures;
directing the test mixtures through a detection zone; and
determining whether the one or more antibodies are
bound to the one or more polypeptides as the solid
supports are flowing through the detection zone.
In one aspect of the fourth embodiment, a plurality of
antibodies which specifically bind to a plurality of polypep-
tides are fixed to uniquely identifiable solid supports. The
determining step may comprise detecting a signal from an
individual solid support wherein the signal indicates that the
antibodies have bound to the molecules. The signal may be
generated by detectably labeled secondary antibodies which
In one aspect of the second embodiment, the one or more
cellular responses is selected from the group consisting of
activation or inhibition of a receptor mediated response,
activation or inhibition of an ion channel, activation or
inhibition of a non-selective pore, activation or inhibition of
45
bind the molecules.
A fifth embodiment of the present invention is a method
of determining whether a sample contains one or more
nucleic acids comprising: a second messenger pathway at a point downstream of a
receptor or channel, activation or inhibition of apoptosis,
and activation or inhibition of cellular necrosis, and cellular 50
toxicity.
In another aspect of the second embodiment, wherein the
one or more cellular responses is measured by contacting the
cells with one or more response indicating reagents which
generate signals indicative of particular cellular responses. 55
For example, the signal generated by the response indicating
reagents may be fluorescence.
attaching one or more nucleic acid probes which specifi-
cally bind the nucleic acids to solid supports;
using an apparatus to combine the one or more probes
attached to the solid supports with samples to form test
mixtures;
directing the test mixtures through a detection zone; and
determining whether the one or more probes are bound to
the one or more nucleic acids as the solid supports are
flowing through the detection zone.
In one aspect of the fifth embodiment, a plurality of
nucleic acid probes are fixed to uniquely addressable solid
In another aspect of the second embodiment, the step of
directing the test mixtures through a detection zone com-
prises directing the test mixtures through a flow cytometer.
In another aspect of the second embodiment, the suspen-
sion of cells comprises more than one type of cell.
60 supports. The determining step may comprise detecting a
signal from an individual solid support wherein the signal
indicates that the probes have bound to the nucleic acids.
The signal may be generated by detectable nucleic acid In another aspect of the second embodiment, the step of
simultaneously measuring a plurality of cellular responses
comprises measuring the plurality of cellular responses in 65
the suspended cells as a slug is flowing through the detection
zone.
probes which bind the nucleic acids.
A sixth embodiment of the present invention is a method
of determining whether a sample contains one or more
single nucleotide polymorphisms comprising:
US 6,558,916 B2
9
performing an extension reaction on a nucleic acid sample
using one or more nucleic acid primers having a 3' end
immediately adjacent to the polymorphic base of one or
more single nucleotide polymorphisms, thereby incor-
porating one base into the nucleic acid primers;
attaching the extended primers to solid supports;
5
directing the solid supports having primers attached
thereto through a detection zone in an apparatus; and
determining the identities of the bases incorporated in the
10 extension reaction.
In one aspect of the sixth embodiment, a plurality of
primers are fixed to uniquely identifiable solid supports. The
determining step comprises detecting a signal from an
individual solid support wherein the signal is indicative of
15 the identity of the polymorphic base in a SNP. The signal
may be generated by detectably labeled nucleotides incor-
porated in the extension reaction.
In another aspect, the first embodiment further comprises
the steps of:
20
(d) combining a suspension of the cells with one or more
standard compounds having a known effect on the
cellular response of the cells to form standard mixtures;
( e) directing the standard mixtures through the detection
zone; and
(f) measuring the cellular response of the cells to the one
or more standard compounds.
25
In the above aspect of the first embodiment, the one or
more standard compounds and the one or more test com-
pounds may be simultaneously mixed with the cells in the 30
combining steps, and the measuring step detects the known
effect or an alteration of the known effect. The one or more
standard compounds is an antagonist of the cellular
response. The one or more standard compounds is an agonist
10
A ninth embodiment of the present invention is an appa-
ratus comprising:
a test compound source;
a test substrate source;
a mixing chamber in fluid communication with said test
compound source and said test substrate source,
wherein said mixing chamber is adapted for combining
a test compound received from said test compound
source with a test substrate received from said test
substrate source to generate a mixture; and
a detector in fluid communication with said mixing
chamber, said detector being capable of detecting an
interaction between said test compound and said test
substrate while said mixture is passing through said
detector.
A tenth embodiment of the present invention is an appa-
ratus comprising:
one or more sample inputs for sequentially providing
multiple samples, the samples comprising one or more
test compounds to be evaluated for the ability to
produce a cellular response or a solution to be evalu-
ated for the presence of molecules;
one or more cell or particle inputs for providing a cell
suspension or particles;
a mixing zone, coupled to the sample input, for receiving
the samples, receiving the cell suspension or particles
from the cell or particle input and mixing each sample
with the cell suspension or particles; and
a detector capable of detecting a plurality of signals
simultaneously, the detector being coupled to the mix-
ing zone and being capable of simultaneously measur-
ing a plurality of cellular responses in the suspended
cells or simultaneously determining whether a plurality
of molecules are present in the samples.
of the cellular response. The suspension of cells may com-
prise more than one cell type. The step of directing the test
mixtures through a detection zone may compri<>e directing
the test mixtures through a flow cytometer.
35 In one aspect of the tenth embodiment, the detector detects
a signal from a single cell in the cell suspension or from a
single particle.
A seventh embodiment of the present invention is a
method of obtaining cells having a desired phenotype or 40
cellular response profile comprising the steps of:
In another aspect of the tenth embodiment, the detector is
a flow cvtometer.
In an~ther aspect of the tenth embodiment, the apparatus
further comprises a coupler disposed between the mixing
zone and the detector. The coupler may deliver slugs com-
prising the samples and the cell suspension or particles to the
detector. In some embodiments the coupler delivers substan-
(a) using an apparatus to combine a suspension of cells
with one or more compounds which produce a cellular
response to form test mixtures;
(b) directing the test mixtures through a detection zone,
the detection zone being capable of detecting a plurality
45 tially undiluted slugs to the detector.
In another aspect of the tenth embodiment, the detector
delivers cells having a desired phenotype or cellular
response to a receptacle. of cellular parameters simultaneously;
In another aspect of the tenth embodiment, the sample
input is an automated robotic sampler capable of selecting a
(c) simultaneously measuring a plurality of cellular
parameters in the suspended cells as the test mixtures
are flowing through the detection zone; and SD specified test compound from a library of test compounds.
(d) delivering cells having a desired phenotype or cellular
response profile into a receptacle.
An eighth embodiment of the present invention is a
method of obtaining cells having a desired phenotype or
55
cellular response profile comprising the steps of:
(a) using an apparatus to combine a suspension of cells
with one or more compounds which produce a cellular
response to form test mixtures;
(b) directing the test mixtures through a detection zone, 60
the detection zone being capable of detecting cellular
responses in individual cells;
(c) simultaneously measuring a plurality of cellular
parameters in the suspended cells as the test mixtures
are flowing through the detection zone; and
(d) delivering cells having a desired phenotype or cellular
response profile into a receptacle.
65
The device may further comprise a controller, coupled to the
sample input, for controlling the operation of the test com-
pound sampler; and
a computer, coupled to the controller, for sending com-
mand signals to the controller in accordance with a
software program implemented by the computer,
thereby controlling the selection and retrieval of test
compounds by the sample input from the test com-
pound library. The apparatus may further comprise a
gradient pump having an input and an output, coupled
to the sample input, for adjusting the concentration
level of the test compound transferred to the mixing
zone from the sample input, wherein:
the sample input comprises:
a first intake nozzle for receiving the specified test com-
pound;
a second intake nozzle for receiving a buffer solution; and
US 6,558,916 B2
11
wherein the gradient pump is coupled to the first and
second intake nozzles and receives specified concen-
trations of the test compound by adjusting the amount
12
from the cell or particle input and mixing each sample
with the cell suspension or particles; and
a detector which detects one or more signals from a single
cell or particle, the detector being coupled to the mixing
zone and being capable of measuring one or more
cellular responses in the suspended cells or determining
whether one or more molecules are present in the
samples.
In one aspect of the elventh embodiment, the apparatus
of test compound and buffer solution received by the
first and second intake nozzles, respectively, wherein 5
the buffer solution is a diluting agent of the test
compound. The apparatus may further comprise a sec-
ond pump, coupled to an input of the mixing zone, for
pumping the suspension of cells or the particles from
the cell or particle input into the mixing zone. The
apparatus may further comprise a reaction developing
line, having an input coupled to an output of the mixing
zone and an output in fluid communication with the
detector wherein the reaction developing line provides
rn further comprises a coupler disposed between the mixing
zone and the detector. The coupler may deliver slugs com-
prising the samples and the cell suspension or particles to the
detector. The coupler may deliver substantially undiluted
slugs to the detector. The apparatus may further comprise an a reaction time delay for a mixture received from the
mixing zone. The apparatus may further comprise: 15 input system for inputting solutions into the mixing zone.
a pump, coupled to the output of the detector, for
providing negative pressure to the apparatus;
a proportionating valve, coupled to the sample input,
for adjusting the concentration level of the test
compound transferred to the mixing zone from the 20
sample input, wherein the sample input further com-
prises:
a first intake nozzle for receiving the specified test
compound;
a second intake nozzle for receiving a buffer solu- 25
tion; and
the proportionating valve receives specified concen-
trations of the test compound by adjusting the
amount of test compound and buffer solution
received by the first and second intake nozzles, 30
respectively, wherein the buffer solution is a dilut-
ing agent of the test compound.
In another aspect of the tenth embodiment, the apparatus
further comprises a plurality of cell suspension reservoirs.
In another aspect of the tenth embodiment, the apparatus 35
further comprises a standard compound sampler, coupled to
the mixing zone, for providing one or more standard com-
pounds having a known effect on the cellular response of the
suspended cells or a known interaction with the particles,
wherein the mixing zone receives the one or more standard 40
compounds from the standard compound sampler and mixes
the one or more standard compounds with the suspended
cells or particles and the detector measures the cellular
response of the suspended cells to the one or more standard
compounds or the interaction between the one or more 45
standard compounds and the particles. The mixing zone may
simultaneously mix the sample and the one or more standard
compounds with the suspended cells or the particles and the
detector detects the known effect or an alteration of the
known effect on the cellular responses of the suspended cells 50
or the known interaction between the standard compound
and the particles or an alteration of the known interaction
between the standard compound and the particles. The
standard compound sampler may be an automated robotic
sampler capable of selecting a specified standard compound 55
from a library of standard compounds.
A elventh embodiment of the present invention is an
apparatus comprising:
The detector may deliver cells having a desired phenotype or
cellular response to a receptacle.
An twelfth embodiment of the present invention is an
apparatus comprising:
a sample input for sequentially providing multiple
samples, the samples comprising one or more test
compounds to be evaluated for the ability to produce a
cellular response or a solution to be evaluated for the
presence of molecules;
a cell or particle input for providing a cell suspension or
particles;
a mixing zone, coupled to the sample input, for receiving
the samples, receiving the cell suspension or particles
from the cell or particle input and mixing each sample
with the cell suspension or particles; and
a detector capable of detecting a plurality of signals
simultaneously, the detector being coupled to the mix-
ing zone and being capable of simultaneously measur-
ing a plurality of cellular responses in the suspended
cells or simultaneously determining whether a plurality
of molecules are present in the samples.
In one aspect of the twelfth embodiment, the detector
detects a signal from a single cell in the cell suspension or
from a single particle.
In another aspect of the twelfth embodiment, the detector
is a flow cytometer.
In another aspect of the twelfth embodiment, the appara-
tus further comprises a coupler disposed between the mixing
zone and the detector. The coupler may deliver slugs com-
prising the samples and the cell suspension or particles to the
detector.
The coupler may deliver substantially undiluted slugs to
the detector.
In another aspect of the twelfth embodiment, the appara-
tus further comprises an input system for inputting solutions
into the mixing zone.
In another aspect of the twelfth embodiment, the detector
delivers cells having a desired phenotype or cellular
response to a receptacle.
In another aspect of the twelfth embodiment, the appara-
tus further comprises a standard compound sampler, coupled
to the mixing zone, for providing one or more standard
compounds having a known effect on the cellular response a sample input for sequentially providing multiple
samples, the samples comprising one or more test
compounds to be evaluated for the ability to produce a
cellular response or a solution to be evaluated for the
presence of molecules;
a cell or particle input for providing a cell suspension or
particles;
60 of the suspended cells or a known interaction with the
particles, wherein the mixing zone receives the one or more
standard compounds from the standard compound sampler
and mixes the one or more standard compounds with the
suspended cells or particles and the detector measures the
a mixing zone, coupled to the sample input, for receiving
the samples, receiving the cell suspension or particles
65 cellular response of the suspended cells to the one or more
standard compounds or the interaction between the one or
more standard compounds and the particles. The mixing
US 6,558,916 B2
13
zone may simultaneously mix the sample and the one or
more standard compounds with the suspended cells or the
particles and the detector detects the known effect or an
alteration of the known effect on the cellular responses of the
suspended cells or the known interaction between the stan- 5
::imal cell response or maximum interaction of
particles with a molecule when mixed with the cell 5
suspension or particles;
a calibration minimum solution which provides for
minimal cell response or minimal interaction of a
particle with a molecule when mixed with the cell
suspension or particles;
10
a diverting valve having a first input coupled to the
calibration maximum solution and a second input
coupled to the calibration minimum solution, for
switching between the flow of either the calibra-
tion maximum solution or calibration minimum
solution; and 15
a pump, coupled to the output of the diverting valve
and an input of the switching valve, for pumping
either the calibration maximum or calibration
minimum solution from the diverting valve into
the switching valve. The apparatus may further 20
comprise a second pump, coupled to an input of
the first mixing zone, for pumping the suspension
of cells or the particles from the cell or particle
input into the first mixing zone. The apparatus
may further comprise a reaction developing line, 25
having an input coupled to an output of the first
mixing zone and an output coupled to an input of
the detector, for providing a flow path and a
reaction time delay for a mixture received from
the first mixing zone and for providing the mixture 30
to the detector. The apparatus may further com-
pri<>e:
a controller, coupled to the first and second
gradient pumps, the sample input, the standard
compound sampler and the switching valve, 35
the first and second mixing zones, the first and
second pumps and the diverting valve for con-
trolling their operation; and
a computer, coupled to the controller, for sending
command signals to the controller in accor- 40
dance with a software program implemented
by the computer, wherein the computer is also
coupled to the detector in order to send and
receive signals indicative of a cellular response
or the presence of a molecule in a sample to 45
and from the detector. The detector may detect
a plurality of cellular responses including a
cellular response selected from the group con-
sisting of activation or inhibition of a receptor
mediated response, activation or inhibition of 50
an ion channel, activation or inhibition of a
non-selective pore, activation or inhibition of a
second messenger pathway at a point down-
stream of a receptor or channel, activation or
inhibition of apoptosis, activation or inhibition 55
of cellular necrosis, and cellular toxicity.
The apparatus may further comprise:
a pump, coupled to the output of the detector, for provid-
ing negative pressure to the apparatus;
a proportionating valve, coupled to the sample input, for 60
adjusting the concentration level of the test compound
transferred to the mixing zone from the sample input,
wherein the sample input further comprises:
a first intake nozzle for receiving the specified test
compound; 65
a second intake nozzle for receiving a buffer solution;
and
16
the proportionating valve receives specified concentra-
tions of the test compound by adjusting the amount of
test compound and buffer solution received by the first
and second intake nozzles, respectively, wherein the
buffer solution is a diluting agent of the test compound.
The apparatus may further comprise:
an automated standard compound sampler capable of
selecting a specified standard compound from a
library of standard compounds, the standard com-
pound sampler including a third intake nozzle for
receiving the specified standard compound and a
fourth intake nozzle for receiving a buffer solution;
and
a second proportionating valve, coupled to the third and
fourth intake nozzles, for receiving specified concen-
trations of the standard compound by adjusting the
amount of standard compound and buffer solution
received by the third and fourth intake nozzles,
respectively, wherein the buffer solution is a diluting
agent of the standard compound. The apparatus may
further comprise:
a first priming valve, coupled to the output of the first
proportionating valve, for receiving the specified
concentration of the test compound and providing
the test compound to the mixing zone;
a second priming valve, coupled to the output of the
second proportionating valve, for receiving the
specified concentration of the standard compound
and providing the standard compound to the mixing
zone. The apparatus may further comprise:
a calibration unit including a calibration maximum
solution which provides for maximal cell response
or maximum interaction with particles when
mixed with the cell suspension or particles and a
calibration minimum solution which provides for
minimal cell response or minimal interaction with
particles when mixed with the cell suspension;
a first diverting valve, having a first input coupled to
the calibration maximum solution and a second
input coupled to the calibration minimum
solution, for switching between the flow of either
the calibration maximum solution or calibration
minimum solution;
a second diverting valve, having a first input coupled
to the output of the mixing zone and a second
input coupled to the output of the first diverting
valve, for switching between the flow of either a
calibration solution from the first diverting valve
or a mixture from the mixing zone;
a third priming valve, coupled to the output of
second diverting valve, for receiving a mixture
from the second diverting valve; and
a second mixing zone, coupled to the output of the
third priming valve, for mixing a mixture provided
by the third priming valve with the cell suspension
or particles, wherein the cell or particle input and
the detector are coupled to the second mixing zone
instead of the first mixing zone. The apparatus
may further comprise a reaction developing line,
having an input coupled to the output of the
second mixing zone and an output coupled to an
input of the detector, for providing a flow path and
a reaction time delay for a mixture received from
the second mixing zone before the mixture reaches
US 6,558,916 B2
17
the detector. The cell or particle input may com-
prise:
a cell suspension or particle reservoir;
a buffer reservoir;
a third diverting valve, having a first input coupled to 5
the cell suspension or particle reservoir and a
second input coupled to the buffer reservoir, for
adjusting the concentration of the cell suspension
18
the interaction between the one or more standard compounds
and the particles. The mixing zone may simultaneously mix
the one or more test compounds and the one or more
standard compounds with the suspended cells or the par-
ticles and the detector detects the known effect or an
alteration of the known effect on the cellular responses of the
suspended cells or the known interaction between the one or
more standard compounds and the particles or an alteration
of the known interaction between the one or more standard or the particles, wherein the buffer is a diluting
agent of the cell suspension or particles; and
10
compounds and the particles. The apparatus may further
comprise: a fourth priming valve, coupled to the output of the
third diverting valve, for receiving the cell sus-
pension or particle mixture from the third divert-
ing valve and providing this mixture to the second
mixing zone. The detector may detect a plurality
15
of cellular responses including a cellular response
selected from the group consisting of activation or
inhibition of a receptor mediated response, acti-
vation or inhibition of an ion channel, activation
or inhibition of a non-selective pore, activation or
20
inhibition of a second messenger pathway at a
point downstream of a receptor or channel, acti-
vation or inhibition of apoptosis, activation or
inhibition of cellular necrosis, and cellular toxic-
ity. The apparatus may further comprise a plurality
25
of cell suspension reservoirs.
A thirteenth embodiment of the present invention is an
apparatus comprising:
a sample input for sequentially providing multiple
samples, the samples comprising one or more test 30
compounds to be evaluated for the ability to produce a
cellular response or a solution to be evaluated for the
presence of molecules;
a cell or particle input for providing a cell suspension or
particles;
a mixing zone, coupled to the sample input, for receiving
the samples, receiving the cell suspension or particles
from the cell or particle input and mixing each sample
with the cell suspension or particles; and
35
a detector which detects one or more signals from a single 40
cell or particle, the detector being coupled to the mixing
zone and being capable of measuring one or more
cellular responses in the suspended cells or determining
whether one or more molecules are present in the
samples. 45
In one aspect of the thirteenth embodiment, the detector
is a flow cytometer.
In another aspect of the thirteenth embodiment, the detec-
tor delivers cells having a desired phenotype or cellular
response to a receptacle. SD
In another aspect of the thirteenth embodiment, further
comprising a coupler disposed between the mixing zone and
the detector. The coupler may deliver slugs comprising the
samples and the cell suspension or particles to the detector.
The coupler may deliver substantially undiluted slugs to the 55
detector.
In another aspect of the thirteenth embodiment, the appa-
ratus further comprises a standard compound sampler,
coupled to the mixing zone, for providing a sample of one
or more standard compounds having a known effect on the 60
cellular response of the suspended cells or a known inter-
action with the particles, wherein the mixing zone receives
the sample of the one or more standard compounds from the
standard compound sampler and mixes the one or more
standard compounds with the suspended cells or particles 65
and the detector measures the cellular response of the
suspended cells to the one or more standard compounds or
a first gradient device, coupled to the sample input, for
automatically adjusting the concentration level of the
one or more test compounds transferred to the mixing
zone from the sample input; and
a second gradient device, coupled to the stand compound
sampler, for automatically adjusting the concentration
level of the one or more standard compounds trans-
ferred to the mixing zone from the standard compound
sampler. The apparatus may further comprise a switch-
ing valve, coupled to the first and second gradient
devices at an input of the switching valve and coupled
to the mixing zone at an output of the switching valve,
for selectively switching the flow of a concentration of
the one or more test compounds or a concentration of
the standard compound or both to the mixing zone
where the one or more test compounds and/or the one
or more standard compounds are then mixed with the
suspension of cells or the particles. The apparatus may
further comprise a calibration unit, coupled to the
sv-ritching valve, wherein the switching valve also
selectively switches the flow of a calibration solution
provided by the calibration unit into the mixing zone
where the calibration solution is mixed with the sus-
pension of cells or the particles. The apparatus may
further comprise reaction developing lines coupled to
the output of the mixing zone, for receiving a mixture
of the cell suspension or the particles mixed with either
the one or more test compounds, the one or more
standard compounds or the calibration solution, and
providing a flow path for the mixture such that there is
adequate time for the suspended cells or particles to
react with the one or more test compounds, the standard
compound or the calibration solution, wherein the
reaction developing lines is further coupled to the input
of the detector which receives the mixture from the
reaction developing lines. The detector may detect one
or more cellular responses selected from the group
consisting of are selected from the group consisting of
activation or inhibition of a receptor mediated
response, activation or inhibition of an ion channel,
activation or inhibition of a non-selective pore, activa-
tion or inhibition of a second messenger pathway at a
point downstream of a receptor or channel, activation
or inhibition of apoptosis, activation or inhibition of
cellular necrosis, and cellular toxicity. The apparatus
may further comprise:
a controller, coupled to the first and second gradient
devices, the sample input, the standard compound
sampler, the sv-ritching valve, and the coupler for
controlling their operation; and
a computer, coupled to the controller, for sending
command signals to the controller in accordance
with a software program implemented by the
computer, wherein the computer is also coupled to
the detector in order to send and receive signals
indicative of a cellular response or the presence of a
molecule in the sample to and from the detector.
US 6,558,916 B2
19
In another aspect of the thirteenth embodiment, the
sample input is an automated robotic sampler capable of
selecting a specified test compound from a library of test
compounds. The apparatus may further comprise:
a controller, coupled to the sample input, for controlling 5
the operation of the test compound sampler; and
a computer, coupled to the controller, for sending com-
mand signals to the controller in accordance with a
software program implemented by the computer,
thereby controlling the selection and retrieval of test 10
compounds by the sample input from the test com-
pound library. The apparatus may further comprise a
gradient pump having an input and an output, coupled
to the sample input, for adjusting the concentration
level of the test compound transferred to the mixing 15
zone from the sample input, wherein:
the sample input comprises:
a first intake nozzle for receiving the specified test
compound;
a second intake nozzle for receiving a buffer solu- 20
tion; and
wherein the gradient pump is coupled to the first and
second intake nozzles and receives specified concen-
trations of the test compound by adjusting the
amount of test compound and buffer solution 25
received by the first and second intake nozzles,
respectively, wherein the buffer solution is a diluting
agent of the test compound.
The apparatus may further comprise:
30
a pump, coupled to the output of the detector, for provid-
ing negative pressure to the apparatus;
a proportionating valve, coupled to the sample input, for
adjusting the concentration level of the test compound
transferred to the mixing zone from the sample input, 35
wherein the sample input further comprises:
a first intake nozzle for receiving the specified test
compound;
a second intake nozzle for receiving a buffer solution;
and 40
the proportionating valve receives specified concentra-
tions of the test compound by adjusting the amount of
test compound and buffer solution received by the first
and second intake nozzles, respectively, wherein the
buffer solution is a diluting agent of the test compound. 45
The apparatus may further comprise:
an automated standard compound sampler capable of
selecting a specified standard compound from a
library of standard compounds, the standard com-
pound sampler including a third intake nozzle for 50
receiving the specified standard compound and a
fourth intake nozzle for receiving a buffer solution;
and
a second proportionating valve, coupled to the third and
fourth intake nozzles, for receiving specified concen- 55
trations of the standard compound by adjusting the
amount of standard compound and buffer solution
received by the third and fourth intake nozzles,
respectively, wherein the buffer solution is a diluting
agent of the standard compound. The apparatus may 60
further comprise:
a first priming valve, coupled to the output of the first
proportionating valve, for receiving the specified
concentration of the test compound and providing
the test compound to the mixing zone; 65
a second priming valve, coupled to the output of the
second proportionating valve, for receiving the
20
specified concentration of the standard compound
and providing the standard compound to the mixing
zone. The apparatus may further comprise:
a calibration unit including a calibration maximum
solution which provides for maximal cell response or
maximum interaction with particles when mixed
with the cell suspension or particles and a calibration
minimum solution which provides for minimal cell
response or minimal interaction with particles when
mixed with the cell suspension;
a first diverting valve, having a first input coupled to the
calibration maximum solution and a second input
coupled to the calibration minimum solution, for
switching between the flow of either the calibration
maximum solution or calibration minimum solution;
a second diverting valve, having a first input coupled to
the output of the mixing zone and a second input
coupled to the output of the first diverting valve, for
switching between the flow of either a calibration
solution from the first diverting valve or a mixture
from the mixing zone;
a third priming valve, coupled to the output of the
second diverting valve, for receiving a mixture from
the second diverting valve; and
a second mixing zone, coupled to the output of the third
priming valve, for mixing a mixture provided by the
third priming valve with the cell suspension or
particles, wherein the cell or particle input and the
detector are coupled to the second mixing zone
instead of the first mixing zone. The apparatus may
further comprise a reaction developing line, having
an input coupled to the output of the second mixing
zone and an output coupled to an input of the
detector, for providing a flow path and a reaction
time delay for a mixture received from the second
mixing zone before the mixture reaches the detector.
The cell or particle input may comprise:
a cell suspension or particle reservoir;
a buffer reservoir;
a third diverting valve, having a first input coupled to
the cell suspension or particle reservoir and a second
input coupled to the buffer reservoir, for adjusting
the concentration of the cell suspension or the
particles, wherein the buffer is a diluting agent of the
cell suspension or particles; and
a fourth priming valve, coupled to the output of the
third diverting valve, for receiving the cell suspen-
sion or particle mixture from the third diverting
valve and providing this mixture to the second
mixing zone. The detector may detect a plurality of
cellular responses including a cellular response
selected from the group consisting of activation or
inhibition of a receptor mediated response, activation
or inhibition of an ion channel, activation or inhibi-
tion of a non-selective pore, activation or inhibition
of a second messenger pathway at a point down-
stream of a receptor or channel, activation or inhi-
bition of apoptosis, activation or inhibition of cellu-
lar necrosis, and cellular toxicity. The apparatus may
further comprise a plurality of cell suspension res-
ervoirs.
In another aspect, the invention relates to a method for
determining the effect of each of a plurality of test agents on
cells from a subject comprising:
(a) obtaining cells from the subject;
(b) combining each of a plurality of samples comprising
the cells with one or more of the test agents to form
each of a plurality of test mixtures; and
US 6,558,916 B2
21
(c) sequentially directing each of said plurality of test
mixtures through a detection zone in an apparatus, said
detection zone being capable of detecting the effect of
said agents on said cells.
22
Further, the above cellular analysis may comprise measuring
the level of activity of one or more multidrug resistance
transporters in said cells.
In an embodiment, the above method further comprises 5
determining whether said one or more test agents has a
desired effect on said cells from said subject.
In another embodiment, the test agent of the above
methods comprises test cells. Within this embodiment, the
subject may be a mammal. The mammal may be selected
from the group consisting of mouse, rat, rabbit, dog, cat,
sheep, goat, cattle, pig, monkey, and human. In another embodiment, the above method further com-
prises using an automated and computer controlled appara-
tus to sequentially combine each of a plurality of samples
comprising said cells with one or more said agents.
In a further embodiment, the agents in the above method
are selected from the group consisting of chemical
compounds, biological molecules, and test cells. The agents
may be chemical compounds.
In an embodiment, the subject in the above method is a
mammal. The mammal may be selected from the group
consisting of mouse, rat, rabbit, dog, cat, sheep, goat, cattle,
pig, monkey, and human. In other embodiments, the mam-
mal is a human.
In another embodiment, the cells are normal cells. The
normal cells may be selected from the group consisting of
cells involved in generating or modulating an immune
system.
In another embodiment, however, the cells are abnormal
cells. The abnormal cells are selected from the group con-
sisting of cancer cells.
In yet another embodiment, the test agents are selected
from the group consisting of agents for treating cancer,
immunosuppressive drugs, antibiotics, anti-inflammatory
drugs, neurotransmitters, growth hormones, and analgesics.
An embodiment of the invention provides for the test
mixtures of the above method to further comprise one or
more response indicating agents. The response indicating
agents may indicate a decrease or cessation of replication, or
thev mav indicate cell death.
In an~ther embodiment, the plurality of test mixtures vary
according to a condition selected from the group consisting
of the concentration of said one or more test agents, incu-
bation condition, type of cell, type of test agent, and number
of test agents, or a combination thereof.
In further embodiments, the detection zone is capable of
detecting a plurality of cellular responses simultaneously.
In an embodiment, the determining step comprises simul-
taneously measuring a plurality of said extent of response of
said cells to said one or more test compounds as each of said
plurality of test mixtures are flowing through said detection
zone.
In another embodiment, the detection zone is capable of
detecting a cellular response as a single cell is flowing
through said detection zone. The detection zone may com-
prise a flow cytometer.
In another of the invention, the above method further
comprises conducting a genetic analysis to determine
whether said subject is likely to have a desirable or adverse
response to said one or more test agents. The genetic
analysis may comprise determining whether said subject has
an allele of one or more single nucleotide polymorphisms
which indicates that said subject will have said desirable or
adverse response.
Another embodiment of the invention provides for the
above method further comprising conducting a cellular
analysis to determine whether said subject is likely to have
a desirable or adverse response to said one or more test
compounds. The above cellular analysis may comprise
determining whether said one or more test compounds will
be maintained at effective concentrations in said cells.
In another embodiment, the cells are normal cells. These
normal cells may be cells involved in generating or modu-rn
lating an immune response.
In vet another embodiment, the cells are abnormal cells.
Thes.; abnormal cells may be cancer cells.
In an embodiment, the test agents are selected from the
group consisting of agents for treating cancer, immunosup-
15 pressive drugs, antibiotics, anti-inflammatory drugs,
neurotransmitters, growth hormones, and analgesics.
In another embodiment, the test mixtures further comprise
one or more response indicating agents. The response indi-
cating agents may indicate a decrease or cessation of repli-
20 cation. The response indicating agents may instead indicate
cell death.
In an embodiment, the plurality of test mixtures vary
according to a condition selected from the group consisting
of the concentration of said one or more test agents, incu-
25 bation condition, type of cell, type of test agent, and number
of test agents, or a combination thereof.
In a further embodiment, the detection zone is capable of
detecting a plurality of cellular responses simultaneously.
In still another embodiment, the determining step com-
30 prises simultaneously measuring a plurality of said extent of
response of said cells to said one or more test compounds as
each of said plurality of test mixtures are flowing through
said detection zone.
In vet other embodiments, the detection zone is capable of
35 detecting a cellular response as a single cell is flowing
through said detection zone. The detection zone may com-
prise a flow cytometer.
In another embodiment, the above method further com-
prises conducting a genetic analysis to determine whether
40 said subject is likely to have a desirable or adverse response
to said one or more test agents. Another embodiment relates
to the above method further comprising conducting a genetic
analysis to determine whether said subject is likely to have
a desirable or adverse response to said test cells. The genetic
45 analysis may comprise determining whether said subject has
an allele of one or more single nucleotide polymorphisms
which indicates that said subject will have said desirable or
adverse response.
In an embodiment, the above method further comprising
50 conducting a cellular analysis to determine whether said
subject is likely to have a desirable or undesirable response
to said test cells. The cellular analysis may comprise deter-
mining whether said test cells express one or molecules on
their surface which will cause a desirable or undesirable
55 response. The one or molecules may comprise major histo-
compatibility molecules.
60
In vet another embodiment, the test agents are test cells
from 'a candidate organ or tissue to be tested for use in an
organ or tissue transplant.
Further details on the latter aspects of the invention are set
forth in Example 22.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. lA and lB are block-diagrams of one embodiment
65 of the combinatorial screening apparatus of the present
invention which do not include or do include a coupling
system.
US 6,558,916 B2
23
FIG. 2 illustrates the multiparametric capacity of the
FCM, and indicates the flow of information obtained from
the FCM
24
FIG. 22 is a flow diagram of a method for analyzing the
activity of ion channels or non-selective pores.
FIGS. 3A and 3B are block-diagrams of a positive pres-
sure fluidic system which do not include or do include a 5
coupling system and which may be used in a combinatorial
screening apparatus of the present invention.
FIG. 23 is a flow diagram of a method for measuring the
activity of second messengers.
FIG. 24 is a flow diagram of a method for measuring
cellular apoptosis or cellular toxicity.
FIG. 25 is a flow diagram of a method for measuring
cellular necrosis. FIGS. 4A and 4B are block-diagrams of one embodiment
FIG. 26 is a flow diagram of a method for measuring
cellular necrosis and cellular apoptosis or cellular toxicity.
of a preferred system which do not include or do include a
coupling system and which may be used in a combinatorial 10
screening apparatus of the present invention.
FIG. 27 is a flow diagram of a method for obtaining or
cloning populations of cells having a desired phenotype or
cellular response profile obtain or clone populations of cells
15 having a desired phenotype or cellular response profile
FIG. 28 is a flow diagram of a method for detecting the
presence of one or more polypeptides in a sample.
FIG. 5 represents a simplified algorithm of a screening
mode which may be utilized in a combinatorial screening
apparatus of the present invention.
FIG. 6 represents a simplified algorithm for a potency
mode which may be utilized in a combinatorial screening
apparatus of the present invention.
FIG. 7 is a flow diagram of a preferred primary mode
operation which may be implemented by a combinatorial 20
screening apparatus of the present invention. FIG. 7 com-
prises the combination of FIGS. 7a-7d.
FIG. 29 is a flow diagram of a method for detecting the
presence of one or more nucleic acids in a sample.
FIG. 30 is a flow diagram of a method for detecting the
identities of one or more polymorphic nucleotides in a
nucleic acid sample. FIG. 8 is a flow diagram of a preferred screening mode
operation which may be implemented by a combinatorial
screening apparatus of the present invention. FIG. 8 com-
prises the combination of FIGS. 8a-8g.
FIG. 9 is a flow diagram of a preferred potency mode
operation which may be implemented by a combinatorial
screening apparatus of the present invention. FIG. 9 com-
prises the combination of FIGS. 9a-9e.
FIG. 10 represents the experimental results of ET-1 dose
dependent Ca2 + mobilization in the presence of BQ-123.
FIG. 11 represents the semi logarithmic transformation of
the data of the FIG. 9.
FIG. 12 represents the experimental results of dose depen-
dent inhibition of ET-1 induced Ca2 + mobilization in the
TE-671 cells with BQ-123.
FIG. 31 is a flow diagram of a method for analyzing
25 enzymatice activities.
30
FIG. 32 is a flow diagram of a method for performing
molecular assembly assays.
FIG. 33 is a flow diagram of a method for evaluating the
interactions of receptors with ligands.
FIG. 34 is a bar graph of the frequency of abnormal events
detected by changes in intracellular calcium, glutathione,
plasma membrane integrity(PI and TO-PR03 exclusion) and
forward and side light scatter for HL-60 cells treated with a
Oubain(12 uM), Actinomycin D (400 nM), Ibuprofen (70
35 uM), Etopside (5 uM), Acetaminophen (70 uM), FCCP (12
uM) and untreated control cells.
FIG. 35 is a dose response curve for actinomycin D.
FIG. 13 represents the experimental results of ET-1 dose
dependent Ca2 + mobilization in TE-671 cells with the sub- 40
sequent inhibition with the BQ-123.
DESCRIPTION OF THE PREFERRED
EMBODIMENTS
FIG. 14 represents a simplified algorithm for cell mapping
mode which may be utilized in a combinatorial screening
apparatus of the present invention.
FIG. 15 represents a simplified algorithm for functional 45
characterization of orphaned receptors mode which may be
utilized in a combinatorial screening apparatus of the present
The present invention provides for real-time, continuous
monitoring and detection of the physiological or pharmaco-
logical effect of a test compound on a series of cell types or
on a single cell type. In its simplest embodiment, the present
invention comprises a method and apparatus for continu-
ously contacting a single cell suspension, a series of cell
suspensions, each of which contains a single cell type
included in the series of cell types to be tested, a single cell
invention.
FIG. 16 represents a flow diagram of a preferred cell
mapping mode operation which may be implemented by a
combinatorial screening apparatus of the present invention.
FIG. 16 comprises the combination of FIGS. l6a-l6f
FIG. 17 represents a flow diagram of a preferred orphaned
receptor determination mode operation which may be imple-
mented by a combinatorial screening apparatus of the
present invention. FIG. 17 comprises the combination of
FIGS. 17a-17g.
FIG. 18 is a flow diagram of a method for measuring
cellular responses.
FIG. 19 is a flow diagram of a method for measuring
cellular responses in cell populations containing more than
one type of cell.
FIG. 20 is a flow diagram of a method for measuring a
plurality of cellular responses simultaneously.
FIG. 21 is a flow diagram of a method for analyzing
receptor activity.
50 suspension containing more than one cell type, or a series of
cell suspensions containing more than one cell type with a
predetermined concentration of at least one potentially
active compound, preferably with predetermined concentra-
tions of at least two active compounds. Then, intracellular
55 changes that occur in response to contact between the cells
and the active compounds are continuously measured as the
suspensions pass a detector. In some embodiments, a plu-
rality of cellular responses can be simultaneously evaluated.
In some embodiments, one or more cellular responses are
60 evaluated in a single cell.
In other embodiments, the present invention comprises a
method and apparatus for continuously contacting particles,
or a series of particles, each of which contains a particle type
included in the series of particles to be tested, a single
65 particle preparation containing more than one type of
particle, or a series of particle preparations containing more
than one type of particle with a predetermined concentration
US 6,558,916 B2
25
of at east one molecule to be tested for interaction with the
particles. In some embodiments, the particles are contacted
with predetermined concentrations of at least two molecules.
Then, interactions between the particles and the molecules
are continuously measured as the particles and the test 5
molecules pass a detector. In some embodiments, a plurality
of interactions can be simultaneously evaluated. In some
embodiments, one or more interactions are evaluated in a
particle.
26
controller. The controller can continuously monitor the
results of each step of the process, and can automatically
alter the testing paradigm in response to those results.
The incubation period after mixing of the compound or
compounds and the cells can advantageously be controlled
by passing that mixture through a length of tubing connect-
ing the mixing zone with the detector. After mixing with the
test compound or compounds, the cell suspension or par-
ticles may be delivered to the detector, such as a flow
In other embodiments, the present invention allows the
monitoring of the physiological effects of test compounds,
the pharmacological effects of test compounds, or the bio-
chemical properties of cells to be measured at the level of
individual cells or in each of the cell types in a heteroge-
neous population of cells. In further embodiments, the
present invention allows the simultaneous examination of
multiple characteristics of each cell or cell type or the
examination of heterogeneous or mixed cell populations.
rn cytometer, directly through the tubing. Alternatively, after
mixing with the test compound or compounds, the cell
suspension or particles may be delivered to the detector,
such as a flow cytometer, via a coupling system.
The coupling system is disposed between the mixing
15 chamber and the detector as illustrated in FIGS. lB, 3B, and
4B. Preferably, the coupling system rapidly provides the
samples to the detector to optimize the number of samples
which can be analyzed in a given time period. A variety of
It is contemplated that the present invention will be of
major value in high-throughput screening; e.g., in screening 20
a large number of candidate compounds for activity against
one or more cell types. It has particular value, for example,
coupling systems are known to those skilled in the art.
More preferably, the coupler is a device which captures a
slug comprising the cell and test compound(s) mixture from
the output tubing connected to the mixing chamber and
delivers the slug to the detector under conditions which
minimize oscillations. Such a device will be referred to
in screening synthetic or natural product libraries for active
compounds or biochemical characterization.
It is also contemplated that the present invention will be
of major value in high-throughput screening of a sample for
a plurality of molecules, such as biological molecules. The
present invention can be used to screen a sample for the
presence of a large number of biological molecules such as
polypeptides, receptor ligands, enzymatic substrates, ago-
ni<>ts or antagonists of enzymatic or receptor activity, or
nucleic acids.
25 hereinafter as a Plug Flow Coupler (PFC). Preferably, the
slug is isolated from the output tubing from the mixing
chamber to a separate piece of tubing and rapidly delivered
to the detector, such as a flow cytometer. Isolating the
In one preferred embodiment, a test compound, a standard
compound, and a cell suspension containing the cell type to
30
sample slug from the output tubing connected to the mixing
chamber helps to reduce the effects of the oscillatory hydro-
dynamics of the peristaltic pump-driven output tubing,
thereby enhancing the quality of the data. For example, the
PFC may deliver the sample under positive air pressure.
35
Preferably, the PFC selects a substantially undiluted por-
tion of the sample slug for analysis instead of analyzing the
entire slug. During transit through the tubing from the
mixing point, diffusion of the cells and test compound(s)
occurs resulting in dilution of the test compound(s) con-
be tested or particle preparation, or a member of the series
40 tacted by the cells. Thus, in this preferred embodiment,
diluted portions of the slug are not analyzed. Timing and
other activities of the PFC may be controlled through a
special section of software that will be incorporated within
of cell types or series of particles to be tested are continu-
ously mixed together and, after an incubation period, are
passed by a detector that measures one or more cellular
responses, one or more interactions between the molecules
and the particles, or the concentration in the cells or in the
intracellular medium of at least one analyte. In a preferred
embodiment, the detector comprises a device capable of
analyzing individual cells, individual cell types in a cell
suspension, individual particles in a sample, individual 45
particle types in a sample, individual components in a
sample, or individual types of components in a sample. In
some embodiments, the detector is capable of evaluating a
plurality of cellular responses or a plurality of interactions
between particles and molecules simultaneously. Preferably,
the detector is a flow cytometer (FCM) which permits the
analysis or characterization of individual cells, individual
cell types in a cell suspension, individual particles in a
sample, individual particle types in a sample, individual
components in a sample, or individual types of components 55
in a sample. In some embodiments, the detector may be
capable of delivering cells exhibiting a desired set of param-
eters to a receptacle, thereby separating desired cells from
undesired cells. For example, the detector may be a fluo-
rescence activated cell sorter. Alternatively, the detector may 60
comprise a microscope based imaging system such as those
available from Bio-Rad (Eugene, OR) or Zeiss(, Germany).
In one embodiment, the concentration of the test compound
and/or the standard compound is varied over time to gen-
erate dose/response curves as output from the detector.
It is preferred that the apparatus of the present invention
is under the control of a computer or other programmable
the software that controls the overall system.
Preferably, the PFC is the PFC described in the U.S.
Patent Application entitled "Plug-Flow Cytometry for High
Throughput Screening and Drug Discovery", filed Jun. 10,
1999, the corresponding PCT application of which is pub-
lished as WO 00/20873, which is incorporated herein by
50 reference in its entirety, including any drawings. However,
it will be appreciated that any device which provides a
sample slug may be used.
In embodiments in which the detector is directly coupled
to the mixing chamber via the reaction lines, the incubation
period will thus be determined by the flow rate and the
length of the tubing. In embodiments where a coupling
system is used, the incubation period is determined by the
amount of time it takes for the cell suspension or particles to
pass from the mixing chamber, through the coupling system,
and into the detector. Incubation periods can vary by several
orders of magnitude, depending on the particular analyte and
the resultant reaction time. For example, the incubation
period could be as little as one second or a fraction of a
second for rapid or short-lived physiological responses, or as
65 long as several minutes or even hours.
The analyte can be any analyte that is readily detectable
by detectors, such as flow cytometers, and/or detector/
US 6,558,916 B2
27
chemistry combinations. Thus, various ion or electrolyte
concentrations, colorimetric changes, optical density
changes, fluorescence, luminescence, pH, gas production,
and the like are all readily adaptable for use in the present
method and apparatus. 5
28
more particle types to be tested); a calibration unit 117, for
supplying calibration solutions to the switching valve 111 so
that the calibration solutions may be mixed with the cell
suspension or particles in predetermined ratios; a reaction
In detecting ion or electrolyte changes, calorimetric or
fluorescent dyes are one particularly preferred embodiment.
For example, calcium ion is detectable by such probes as
Fura-2, Indo-1, Fura Red or Quin-2, sodium ions by SBFL,
proton ions by BCECF, SNAFL, DM-NERF, magnesium 10
ions by Mag-Fura-2 or Mag-Fura-5, chloride ions by SPO,
SPA or MOAE. All these dyes are commercially available,
developing lines unit 115, for receiving the cell suspensions
or particles mixed with either the compound/standard solu-
tion mixture or the calibration solution and for transferring
this mixture to a detector 119; and a drain 121 for receiving
and draining the mixture from detector 119 after the cell
response or particle interaction has been detected and mea-
sured by detector 119. A controller 109 is coupled to the test
compound sampler 101, the standards sampler 103, gradient
devices 105 and 107 and switching valve 111. The controller
controls the above devices by receiving command signals
from a computer 123 which in turn generates, sends and
receives signals in accordance with a software program 125.
for example, from Molecular Probes, Inc., Oregon.
In some embodiments, it may be desirable to correlate the
parameter of transit time from the final mixing point with 15
compound concentration in order to conduct compound
concentration-cell response measurements. In such
embodiments, the device may include a calibration unit
between the mixing point and the detector to determine the
amount of signal generated by different concentrations of 20
compounds. For example, the calibration unit may deter-
mine the amount of fluorescence generated by different
concentrations of fluorescent materials. In embodiments
which include a PFC, the calibration unit may be inserted in
the output tubing from the mixing point just upstream of the 25
PFC.
The mixing zone 112 can constitute a chamber, a tube, a
series of baffles, or any other structure in which mixing can
occur. In some embodiments, the computer 123 may also
control the operation of the coupling system 120. In further
embodiments, the computer 123 may synchronize the cou-
pling system 120 with the test compound sampler 101 such
that the next sample slug is delivered to the detector shortly
after the analysis of the current sample slug is completed,
thereby optimizing the number of samples which may be
analyzed in a given time period. In further embodiments, the
In one embodiment, the calibration unit may comprise a
light emitting diode (LED), a glass cuvette and a fluores-
cence detector in line immediately before the PFC. The glass
cuvette is inserted into the output line from the mixing point
just upstream of the PFC. The LED provides light to the
cuvette. A light sensing device, such as a fiber optic based
microfluorimeter, is aligned at right angles to the LED
source to collect fluorescence emissions from the cuvette.
The single step and gradient forming systems of the mixing
apparatus are used to form a pre-gradient slug and a con-
centration gradient of a solution of fluorescent material such
as fluorescein. As the gradient of fluorescent material passes
through the cuvette, its fluorescent signature is registered by
the microfluorimeter. The fluorescence intensity is an index
of the concentration of the fluorescent material. The con-
centration of the fluorescent material is correlated with its
time of transit to calibrate the gradient formed by the mixing
apparatus.
FIGS. lA and lB illustrate schematic configurations of
some embodiments of the apparatus of the present invention.
The embodiments shown in FIGS. lA and lB consist of a
test compound sampler 101, for receiving and holding a
sample of a compound to be tested; a standards sampler 103,
for receiving and holding a sample of a standard solution
( e.g, a buffer and/or a known agonist or antagonist) to be
mixed with the compound to be tested; a concentration
gradient device 105 connected to compound sampler 101 for
controlling the flow volume of the compound to be tested; a
concentration gradient device 107, connected to standards
sampler 103, for controlling the flow volume of the standard
solution; a switching valve 111 for receiving a compound/
standard solution mixture or a calibration solution and for
directing them to the mixing zone 112 for mixing one of
these solutions with cell suspensions containing one or more
cell types to be tested, a member of a series of one or more
cell types to be tested, or one or more types of particles to
be tested; at least one cell suspension or particle reservoir
113 (and, where a series of cell types or particles is to be
examined, a plurality of cell suspension or particle reservoirs
each of which contains a cell suspension of one of the cell
types in the series to be examined or a preparation of one or
computer 123 may also control the operation of the stan-
dards sampler 103. It will also be appreciated that the
operation of the preceding devices may be controlled by
30
more than one computer.
While the embodiments shown in FIGS. lA and lB
include a standards sampler 103, gradient device 107,
switching valve 111, and calibration unit 117, it will be
appreciated that these aspects of the device need not be
35 present if the analysis being performed does not require the
use of a standard compound(s) or calibration solution(s).
Likewise, when the analysis being performed does not
require a concentration gradient of the test compound(s), the
device need not include a gradient device 105 connected to
40 the test compound sampler 101.
After a compound and/or standard is mixed with cells or
particles in mixing zone 112, which is readily commercially
available and in the preferred embodiment is the static mixer
125-1345 (Bio Rad), this mixture is then sent through
45 reaction developing lines 115 so that it may be mixed more
thoroughly and provided with enough time for its reagents to
thoroughly react with one another. The detector 119, is
connected to the reaction developing lines unit 115, and
receives the cell or particle/compound/standard mixture
50 from the reaction developing lines 115.
In a preferred embodiment, each of the samplers 101 and
103 include a sipper nozzle which may be positioned into a
vial containing a respective compound or standard solution.
These samplers are well-known in the art and are readily
55 available commercial products, e.g A/S 300 Autosampler,
Catalog# 15006330, Scientific Measurement Systems, Inc.,
Colorado. For handling multiple samples, conventional
automated or robotic equipment (not shown) may be used to
supply the apparatus with the samples. Banks of different
60 samples, arranged in 48 or 96 well plate format for example,
can ea-;ily be accommodated by such automated sampling
equipment.
The gradient devices 105 and 107 are also readily avail-
able commercial devices, e.g, GP40 Gradient Pump
65 (Dionex). Depending on the mode of action, the correspond-
ing concentration gradient devices 105 and 107 can prepare
either discrete concentrations or continuous gradients of
US 6,558,916 B2
29
concentrations of the compound to be tested or the standard
substance (agonist or antagonist) by diluting them with
buffer. For example, if a continuous curve of cell response
or particle interactions versus compound concentration in
the presence of a standard substance is desired, the computer 5
123 will instruct the controller 109 to control the gradient
devices 105 and 107 in such a way that continuous gradients
of respective compound and a predetermined constant con-
centration of standard solution are provided to switching
valve 111. Alternatively, if a continuous curve of cell
10
response or particle interaction versus standard solution
concentration in the presence of a compound is desired, the
computer 123 will instruct the controller 109 to control the
gradient devices 105 and 107 such a way that continuous
gradients of the respective standard solution and a prede-
15
termined constant concentration of the respective compound
are provided to switching valve 111.
The calibration unit 117, connected to the switching valve
111, supplies calibration solutions needed to calibrate the
output signal, MAX and MIN, of the apparatus. In a pre- 20
ferred embodiment, this calibration unit consists of a divert-
ing valve which is a readily available commercial device,
e.g.,. the SV3-2 Diverter Valve, (Bio-Rad). The diverter
valve alternates supply of two calibration solutions to the
switching valve 111. The switching valve 111 combines 25
outflows from the gradient devices 105 and 107 or,
alternatively, from the calibration unit 117 with a cell flow
or particle flow from a cell suspension or particle reservoir
113 (or from one of a plurality of cell suspension or particle
reservoirs 113 each of which contains one or more cell types 30
or particle preparations each of which contains one or more
particle types in a series of cell types or particle preparations
to be examined) and directs the mixed flow into one of the
reaction developing lines 115. The switching valve 111 is of
a type which is well-known in the industry and in a preferred 35
embodiment is the 3-way microvalve 4-8-900 manufactured
by General Valve Corp. The cell suspension or particle
reservoir(s) 113 are also of a type which is well known in the
art and in a preferred embodiment is a regular glass beaker.
Cells or particles are maintained in suspension pending 40
introduction into the device by simple low shear stirring.
The reaction developing lines 115 are typically tubes
made from a non-corrosive material, having a specified
diameter, through which the above mentioned mixture of
cell suspension or particle preparation, compound and stan- 45
dard solution may flow. For example, polyethylene,
polypropylene, or polytetrafluoroethylene tubing can be
used. Tubing to which cells or particles and other reagents
will not stick is particularly preferred. The diameter of the
tubing is a matter of choice. Capillary tubing having an inner 50
diameter of from about 0.2 mm to about 2 mm is particularly
advantageous, because it allows the use of very small sample
sizes.
This tubing is typically set in a winding configuration so
that as the mixture flows through it, the mixture is thor- 55
oughly agitated and mixed. Even if the mixture is well
mixed before introduction into the tubing, a spiral or wound
configuration allows long tubing lengths in a compact area.
These reaction developing lines 115 are commercially avail-
able and in one embodiment may be formed by Teflon 60
tubing.
The mixture of a cell suspension containing one or more
cell types to be tested, one or more cell types included in a
series of cell types to be tested, particle preparations con-
taining one or more particle types, or particle preparations 65
containing one or more particle types included in a series of
particle preparations to be tested and compound/standard
30
solution, also referred to as "reaction suspension," enters the
detector's flow-through optical cell or other detection zone
with a time delay or incubation period determined by the
length of the reaction developing line 115. Once the reaction
suspension reaches detector 119, the detector 119 can mea-
sure the cell suspension response to the specified concen-
tration of the test compound or the presence of a particular
molecule in the sample.
In a preferred embodiment, the detector 119 measures a
fluorescence signal from the calcium sensitive dye, FURA2,
spectral characteristics of which depend on a concentration
of the intracellular ionized calcium, in order to determine the
level of cell activity. In order to make this measurement, the
detector 119 alternately irradiates the reaction suspension
passing through a flow-through optical quvette, with the
light of the wavelengths 340 nm and 380 nm and measures
fluorescence intensity at 540 nm. The ratio (R) of the
fluorescence intensities registered at 540 nm upon excitation
at 340 nm and 380 nm, respectively, is transmitted to the
computer 123. The computer 123 calculates the concentra-
tion of the intracellular ionized calcium in accordance with
the following equation which is well-known in the art:
where Kd is the dissociation constant of calcium/FURA2
complex and Rm;n and Rmax are the ratios obtained in
the presence of the calibration solutions MIN and
MAX, respectively, and If and lb are the fluorescence
intensities measured at 540 nm upon excitation at 380
nm in the presence of calibration solutions MIN and
MAX respectively.
In another preferred embodiment, the detector irradiates
the mixture of cells or particles with test compound(s) with
laser light of one or more wavelengths. The irradiation and
emission wavelengths may be any wavelengths suitable for
the particular cellular responses or particle interactions to be
measured. It will be appreciated that the detector may detect
any type of signal suitable for detecting the cellular
responses or particle interactions and is not limited to
detectors based on light.
A computer 123, connected to the detector 119, dictates its
operation. The computer 123 is also connected to or is a part
of the controller 109 which controls the first and second
gradient devices 105 and 107, switching valve 111 as well as
test compound sampler 101 and standard sampler 103 in
a=ordance with a software 125 implemented within the
computer 123. The detector 119 is capable of measuring the
particular desired signal, whatever its origin. An optical
detector 119, may be a spectrophotometer,
spectrofluorometer, or a luminometer, each of which have a
flow-through optical cell. These devices are well-known in
the art and are commercially available. For example, one
embodiment of the apparatus of the present invention may
use an AMINCO-Bowman Series 2 Luminescence Spectrof-
luorometer (Fa-256, Spectronic Instruments, Inc.). If the
detector is a direct ion measuring device, it can, for example,
comprise a pH sensor or an ion selective electrode. Sodium,
calcium, and potassium detectors are examples of such
devices. Detectors of this type are commercially available.
In the embodiment of FIG. lB, the apparatus further
includes a coupling system 120, such as the plug flow
coupling systems described above. The coupling system 120
is disposed between the mixing chamber 112 and the detec-
tor 119.
In a preferred embodiment, the detector 119 comprises a
device capable of analyzing individual cells, individual cell
US 6,558,916 B2
31
types in a cell suspension, individual particles in a sample,
individual particle types in a sample, individual components
32
level and flow of a standard solution (agonist or antagonist)
into the mixing zone 227. Each of the gradient pumps, 211
and 225, respectively, may advantageously have two inlet
tubing lines and one outlet tubing line, the outlet tubes being
connected to each other through the mixing zone 227. A
controller (109 of FIGS. lA and lB) which is not shown in
FIGS. 3A and 3B controls the operation of autosamplers 201
and 213 and gradient pumps 211 and 225 by supplying
remote signals to start/stop the pumping. In a preferred
in a sample, or individual types of components in a sample.
Preferably, the detector has the ability to simultaneously
resolve multiple characteristics or properties derived from 5
each particle or cell. For example, in one embodiment, the
detector is a flow cytometer (FCM). The FCM may be any
current or future system, such as the Epics Elite from
Beckman-Coulter or the FACSCalibur from Becton-
Dickinson. The single particle/single cell, multiparametric
properties permit resolution of subsets of particles or cells
within a heterogeneous population. For example, subsets of
cells from within heterogeneous cell populations or mixed
cell populations which respond to a particular test compound
rn embodiment, gradient pumps 211 and 225 may be the GP 40
Gradient Pump manufactured by Dionex.
The mixing zone 227 receives the compound solution
flowing through gradient pump 211 and, optionally, a stan-
dard solution flowing through the gradient pump 225, and
or test compounds may be resolved from the entire popu-
lation by this technique. Alternatively, subsets of particles in
heterogeneous or mixed populations which possess particu-
lar properties may be resolved from the population.
Preferably, the detector also has the ability to measure
properties that can not be resolved using standard fluori-
metric techniques. FIG. 2 illustrates the multiparametric
capacity of the FCM, and indicates the flow of information
obtained from the FCM.
For the simultaneous analysis of multiple parameters, a
sample, such as an individual cell, particle, or components,
or individual cell types, particle types, or component types,
15 mixes the compound and standard substances together. A
diverting valve 235 alternates the supply of either the
calibration solution for maximal response 233 or the cali-
bration solution for minimal response 237. The diverting
valve 235 preferably includes two inlet tubes, one tube for
20 each of the two different calibration solutions 233, 237, and
one outlet tube connected to a pump 231. The diverting
valve 235 is well-known in the art and can be implemented
by the SV-3 Diverter Valve (BioRad). The controller (109 of
FIGS. lA and lB) which is not shown in FIGS.3A and 3B,
25 sends signals to the diverter valve 235 which switches intake
ports to connect one calibration solution 233 or another, 237,
to the intake of the pump 231. The pump 231 for supplying
calibration solutions to the fluidic system is also shown in
FIGS. 3A and 3B. The pump 231 receives either the cali-
is exposed to an agent which produces a signal detectable by
the detector. The signal may be a fluorescent signal or a
non-fluorexcent signal. After the cell or particle samples are
delivered from the mixing system, they are interrogated by
one or more detection agents, such as lasers, and, if the
sample possesses one or more of the parameters being
evaluated, a signal indicative of the presence of that param-
eter is generated and processed to provide a data output
which may be manipulated or stored using conventional 35
procedures.
30 bration max. solution 233 or the calibration min. solution
237 from diverting valve 235 and then pumps the received
calibration solution to switching valve 229. The outlet tube
of pump 231 is connected to an input of the switching valve
229. The pump 231 is advantageously a standard piston
pump which is well-known in the art. In a preferred
embodiment, the pump 231 is the series 1350 Soft-Start
All of the detectors described above can be controlled by
means of a computer 123 during the data acquisition pro-
cess.
The apparatus of the present invention is contemplated in
two different specific embodiments. A positive pressure
system may be created with piston pumps or other suitable
pumps supplying reagents under positive pressure.
Alternatively, a negative pressure system may be created
with a peristaltic pump or other suitable pump drawing
reagents through the system. The negative pressure system
is the simplest embodiment, because a large number of input
sources can be driven by a single downstream pump.
FIGS. 3A and 3B illustrate schematic configurations of
some embodiments of a positive pressure fluidic system. In
a preferred embodiment, this system operates automatically
under the control of a programmable controller, as will be
explained in more detail below. This embodiment includes
an autosampler 201 which hold<> one or more compounds to
be tested 203 and buffer 205 which may be mixed with the
compound 203 in order to provide various concentrations of
the compound 203; intake nozzles, or ports, 207 and 209 for
receiving a compound and buffer, respectively and deliver-
ing the same to a gradient pump 211 which controls the
concentration level and flow of a test compound 203 into a
mixing zone 227; an autosampler 213 which holds one or
more standards (i.e., antagonists 215 and/or agonists 217)
and a buffer 219 which may be mixed with the standard 215
or 217, in order to provide various concentrations of the
standard; intake nozzles, or ports, 221 and 223, for receiving
a standard and buffer, respectively, and providing the same
to a gradient pump 225 which controls the concentration
Pump (BioRad). The switching valve 229 alternates the
supply of either the compound/standard mixture or one of
the calibration solutions to a mixing zone 239. A pump 241
40 supplies cells or particles from a cell suspension or particle
reservoir 243 containing one or more cell types or one or
more particle types to be tested (or from one of a plurality
of cell suspension or particle reservoirs 243 each containing
one or more cell types or one or more particle types included
45 in a series of cell types or particle types to be examined) to
the mixing zone 239. The pump 241 receives cells or
particles from the cell suspension or particle reservoir 243
from an inlet tube and pumps the received cells or particles
through an outlet tube to one intake of the mixing zone 239.
50 The compound/standard solutions or calibration solutions
come through another intake of mixing zone 239. Both
mixing zones 227 and 239, are well known in the art and in
a preferred embodiment may be implemented by the Static
Mixer, 125-1345 (BioRad). The pump 241 can be a standard
55 piston pump which is well-known in the art. In a preferred
embodiment, the pump 241 is the series 1350 Soft-Start
Pump (BioRad). The mixture of cells or particles and
compound/standard solution or cells and a calibration solu-
tion is then fed from mixing zone 239 to reaction developing
60 lines 245. The length of these lines, combined with the flow
rate, determines the incubation period; i.e., the time elapsed
from the point where the cells or particles are mixed with the
compound/standard mixture or the calibration mixture to the
point of reaching detector 247. The detector 247 then
65 measures the amount of cell response or particle interaction
due to the compound/standard mixture or the calibration
solution. After the cell response or particle interaction has
US 6,558,916 B2
33
been measured by detector 247, the mixture is then drained
from the detector via drain 249.
In the embodiment of FIG. 3B, the apparatus further
includes a coupling system 251, such as the plug flow
coupling systems described above. The coupling system 251 5
is disposed between the mixing chamber 239 and the detec-
tor 247.
34
calibration solutions and one outlet tube connected to
another diverting valve 333. The diverting valve 333 can
have two inlet tubes, one for receiving a compound/standard
solution mixture and the other for receiving one of the
calibration solutions 337 or 339. The diverting valve 333
then alternates the supply of either the compound/standard
mixture or one of the calibration solutions through an outlet
tube to a priming valve 341 which then delivers the received
fluid to a mixing zone 343. A third diverting valve 347
As discussed above, while the embodiments shown in
FIGS. 3A and 3B include an autosampler 213, antagonists
215 and/or agonists 217, buffer 219, intake nozzles or ports
221 and 223, gradient pump 225, pump 231, diverting valve
235, calibration solutions 233 and 237, and switching valve
229, it will be appreciated that these aspects of the device
need not be present if the analysis being performed does not
require the use of a standard compound(s) or calibration
solution(s). Likewise, when the analysis being performed
does not require a concentration gradient of the test
compound(s), the device need not include a gradient pump
211, buffer 205 or port 209, but instead may include a pump
for delivering the test compound(s) to the mixing chamber.
rn alternates the supply of either cells or particles from a cell
suspension or particle reservoir 349 containing one or more
cell types or one or more particle types to be tested (or from
one of a plurality of cell suspension or particle reservoirs
each containing one or more cell types or one or more
15 particle types included in a series of cell types or particle
types to be tested) or the buffer solution 305 through an
outlet tubing 346 to a priming valve 345 which then delivers
either the cells or particles or buffer to the mixing zone 343
where it is mixed with either a compound/standard mixture
FIGS. 4A and 4B illustrate schematic configurations of
some embodiments of the negative pressure fluidic system.
As shown in FIGS. 4A and 4B, one preferred embodiment
includes an autosampler 301 which holds one or more
compounds (N;) 303 and a buffer 305, the autosampler 301
further including two intake nozzles, or ports, 307 and 309,
20 or one of the calibration solutions received from priming
valve 341. The diverting valves 333, 335 and 347 may be of
any type which is well known in the art, for example, a SV-3
Diverter Valve (BioRad). This mixture is then sent to reac-
tion developing lines 353 which determine the time elapsed
25 from the point where the cells or particles are mixed \vith the
compound/standard mixture or one of the calibration solu-
tions to the point where the mixture reaches the detector
optical cell 355. As explained above, the reaction developing
lines 353 and the detector 355 may be of any type which are
for receiving the compound (N,) 303 and buffer 305, respec-
tively; a proportionating valve 311 for preparing dilutions of
the compound 303 with the buffer 305 in predetermined or
specified proportions and delivering this mixture to a prim-
ing valve 313; an autosampler 315 which holds standard
antagonists (M) 317 agonists (Lk) 319 and the buffer 305,
the autosampler 315 further including two intake nozzles
323 and 325 for receiving the standard and the buffer,
respectively; a proportionating valve 327 for preparing 35
dilutions of the standard substance 317 or 319 with buffer
30 well-known in the art.
A peristaltic pump 357, used as a negative pressure pump,
can advantageously supply the necessary pressure required
to make the various solutions and mixtures described above
flow through the various valves (311, 313, 327, 329, 333,
335, 341, 345 and 347) mixing zones (331 and 343), the
reaction developing lines 353, the detector 355, and finally
to the drain 359. Suitable peristaltic pumps 357 are well-
known in the industry and in a preferred embodiment may
be implemented by a EP-1 Econo Pump (BioRad). The
305 in predetermined or specified proportions and delivering
this mixture to a priming valve 329. The proportionating
valves 311 and 327 may be of any type which is well-known
in the art. In a preferred embodiment these valves 311 and
327 are the series 4 miniature solenoid type valves ( 4-8-900
General Valve Corp.) working in a proportioning frequency
mode. Similarly, priming valves 313 and 329 may be of any
"normally opened" type which is well-known in the art and,
in a preferred embodiment, are the series 4 miniature sole-
noid type valves ( 4-39-900) manufactured by General Valve
Corp.
40 pumps, valves, detectors, and other active components of the
system are preferably under the automated control of a
programmable controller, as explained in more detail below.
In the embodiment of FIG. 4B, the apparatus further
includes a coupling system 361, such as the plug flow
45 coupling systems described above. The coupling system 361
is disposed between the mixing chamber 343 and the detec-
tor 355.
A mixing zone 331 mixes a compound solution received
from priming valve 313 and a standard solution received
from priming valve 329. The first mixing zone 331, as with 50
the other mixing zones discussed herein, can constitute a
simple "Y" connector; a chamber having a diameter several
times that of the tubing; a length of tubing; a serpentine,
baffled chamber; a chamber containing a mechanical rotat-
ing mixer; or any other suitable structure. Typically, the 55
method will involve very small quantities of liquid (e.g., a
full concentration gradient run can be accomplished with as
little as 0.3 ml of sample). Thus, the volumes to be mixed are
small and the mixing zone should have small internal
volume and high mixing efficacy. These types of mixers are 60
well known in the art and in a preferred embodiment may be
implemented by the Visco JetD Micro-Mixer
(#TCMA0120113T) manufactured by The Lee Company.
A diverting valve 335 alternates the supply of either a
calibration solution for maximal response 337 or a calibra- 65
tion solution for minimal response 339. The diverting valve
335 includes two inlet tubes for receiving the two different
As discussed above, while the embodiments shown in
FIGS. 4A and 4B include components for supplying a
standard(s) and a calibration solution(s), it will be appreci-
ated that these aspects of the device need not be present if
the analysis being performed does not require the use of a
standard compound(s) or calibration solution(s). Likewise,
when the analysis being performed does not require a
concentration gradient of the test compound(s), the device
need not include components which provide a gradient, but
instead may include a pump for delivering the test
compound(s) to the mixing chamber.
The apparatus in the present invention preferably has two
primary modes of action, a screening mode and a potency
mode.
FIG. 5 shows an algorithm that may be used in the
invention to detect one or more cell responses or the
presence of one or more molecules during the screening
mode. First, a cell or particle/compound mixture or a sample
is provided to a detector (step 401). Next, the apparatus
determines if the compound, upon contact with the cells,
US 6,558,916 B2
35
triggers any cell response or whether the sample contains a
molecule (step 403). There are two possibilities: either the
compound does not produce any of the responses being
analyzed or the sample does not contain any of the mol-
ecules being analyzed (NO), or it induces one or more of the 5
cell responses or does contain one or more of the molecules
(YES). Cell response is determined by monitoring the signal
from the detector for the particular analyte or analytes being
detected. Similarly, the presence of one or more molecules
in the sample is determined by monitoring the signal from rn
the detector for the presence of signal which will be pro-
duced if the particular molecule or molecules being analyzed
is present. The control system first calibrates to establish a
baseline and a maximal response, and any signal from the
detector falling between those values is considered to be a 15
positive response.
If the compound causes no cell response or no particle
binding, (NO), as measured by the monitoring part of the
apparatus, the cells or particles are successively and auto-
matically brought into contact with a mixture of the com- 20
pound and the standard substances from a predetermined set
of agonist solutions (step 407). Each solution in the set
contains one or more ingredients that initiates cell response
or that binds to particles in the absence of the test compound,
e.g., through the stimulation of a known cell receptor, ion 25
pump or ion channel molecules or through a known molecu-
lar interaction. Next, a determination is made as to whether
a cell response or particle interaction normally triggered
with a particular agonist is suppressed by the particular
compound (step 411). The apparatus will keep repeating an 30
admixture of different standard agonist substances with the
compound until it detects that the cell response or particle
interactions triggered with a particular standard agonist is
suppressed in the presence of the compound, or until all
agonists available to the machine have been tested. If in step 35
411, it is determined that a cell response or particle inter-
action normally triggered with a particular agonist is sup-
pressed by the particular compound (YES), that compound
is categorized as an antagonist to the receptor Ri (step 415).
After this happens, the instrument is switched over to the 40
potency mode of action if instructed by the software man-
aging the apparatus' performance (step 417).
If the contact of the cells or particles with the compound
does initiate the cell response or particle interaction, (YES),
as measured by the monitoring part of the apparatus, the 45
cells or particles are automatically brought into contact with
successive mixtures of the compound and the standard
substance from a predetermined set of antagonist solutions
(step 405). Each solution in the antagonist set contains one
or more ingredients that block the cell response or particle 50
interaction initiated by at least one known agonist, for
example through the stimulation of a known cell receptor,
ion pump or ion channel molecules. Next, a determination is
made as to whether a cell response or particle interaction
triggered with the compound is suppressed in the presence 55
of a particular standard antagonist (step 409). The apparatus
will keep repeating an admixture of different standard
antagonist substances with the compound until it detects that
the cell response or particle interaction triggered with the
compound is suppressed in the presence of particular stan- 60
dard antagonist, or until all the antagonists have been tested.
If it is determined in step 409, that a cell response or particle
interaction triggered with the compound is suppressed by the
particular antagonist (YES), that compound will be charac-
terized as an agonist to the receptor Ri or other molecule on 65
the cell or particle (step 413). After this happens, the
instrument is switched over to the potency mode of action if
36
instructed by the software managing the apparatus' perfor-
mance (step 417).
By using known sets of standard agonist and antagonist
substances to different receptors, it is possible to screen the
compounds against several receptor types and subtypes for
specificity and selectivity. When a series of cell types or
particle types is to be tested, the process can be repeated for
each of the cell types or particle types included in the series
of cell types or particle types to be tested in order to evaluate
the compounds' activities in a number of cell types or
interactions with a number of particle types.
For example, for the endothelin receptor, stimulation of
which is indicated by an increase in intracellular concentra-
tion of ionized calcium, the following receptor subtype
specific antagonists may be used: BQ-123, BQ-788,
BQ-153, BQ-485, BMS-182874 PD 151, 242, and the
following receptor subtype specific agonists may be used:
endothelin-1, endothelin-2, and endothelin-3. Sarafotoxin
S6c, IRL 1620, BQ-3020.
For calcium channels, there are sets of channel type
specific agonists and antagonists which can be used in a
preferred embodiment. For example, agonists of intracellu-
lar calcium channels are: Ins (1,4,5)P3 , Ryanodine, Caffeine,
Heparine, Perchlorate, and their antagonists are:
Decavanadte, Ruthenium Red and high concentrations of
Ryanodine.
As discussed above, while the embodiments shown in
FIG. 5 include steps for supplying a standard(s), it will be
appreciated that these steps need not be present if the
analysis being performed does not require the use of a
standard compound(s). Instead, the algorithm simply deter-
mines whether the cells responded to the test compound(s)
or whether the sample contains a molecule.
In another embodiment, the screening mode is used for
the characterization of the cell receptor pattern, commonly
known asD receptor fingerprintingD. In accordance \Vith
this embodiment, a cell type to be tested or a series of cell
types are screened against standard substances from a pre-
determined set of agonist solutions. Each solution in the set
contains one or more ingredients that initiates cell response
through the stimulation of a known cell receptor. In this way,
the patterns of receptor expression in two or more cell types
may be evaluated. When one or more of the cell types
respond to the particular agonist substance, the instrument is
switched over to the potency mode of action if instructed by
the software managing the apparatus' performance.
FIG. 6 shows one algorithm that may be used in the
invention in the preferred potency mode. The first step in the
potency mode is to determine whether a particular com-
pound has been categorized as an agonist or antagonist (step
501). The apparatus then prepares continuous concentration
gradients of either the standard substance or the compound
being tested.
If the compound has been categorized as an agonist for
one or more of the cell types or particle interactions to be
tested during the screening mode, the apparatus will mea-
sure and register the concentration dependence of the cell
response in the responding cells or the particle interaction
(step 503). During step 503, the apparatus will generate
continuous experimental activation curves for the activatory
compound. From these curves, one can calculate the potency
of the compound in terms, for example, of EC50 (the
effective concentration of an activator which causes 50% of
the maximal stimulatory response of the cells)(step 505).
This calculation can be performed using well known in the
art curve fitting software. In a preferred embodiment this
software may be implemented by the graphical software
PRISM, manufactured by GraphPad, Inc.
US 6,558,916 B2
37
If the compound is determined to be an antagonist for one
38
A continuous flow diagram of a preferred system priming
process is shown in FIGS. 7a-7d for the preferred negative
pressure fluidics system presented in FIGS. 3A and 3B.
Referring to FIG. 7a, the system priming program begins
from a start state 600 and enters state 602 where it sets all
parameters to their initial values. These initial parameters
include, but are not limited to, the internal initialization of
software variables and subroutines.
Once the initial parameters are determined, autosamplers
301 and 302 (FIGS. 4A and 4B), position their correspond-
ing intake nozzles 307 and 323 at "zero" position (Step 604).
Each autosampler, in its "zero" position, contains a reservoir
305 filled with a washing buffer. During step 604, the power
supply which supplies power to proportioning valves 311
or more of the cell types or particle interactions to be tested
during the screening mode, the apparatus will measure and
register the concentration dependence of cell response or
particle interaction inhibition in these cell types or particle 5
types in the presence of a standard agonist (step 507).
During step 507, the apparatus will generate continuous
experimental inhibition curves for the antagonist compound
taken at constant concentrations of the standard agonist
substance. From these curves, one can calculate the potency
10
of the compound in terms, for example, of IC50 (the effective
concentration of a blocker which causes 50% of the maximal
inhibitory response cells or 50% of the maximal level of
particle interaction) (step 509). The calculation of the IC50
valves can also be implemented in a preferred embodiment
with the PRISM software package. 15 and 327 (FIGS. 4A and 4B), to diverting valves 333, 335 and
347 (FIGS. 4A and 4B), and to priming valves 313, 329, 341
and 345 (FIGS. 4A and 4b) is turned off. In the preferred
embodiment, the priming valves, 313, 329, 341and345, are
If the compound is determined to be an antagoni<>t for one
or more of the cell types or particle types to be tested, the
apparatus will also measure and register the concentration
dependence of the cell response or particle interaction in
these cell types or particle types to a standard agonist 20
substance in the presence of several concentrations of the
antagonist compound (step 511). During step 511, the appa-
ratus will generate a series of continuous experimental
activation curves for the standard agonist substance taken at
different discrete concentrations of the antagonist com- 25
pound. The apparatus will then calculate the affinity and the
potency of the compound in terms, for example, of p~
values and determine whether the antagonist is competitive
or non-competitive (step 513). The p~ value is proportional
normally opened in a non-powered state (off); the propor-
tioning valves, 311 and 327, in the non-powered (off) state,
connect their outlets 312 and 328 with respective "normally
opened" intake ports, 309 and 325; and in the non-powered
(off) state, the diverting valve 335 connects its common
outlet 334 with the "normally opened" intake port 336,
diverting valve 333 connects its common outlet 342 with the
"normally opened" intake port 332, and diverting valve 347
connects its common outlet 346 with the ''normally opened"
intake port 348.
After the system has been initialized, peristaltic pump 357
is started (Step 606) and priming valves 313 and 345 are
turned on (Step 608). In the powered state, the priming
valves 313 and 345 are closed. This forces the buffer flow
through tubing 325, outlet tubing 328 of proportioning valve
327, priming valve 329, mixing zone 331, intake tubing 332
to a negative logarithm of the binding constant of a ligand/
30 receptor complex and is a measure of the affinity of the
ligand to the receptor: the bigger pA2 value, the higher the
compound's affinity to the receptor. Practically, pA2 value
can be calculated from the shift of the activation curves in
the presence of different concentrations of the antagonist
compound and can be implemented by the formula: 35 and outlet tubing 342 of the diverting valve 333, priming
valve 341, mixing zone 343, a reaction developing line 353,
detector 355, pump 357 and then into a drain container 359. pA 2 -Log(R-1)-Log B,
where R is a ratio of equipotent concentrations of the
standard agonist substance measured both in the pres-
ence of discrete concentration (B) of the antagonist
compound and without the antagonist. In a preferred
embodiment the equipotent concentrations of the stan-
dard agonist substance can be found from the PRISM
software activation curves. With the above sets of
experimental curves, one can evaluate the potency and
the pharmacological profile for the compound under
investigation in accordance with Cheng-Prusoff (Cheng
& Prusoff, 1973, incorporated herein by reference),
Gaddum (Gaddum, 1957, incorporated herein by
reference) or Schild (Arunlakshana & Schild, 1959,
incorporated herein by reference) analyses.
A detailed description of the operation of the negative
pressure fluidics system of FIGS. 4A and 4B is given below.
Referring again to FIGS. lA and lB, computer 123
implement<> software code 125 to provide control signals to
40 a controller 109 which controls the various valves, chambers
etc. of the system. This control signal is delayed in order to
provide adequate time for liquid to fill the fluidic lines (Step
610). Next, the proportioning valve 327 is turned on (Step
612). While proportioning valve 327 is in the powered state,
45 the buffer located at "zero" position of the autosampler 315
will flow through tubing 323, outlet tubing 328 of propor-
tioning valve 327, priming valve 329, mixing zone 331,
intake tubing 332 and outlet tubing 342 of diverting valve
333, priming valve 341, mixing zone 343, a reaction devel-
50 oping line 353, detector 355, pump 357 and then into a drain
container 359.
The process is started when a computer prompts an 55
operator to chose a mode of operation from a choice of three
modes: system priming mode, screening mode and potency
profiling mode. In the presently preferred top-level process,
the first choice is to perform system priming. Referring to
FIGS. 4A and 4B, before the priming process starts, the 60
operator is prompted to place the nozzles of intake ports 309,
325 and 351 into a reservoir 305 filled \Vith a buffer, the
nozzles of the intake ports 336 and 338 into reservoirs 337
and 339, respectively, filled with corresponding calibration
solutions, and the nozzle of the intake port 348 into one of 65
the cell suspension or particle reservoirs 349 filled with one
After energizing the proportioning valve 327, the control
signal is delayed again (Step 613) in order to provide the
time needed for the buffer to fill out the fluidics system. After
the delay in Step 613, the control signal will set the
proportioning valve 327 to its off position (Step 614), turn
off (open) priming valve 313 (Step 616) and turn on (close)
the priming valve 329 (Step 618). This will fill out fluidic
lines 309, 312, 332, 342, priming valve 341, mixing zone
343, a reaction developing line 353, detector 355 and pump
357 during a delay (Step 619) which is needed for the lines
to be filled with buffer 305.
Next, the proportioning valve 311 is powered (Step 620)
for a time duration controlled by Step 621. During this delay
period, the same fluidic lines are being fed with the buffer
located at "zero" position of the autosampler 301, coming
through the nozzle 307. or more cell types or one or more particle types to be tested.
US 6,558,916 B2
39
After the delay period (Step 621) the control signal
simultaneously turns off both proportioning valve 311 (Step
622) and priming valve 329 (Step 624) and turns on both
diverting valve 335 and diverting valve 333 (Step 626).
During a delay period, determined by Step 627, the liquid
flow will be directed from intake tubing 338 and outlet
tubing 334 of the diverting valve 335 through the intake
tubing 340 and the outlet tubing 342 of the diverting valve
333 through the priming valve 341, the mixing zone 343 and
further through the reaction developing line 353, the detec-
tor 355, the pump 357 and then to the drain container 359.
Then the control signal turns off diverting valve 335 (Step
628), which allows the same fluidic lines to be fed from
intake tubing 336. The length of time required i<> a function
of the length of the different lines involved and the rate of
fluid flow through the system, which for any particular
system can be readily determined.
After the delay provided by Step 629, the control signal
turns off diverting valve 333 (Step 630), turns off (opens)
priming valve 345 (Step 632) and turns on (closes) priming
valve 341 (Step 634). Under these conditions, the liquid flow
will be directed from the intake tubing 348 to outlet tubing
346 of diverting valve 347, through the priming valve 345,
the mixing zone 343, the reaction developing line 353, the
detector 355, the pump 357 and then to the drain container
359. The time required for the above liquid flow to occur is
provided by a delay period in Step 635. After this delay, the
control signal turns on diverting valve 347 (Step 636), which
allows the same fluidic lines to be filled with the buffer 305
coming from tubing 351, during a delay period provided in
Step 637.
40
occupied by a wash buffer reservoir 305 (Step 704), and
turns off proportioning valves 311 and 327, diverting valves
333, 335 and 347, and priming valves 313, 329, 341and345
(Step 706). In a turned off state, priming valves 313, 329,
5 341 and 345, are normally opened, proportioning valves 311
and 327 connect, respectively, their outlets 312 and 328 \Vith
corresponding "normally opened" intake ports 309 and 325,
diverting valve 335 connects its common outlet 334 with
"normally opened" intake port 336, diverting valve 333
rn connects its common outlet 342 with the "normally opened"
intake port 332, and diverting valve 347 connects its com-
mon outlet 346 with the "normally opened" intake port 348.
After the system has been initialized, pump 357 is started
(Step 708). This will force the wash buffer from the "zero"
15 positioned reservoirs of both autosamplers 301 and 315 to
run through nozzles 309 and 325 and outlet ports 312 and
328 of proportioning valves 311 and 327, respectively,
priming valves 313 and 329, mixing zone 331, intake port
332 and outlet port 342 of diverting valve 333, priming
20 valve 341, mixing zone 343, a reaction developing line 353,
detector 355, pump 357 and then into a drain container 359.
In mixing zone 343 this flow is mixed with cell suspensions
or particle preparations, which contain one or more cell
types or one or more particle types to be tested, from cell
25 suspension or particle reservoir(s) 349 coming from intake
tubing 348 and an outlet port 346 of diverting valve 347
through priming valve 345. Thus, the total flow passing
mixing zone 343 is a sum of three flows, one of each coming
from the proportioning valves 311 and 327 and one coming
30 from diverting valve 347. During this step, both proportion-
ing valves supply wash buffer 305 from the reservoirs
located at "zero" position of each autosampler in such a way
that the final mixture consists of one part cell suspension or
After the delay provided by Step 637, the control signal
turns off (opens) priming valve 341 (Step 638), and turns off
the diverting valve 347 (Step 640). The control signal then
turns off pump 357 (Step 642). Finally, the priming program 35
ends the system priming mode and all components of the
fluidics system are filled with liquids (Step 643).
particle preparation and two parts wash buffer 305.
After the delay determined in step 709, which is needed
for the flow of the mixture of the cell suspension or particle
types containing one or more cell types or particle types to
be tested (or a member of a series of cell types or particle
types to be tested) and wash buffer 305 to stabilize, detector
The state of the valves at each step of the program is
shown in FIG. 7d. Priming valves 345, 341, 313 and 329 are
of a two-way normally opened type. Diverting valves 335,
333 and 347 are of a three-way type with one common outlet
port and one each normally opened and normally closed
intake ports. Proportionating valves 311 and 327 are also of
the three-way type similar to diverting valves 335, 333 and
347. The symbols''-" and"+" indicate turned off and turned
on state of the valves. The state of all the valves after the
priming mode is finished is the same as at the initial state.
After the priming of the system is done, the operator is
prompted to start either the screening or potency profiling
mode. If screening mode is chosen, the operator is prompted
to specify how many compounds are located in the set of
compounds to be tested, N,,,=, and how many antagonists,
M,,,ax' and agonists, L,,,ax' solutions are located in the
respective sets of the standards. If potency profiling mode is
chosen, the operator is prompted to specify which com-
pounds in a set should be measured.
A continuous flow diagram of the presently preferred
screening mode with the negative pressure fluidics system of
FIGS. 4A and 4B is shown on FIGS. 8a-8g.
The screening program begins from a start state 700 and
enters state 702 where it sets all parameters to their initial
values. These initial parameters include, but are not limited
to, the internal initialization of software variables and sub-
routines.
Once the initial parameters are determined, the autosam-
plers 301 and 315 (FIGS. 4A and 4B) position the intake
no=les 307 and 323 at the corresponding "zero" position
40 355 registers a basal signal produced by the cells or particles
alone, BS (Step 710). After the BS signal is registered, it is
saved as a reference (Step 712) and computer 123 (FIGS. lA
and lB) triggers incremental counter N (Step 714).
The numerical content of the incremental counter N is
45 increased by unity each time it is triggered thus determining
the position of the nozzle of intake port 307 of autosampler
301 which samples the sets of compounds 303 to be tested.
Next, the numerical content, N;, of the incremental counter
is checked against an entered maximum number of the
50 compounds to be tested, N,,,ax (Step 716). If N; does not
exceed N,,,ax, autosampler 301 positions nozzle 307 into the
compound reservoir located at the N; position of a rack of
compounds 303. Next, proportioning valve 311 is switched
on to open its "normally closed" intake port 307 to the
55 common output port 312 (Step 720). During Step 720, the
combined flow in mixing zone 343 and afterwards in detec-
tor 355, is composed of one portion of buffer 305 coming
through nozzle 325 of proportioning valve 327, one portion
of the compound 303 to be tested coming from nozzle 307
60 of proportioning valve 311 and one portion of the cell
suspension or particle preparation containing one or more
cell types or one or more particle types to be tested coming
from the intake tubing 348 which is "normally opened" to
the output port 346 of diverting valve 347. After a delay
65 provided by Step 721, which is needed for the mixing
process to stabilize, detector 355 registers a signal, SN;,
produced by the cells or particles in the presence of the given
US 6,558,916 B2
41 42
compound, N; (Step 722). After the SNi signal is registered,
its value is saved as a reference value (Step 723). The value
of the SNi signal is then compared with the value of the
reference basal signal, BS (Step 724). If the SN,. signal is
greater than the BS signal, the computer 123 (FIGS. lA and 5
lB) will "flag" the corresponding compound as "positive"
(Step 725), which means that the compound stimulates the
cell signal or interacts with the particle. If SN; is greater than
BS, the computer 123 controls a set of antagonists and
calculates corresponding coordinates for positioning the rn
no=le 323 of autosampler 315 over the antagonist contain-
ing reservoirs located in rack 317. If SN; is less than BS,
computer 123 will "flag" the corresponding compound as
"negative" (Step 726). In this case, the computer 123
controls a set of agonists and calculates corresponding 15
coordinates for positioning the nozzle 323 of autosampler
315 over the agonist containing reservoirs located in rack
319.
flagged as "positive" and the standard antagonist Mj is
flagged as "negative" (Step 752). After the data is flagged,
the data, containing the ID number of the compound N; with
its flag value and ID number of the standard antagonist Mj
with its flag value is transferred into a database (Step 753).
After the data is saved the program loops back to trigger the
incremental counter M (Step 730), to increase its count by
one and Step 754 is repeated until the condition 732 is
achieved.
When the condition determined in the decision step 732 is
met, that is the compound has been tested against all
standard antagonists in a given set, the program resets the
content of incremental counter M to zero (Step 755), and
both autosampler 301 and autosampler 315 position their
respective nozzles 307 and 323 to "zero" position, where the
reservoirs with a washing buffer 305 are located (Step 756).
Next, both proportioning valves 311 and 327 are turned on
(Step 758). This opens "normally closed" intake ports 307
and 323 to the corresponding outlet port<> 312 and 328. Each time the condition, "SN;>BS", is satisfied, the
program flow will go through loop K-0 of the incremental
counter M (Step 730). The incremental counter, M, increases
the count number by one each time it is triggered and thus
determines the successive positions of nozzle 323 of
autosampler 315 which serves the set of standard antagonists
located on rack 317. Next, computer 123 checks if the
numerical content of the incremental counter (M) exceeds
the maximum number of standards to be tested (M,,,a.J
entered by the operator (Step 732).
If M 1 is less than or equal to Mmax' autosampler will
position nozzle 323 into the standard antagonist reservoir,
located on rack 317 corresponding to the numerical content
of the incremental counter, Mj (Step 734). Next, proportion-
ing valve 327 is switched over to connect its "normally
closed" intake port 323 with the outlet port 328 (Step 736).
During Step 736, the combined flow through mixing zone
343, reaction developing lines 353 and detector 355 is
composed of one portion of standard antagonist coming
from proportioning valve 327, one portion of the compound
to be tested, coming from proportioning valve 311 and one
portion of the cell suspension or particle preparation con-
taining one or more cell types or one or more particle types
to be tested coming from diverting valve 347.
Next, detector 355 registers and saves (Step 738) for
further comparison, a signal, SN;Mj, produced by a given
compound, N,., in the presence of a given standard
antagonist, Mj. Then, both proportioning valves 311and327
are turned off (step 740). This closes intake ports 307 and
323 and opens intake ports 309 and 325 to the corresponding
outlet ports 312 and 328. Autosampler 315 then positions
no=le 323 into "zero" position occupied by wash buffer
reservoir 305. The proportioning valve 327 is then turned on
again to open its "normally closed" intake port 323 into the
outlet port 328 (Step 744) and the content of nozzle 323 is
washed out (Step 746). Next, proportioning valve 327 is
turned off to close intake port 323 and to open "normally
opened" intake port 325. During this process, the fluidic
lines are washed with buffer coming from nozzle 325 of
autosampler 315.
20 During this washing delay period determined by Step 760,
the contents of both nozzle 312 and nozzle 328 are washed
out with buffer 305. After Step 760 is finished, both pro-
portioning valves 311 and 327 are turned off (Step 762).
After the washing delay period (Step 760), the program
25 loops back to the incremental counter N content of which is
incremented by one (Step 714) by triggering signal S,r from
the computer 123 (FIGS. lA and lB). Then, the numerical
content N,. of counter N is compared with the maximum
number, Nmax' of the compounds to be tested. If N,. exceeds
30 N,,,ax' the content of incremental counter N is zeroed (Step
717) and the screening mode is stopped (Step 719).
Otherwise, Step 715 will be repeated with the next com-
pound.
If the condition of the decision process 724, SNi>BS, is
35 not satisfied, the Ni compound is flagged as "negative" (Step
726) and the incremental counter Lis triggered (Step 764).
The incremental counter L is increased by one (Step 764)
each time it is triggered and thus determines the su=essive
positions of nozzle 323 of autosampler 315 relative to the set
40 of standard agonists located on rack 319. Next, the numeri-
cal content, Lb of incremental counter L is compared with
the maximum number, L,,,=, of standard agonists to be used
(step 766).
If Lk is less than or equal to the Lmax' autosampler 315
45 positions nozzle 323 into the standard agonist reservoir in
rack 319, which corresponds to the numerical content, Lk, of
incremental counter L. Next, proportioning valve 327 is
turned on to open its "normally closed" intake port 323 to
outlet port 328 and proportioning valve 311 is turned off to
50 close its intake port 307 and open intake port 309 to outlet
port 312 (Step 770). During step 770, the combined flow
coming through mixing zone 343, reaction developing lines
353 and detector 355 is composed of one portion of standard
agonist 319 coming from inlet port 323 of proportioning
55 valve 327, one portion of buffer 305, coming from inlet port
309 of proportioning valve 311 and one portion of the cell
suspension or particle preparation containing one or more
cell types or one or more particle types to be tested coming
from the diverting valve 347.
After a delay period provided by Step 772, which is
needed for the mixing process to stabilize, a signal, SLk,
produced by a given standard agonist, Lk is registered and
saved (Step 774). Next, proportioning valve 311 is turned on
to open its "normally closed" intake port 307 to outlet port
Next, Computer 123 (FIGS. lA and lB) compares signal,
SN;, measured in the presence of a compound N; alone, with 60
the signal, SN;Mj, measured in the presence of both a
compound Ni and a standard antagonist, Mj (Step 750). If the
signal in the presence of a standard antagonist, Mpis lower
than the signal generated by a compound N; alone,
SN;Mj set forth in Example 1.)
15 valve 341 into the mixing zone 343, where it was mixed with
a suspension of TE-671 cells to provide a final concentration
of cells 4x105 cells/ml. At the same time, BQ-123, as a
standard antagonist solution, was also directed to the mixing
zone 331 at a predetermined concentration prepared by
20 mixing BQ-123 with DPBS in the proportioning valve 327,
in a ratio of 1:1 with the ET-1 solution. In mixing zone 343,
the mixed flow of BQ-123 at constant concentration, and
ET-1 at a continuously changing in time concentration is
mixed with cells in a ration of 2: 1. This gives three fold final
Alternatively, if one wishes to determine the pattern of
natural expression of receptors responsible for Ca2 + signal-
ing pathway, then one may use the cells of particular interest
and then using a set of agonists known to exert their activity
through Ca2 + mobilization, to characterize the cells by what
type of the receptors are expressed in these particular cells.
This set of agonists may consist of acethylcholin, adrenaline,
noradrenaline, 5-hydroxitriptamine, DOPA, NMDA,
AMPA, Angiotensin II, Bradykinin, Bombesin, Opioid,
Endothelin-1 Neuropeptide Y, TNF, PDGF, FGF and so on.
The following examples illustrate specific, non-limiting
experiments, or compound profiling operations, in accor-
dance with the present invention.
EXAMPLE 1
TE-671 cells, human medulloblastoma (ATCC CRL
8805), naturally express endothelin subtype A receptor
E'J'."R. This receptor belongs to the family of seven
transmembrane-spanning G-protein coupled receptors and is
known to activate calcium mobilization in the cell cytoplasm
upon binding to its specific agonist endothelin-1, a 21-amino
acid peptide. To characterize the affinity of the agonist for
the receptor, conventional methods measure the physiologi-
cal response of the cell in the presence of several concen-
trations of the agonist or antagonist (Sakamoto, A. et al.,
1994, incorporated herein by reference).
FIG. 10 illustrates a measurement of the activation of
intracellular calcium mobilization (Ca2 + measured in nano-
molar concentration) as a function of the concentration of
endothelin-1 (ET-1, measured in picomolar concentration),
using the apparatus and method of the present invention. The
apparatus was a negative pressure computer controlled unit
as described above. This run was performed in the presence
of several concentrations of E'J'."R specific competitive
antagonist BQ-123 (shown in nanomolar concentration).
25 dilution for each component of the flow passing through
mixing zone 343 and eventually, detector 355. The propor-
tioning valve 327, was programmed for the first, second and
third runs to give fmal BQ-123 concentration of zero, 10 nM
and 40 nM, respectively. The proportioning valve 311 was
30 preprogrammed to give a final concentration of ET-1 in each
run from 0 to 100 pM. Changes in intracellular calcium ion
concentration in the TE-671 cells passing through the detec-
tor 315, were measured in real time as a function of ET-1
concentration using fluorescent dye FURA-2 as described
35 above.
The flow rate and the length of the reaction developing
line were chosen so that the time interval from the point of
mixing the cells with the compound to the point of signal
detection in the flow-through optical cell was 40 seconds.
40 The time required for the complete concentration gradient
run to generate the curves in FIG. 10 was five minutes using
the present invention. In contrast, in a conventional assay
using the phosphatidylinositol turnover rate as an indicator
of ETAR stimulation, it usually takes several hours to get
45 only a few concentration points on the response curve, and
it would take an inordinate amount of time to generate a
complete curve as shown in FIG. 10.
FIG. 11 is a logarithmic transformation of the data in FIG.
10, which is used in the art to calculate maximal cell
SD response in the presence of antagonist as well as EC50 value
for agonist and pA2 value for antagonist. The EC50 value
calculated from the activation curve without BQ-123 is
equal to 10 pM and one can see that the activation curves in
FIG. 11 are parallel shifted to the right in the presence of
55 BQ-123, which is known in the art as characteristic for
competitive type of inhibition. The average pA2 value
calculated from the two different concentrations of BQ-123
is equal to 7.96. This corresponds to inhibition constant, K 1 ,
for BQ-123 of 11 nM. The EC50 and pA2 values are in close
agreement with the literature data (Masaki Ihara et al., 1992,
is incorporated herein by reference).
EXAMPLE 2
FIG. 12 illustrates the inhibition of ET-1 induced intrac-
TE-671 cells were prepared for use in the cell physiom- 60
eter of the present invention by growing them in a T75 flask
until confluence. The growth media was decanted and the
cells were washed twice with Dulbecco's phosphate buff-
ered saline (DPBS) and supplemented with a fresh DPBS
containing 2 ,uM FURA-2AM, which easily penetrates into
cell cytoplasm and is hydrolyzed there with nonspecific
esterases to form FURA 2, a dye which is sensitive to the
65 ellular calcium mobilization with BQ-123 in TE-671 cells.
The same cell physiometer apparatus and cell preparation
procedure was used here as in Example 1.
US 6,558,916 B2
49
In this experiment, ET-1 was introduced at a constant
concentration from the "standard" sipper nozzle of the
inventive apparatus and BQ-123 was introduced through the
"compound" no=le of the apparatus. In this example, as in
Example 1, the concentration run was five minutes and the 5
reaction time (from mixing to detection) was forty seconds.
Briefly, after calibration of the device of FIG. 4A with
minimum and maximum standards (337, 339), ET-1
solution, an agonist standard solution, was directed at a
predetermined concentration prepared by proportioning rn
valve 327 by mixing it with DPBS buffer, through priming
valve 329, mixing zone 331, diverting valve 333 and prim-
ing valve 341 and then into mixing zone 343, where it was
mixed with a suspension of TE-671 cells to provide a final
concentration of cells of 4x105 cells/ml. At the same time, 15
DPB in volumetric ratio of 1:1 to ET-1 solution was directed
through intake port 309 and outlet port 312 of proportioning
valve 311, priming valve 313 to mixing zone 331 and further
through fluidics system to the detector 355. After the intra-
cellular calcium concentration in the presence of the stan- 20
, mixtures of a known agonist and a known
antagonist, or compounds whose activity is unknown as
described above. This process is performed for each of the
cell types to be tested to determine the nature and magnitude
20
of the cellular response in each cell type. To facilitate the
analysis of multiple cell types, each of the cell types may be
placed in one of a plurality of cell suspension reservoirs in
the devices described above.
FIG. 14 shows an algorithm that may be used in the
invention to perform cell mapping and cell receptor "fin-
gerprinting". First, cells are mixed with standard agonist
(step 1301) and the mixture is provided to a detector (step
1302). Next, the apparatus determines if this standard
agonist, upon contact v-rith the cells, triggers any cell
response (step 1303). There are two possibilities: either the
standard agonist does not produce any response (NO), or it
induces the cell response (YES). Cell response is determined
by monitoring the signal from the detector for the particular
standard agonist being evaluated. The control system first
calibrates to establish a baseline and a maximal response,
and any signal from the detector falling between those
values is considered to be a positive response.
If the standard agonist causes no cell response, (NO), as
measured by the monitoring part of the apparatus, the system
increments an agonist counter (step 1309) which indicates
that a next standard agonist in a specified set of agonists will
be tested with a particular cell line. The system then deter-
mines if all available agonists have been tested (step 1305).
15 are then repeated. In one embodiment, the apparatus will
keep repeating an admixture of different standard agonist
substances with the cells until it detects that all agonists
available to the apparatus have been tested with all cell lines
available to the apparatus.
FIG. 15 shows an algorithm that may be used in the
invention to perform transfected receptor characterization,
particularly, of an orphaned receptor. First, in step 1401, an
apparatus supplies cells transfected with a gene which
produces a known orphan receptor, and mixes them with a
25 standard agonist (step 1402). The mixture is provided to a
detector (step 1403). Next, the apparatus determines if this
standard agonist, upon contact with the cells, triggers any
cell response (step 1404). There are two possibilities: either
the standard agonist does not produce any response (NO), or
30 it induces the cell response (YES). Cell response is deter-
mined by monitoring the signal from the detector for the
particular standard agonist being evaluated. The control
system first calibrates to establish a baseline and a maximal
response, and any signal from the detector falling between
35 those values is considered to be a positive response.
If the standard agonist causes no cell response, (NO), as
measured by the monitoring part of the apparatus, the system
increments a standard agonist counter (step 1405) which
indicates that a next standard agonist in a specified set of
40 standard agonists will be tested with the transfected cells.
The system then determines whether all available standard
agon~ts have already been tested v-rith the tranfected cells
(step 1406). If not, the process moves back to step 1401 in
which fresh cells from the same transfected cell line are
If all standard agonists have not been tested with a particular 45
cell line, fresh cells from the original cell line are then
supplied to the system (step 1306). These new cells are then
automatically brought into contact with the next standard
agonist from the predetermined set of agonist solutions (step
1301). Each agonist solution in the set contains one or more 50
ingredients that are known to initiate cell response through
the stimulation of a known cell receptor, ion pump or ion
channel molecules. The apparatus will keep repeating an
admixture of different standard agonist substances with the
cells until it detects that the cell response is triggered v-rith 55
a particular standard agonist, or until all agonists available
automatically brought into contact with the next standard
agonist from the predetermined set of agonist solutions (step
1402). Each standard agonist solution in the set contains one
or more ingredients that are known to initiate cell response
through the stimulation of a known cell receptor, ion pump
or ion channel molecule. The apparatus will keep repeating
an admixture of different standard agonist substances with
the transfected cells until it detects that the cell response is
triggered with a particular standard agonist, or until all
agonists available to the machine have been tested.
If in step 1404 it is determined that the contact of the
transfected cells v-rith the standard agonist does initiate the
cell response, (YES), as measured by the monitoring part of
the apparatus, the process then supplies host cells (step
1407) to be mixed v-rith the same standard agonist (step
to the machine have been tested.
If in step 1303, it is determined that a particular agonist
triggers a cell response, the process is switched over to the
potency mode of action (step 1306) in which the cellular
response at various concentration levels of the standard
agonist is recorded. After step 1306 has been completed, the
process then records the potency parameters, or profile,
representing the level of cellular response produced in a
given cell line at various concentration levels of the standard
agonist (step 1307). The process then once again determines
if all available standard agonists have been tested (step
60 1408). This mixture is then supplied to the detector (step
1409) for measuring the cellular response produced by the
standard agonist on the host cells. Next, the system deter-
mines if the standard agonist produces a cellular response in
the host cell (step 1410). If a cell response i<> detected (YES)
65 the system then increments the standard agonist counter
(step 1405) after which the system determines if all available
agonists have been tested (step 1410). If all available ago-
US 6,558,916 B2
53
nists have been tested, the process is complete (step 1412).
If all available standard agonists have not been tested, the
process moves back to step 1401 where fresh transfected
cells are supplied to be mixed with the next standard agonist
in the set of standard agonists (step 1402), and the process 5
steps described above are repeated.
If in step 1410, no cell response is produced in the host
cell by the standard agonist the process has detected a
difference bet\veen the host cell and the transfected cell. At
this point, the process is complete (step 1412). In one rn
embodiment, the apparatus will keep repeating an admixture
of different standard agonist substances with the cells until
it detects that a particular agonist stimulates a response in the
cells transfected with an orphaned receptor, and do not
stimulate a response on host cells or until all standard 15
solutions available to the machine have been tested.
54
a first flow coming from the proportioning valve 311, a
second flow coming from the valve 327 and a third flow
coming from the multichannel diverting valve 347. During
this step, both proportioning valves supply wash buffer 305
from the reservoirs located at "zero" position of each
autosampler in such a way that the final flowing mixture
consi-;ts of one part of cell suspension 349 and two parts of
wash buffer 305.
After a delay determined in step 1509, which is needed for
the flow of the mixture of cell suspension 349 and wash
buffer 305 to stabilize, detector 355 registers a basal signal
produced by the cells alone (Step 1510). After the basal
signal is registered, it is saved as a reference signal, BS;
(Step 1512) and computer 123 (FIGS. lA and lB) incre-
ments counter L (Step 1514).
The numerical content of the incremental counter L is
increased by one each time it is triggered. The numeric value
of the counter determines the position of the nozzle of intake
port 323 of autosampler 315 that samples the sets of standard
20 agonists 319 to be tested. Next, in step 1516, the numerical
content, Lk, of the incremental counter L is compared with
the previously entered (Step 1503) value of maximal number
of the standard agonists to be tested, L,,,ax·
A continuous flow diagram of the presently preferred cell
mapping and cell receptor "fingerprinting" mode with the
negative pressure fluidics system of FIGS. 4A and 4B is
shown on FIGS. 16a-16f. To practice this mode, diverting
valve 347 is replaced by a multichannel diverting valve 347
which allows multiple reservoirs, each with a different cell
suspension, to be connected separately to the common outlet
346 so that once an experiment with a particular cell line is
complete, the valve 347 may switch over to a next cell 25
reservoir.
The screening program starts from a state 1500 and enters
state 1502 where it sets all parameters to their initial values.
These initial parameters include, but are not limited to,
zeroing counters, the internal initialization of software vari-
ables and subroutines.
Once the initial parameters are set, the software requests
an operator to enter values for a maximum number of
standard agonists, LMAX, and a maximum number of cell
lines, C,\L4X, to be used in the experiment (Step 1503). The
autosaniplers 301 and 315 (FIGS. 4A and 4B) position the
intake nozzles 307 and 323 at the corresponding "zero"
positions occupied by a wash buffer reservoir 305 (Step
1504), and turns off proportioning valves 311 and 327,
diverting valves 333, 335 and 347, as well as priming valves
313, 329, 341 and 345 (Step 1506). In a turned off state,
priming valves 313, 329, 341and345, are normally opened,
proportioning valves 311 and 327 connect, respectively,
their outlets 312 and 328 with corresponding "normally
opened" intake ports 309 and 325, diverting valve 335
connects its common outlet 334 with "normally opened"
intake port 336, diverting valve 333 connects its common
outlet 342 with the "normally opened" intake port 33 2, and
multichannel diverting valve 347 connects its common
outlet 346 with the first intake port 348 connected with the
reservoir containing first cell suspension under investiga-
tion.
If Lk does not exceed L,,,ax' autosampler 315 positions
nozzle 323 into the standard agonist reservoir located at the
Lk position of a rack of standard agonists 319 (Step 1518).
Next, proportioning valve 327 is switched ON to open its
"normally closed" intake port 323 to the common output
port 328 (Step 1520). During Step 1520, the combined flow
30 in mixing zone 343 and afterwards in detector 355, is
composed of one portion of buffer 305 coming through
nozzle 309 of proportioning valve 311, one portion of the
standard agonist 319 to be tested, coming from nozzle 323
of proportioning valve 327 and one portion of particular cell
35 suspension 349 coming from one of the multitude of the cell
reservoirs through the intake tubing 348 to the output port
346 of multichannel diverting valve 347. After a delay
provided by Step 1521, which is needed for the mixing
process to stabilize, detector 355 registers a standard agonist
40 induced signal, SLk, produced by given cells in the presence
of the given standard agonist, Lk (Step 1522). After the SLk
signal is registered, its value is saved as a value, SLk (Step
1523) and the apparatus proceeds to step 1530 where the
proportioning valve 327 is turned OFF, thus connecting
45 normally opened intake port 325 with buffer 305. In step
1532, autosampler 315 moves its nozzle 323 to a zero
position occupied with reservoir filled with washing buffer.
Proportioning valve 327 is turned ON Step 1534), connect-
ing the normally closed inlet port 323 with outlet port 328.
50 Delay, determined in step 1536, allows for the flow path
composed of intake port 323 and outlet port 328 of the
proportioning valve 327, priming valve 329, mixing zone
331, intake port 332 and outlet port 342 of the diverting
valve 333, priming valve 341, mixing zone 343, reaction After the system has been initialized, pump 357 is started
(Step 1508). This will force the wash buffer from the "zero"
positioned reservoirs of both autosamplers 301 and 315 to
run through no=les 309 and 325 and outlet ports 312 and
328 of proportioning valves 311 and 327, respectively,
priming valves 313 and 329, mixing zone 331, intake port
332 and outlet port 342 of diverting valve 333, priming 60
valve 341, mixing zone 343, a reaction developing line 353,
detector 355, pump 357 and then into a drain container 359.
55 developing lines 353 and detector 355 to be washed clean of
the remains of the previous compound. After the delay, the
proportioning valve 327 is turned OFF in the step 1538, thus
bringing the system into original state.
Next, the value of the standard agonist induced signal,
SLb is compared with the value of the reference basal signal,
BS; (Step 1544). If the SLk signal is greater than the BS;
signal, the computer 123 (FIGS. lA and lB) will "flag" the
corresponding standard agonist as "positive" (Step 1540),
which means that the particular cells express a receptor
In mixing zone 343 this flow is mixed with the cell suspen-
sion 349 coming from intake tubing 348 connected with the
first cell line, and an outlet port 346 of the multichannel 65
diverting valve 347 through priming valve 345. Thus, the
total flow passing mixing zone 343, is a sum of three flows,
which is stimulated by the particular agonist. If SLk is not
greater than BS;, computer 123 will "flag" the corresponding
standard agonist as "negative" (Step 1542), which means
US 6,558,916 B2
55
that the particular cells do not contain a receptor which can
be stimulated by the particular agonist.
56
respectively, with corresponding "normally opened" intake
ports 309 and 325, outlet 334 of diverting valve 335 is
connected with "normally opened" intake port 336, common
outlet 342 of diverting valve 333 is connected with the
Each time the condition, "SL,;>BS/', is satisfied, the
program flow will go through loop K-0 initiating the
gradient mode of action. In the gradient mode, step 1550
moves autosampler 315 at a position corresponding to the Lk
value determined in step 1514. After that, the proportioning
valve 327 is turned on to operate in the gradient mode (step
1552). In step 1554, the dose response curve is registered for
the standard agonist stimulated signal. Step 1556 calculates
a potency parameter that could be determined as some
specific concentration of the agonist causing a specific level
5 "normally opened" intake port 332.
After the system has been initialized, diverting valve 347
is set to connect its common outlet 346 with the reservoir
containing host cells, position 1, (Step 1608). The pump 357
is started to pump liquid (Step 1610). This will force the
of a signal. The data are saved in step 1558.
After the data are saved, the proportioning valve 327 is
turned OFF (step 1560), autosampler 315 is moved to a zero
position (step 1562), proportioning valve 327 is turned ON
(step 1564) and, after a washing delay period determined in
step 1566, the valve 327 is turned OFF again (step 1568).
After step 1568 has been performed, the process goes back,
through line 0, to the incremental counter L (step 1514).
10 wash buffer from the "zero" positioned reservoirs of both
autosamplers 301 and 315 to run through nozzles 309 and
325 and outlet ports 312 and 328 of proportioning valves 311
and 327, respectively, priming valves 313 and 329, mixing
zone 331, intake port 332 and outlet port 342 of diverting
15 valve 333, priming valve 341, mixing zone 343, a reaction
developing line 353, detector 355, pump 357 and into a drain
container 359. In mixing zone 343, this flow is mixed \Vith
the host cell suspension coming from intake tubing 348 and
an outlet port 346 of the multichannel diverting valve 347
If in step 1544, the SLk signal is not larger then BS;, then
step 1542 flags the standard agonist asAnegative=, meaning
that the particular cells do not have the particular receptor
expressed, and the program flow is returned, through line M,
to the step 1514.
20 through priming valve 345. Thus, the total flow passing
through mixing zone 343 is a sum of three flows, a first flow
coming from the proportioning valves 311, a second flow
coming from proportioning valve 327 and a third flow
coming from the multichannel diverting valve 347. During
25 this step, both proportioning valves supply washing buffer
305 from the reservoirs located at "zero" position of each
autosampler in such a way that the final mixture consists of
one part of the host cell suspension and two parts of washing
If in step 1516 (FIG. 16b), Lk exceeds Lm=' indicating
that all agonists have been tested, the process moves through
line N to step 1570 were a computer 123 increments a cell
counter C. The numeric value of the counter is compared
30
(step 1572) with the maximum number of cells entered in
step 1503. If the counter value, C; does not exceed CMAX,
then controller L is set to zero in step 1574, multichannel
diverting valve 347 is switched to the next position, con-
necting a new cell reservoir with the output 346, and the
35
process returns to step 1509 (FIG. 16a) through line P. If in
step 1572 the C; value is higher then CMAX, then the process
moves to step 1578, where autosampler 315 is moved to its
zero position and, proportioning valve 327 is turned ON
(Step 1580). After a washing delay, determined in step 1582,
40
the valve 327 is turned OFF (Step 1584) and pump 357 is
turned OFF (Step 1586), and the whole program is stopped
in step 1588.
A continuous flow diagram of the presently preferred
orphaned receptor detection mode with the negative pressure 45
fluidics system of FIGS. 4A and 4B is shown on FIGS.
17a-17g. To practice this mode, diverting valve 347 is
replaced by a multichannel diverting valve 347 which allows
for at least two reservoirs 349, containing host cells, native
or transfected with an empty vector, and cells transfected 50
with the orphaned receptor of interest, to be connected
separately to the common outlet 346.
The program begins from a start state 1600 and enters
state 1602 where it sets all parameters to their initial values.
These initial parameters include, but are not limited to, the 55
internal initialization of software variables and subroutines.
Then the program requests an operator to enter a maximum
number of agonists to be tested (Step 1603).
Once the initial parameters are set up, the autosamplers
301 and 315 (FIGS. 4A and 4B) move their intake nozzles 60
307 and 323 to the corresponding "zero" positions occupied
by a wash buffer reservoir 305 (Step 1604). Proportioning
valves 311 and 327, diverting valves 333, 335 and 347, and
priming valves 313, 329, 341 and 345 are turned OFF (Step
1606). In that turned off state, priming valves 313, 329, 341 65
and 345, are normally opened, the outlets 312 and 328 of
proportioning valves 311 and 327 are connected,
buffer.
After a delay determined in step 1612, which is needed for
the flow of the mixture of cell suspension and wash buffer
to stabilize, detector 355 registers a basal signal produced by
the host cells (Step 1614). After the basal signal is registered,
it is saved as a reference signal, (BS)Hc (Step 1616). In step
1620, multichannel diverting valve 347 is switched over to
position 2 to supply the outlet 346 with the suspension of the
transfected cells. After a delay determined in step 1622,
which is needed for the flow of the mixture of new cell
suspension and wash buffer to stabilize, detector 355 regis-
ters a second basal signal produced by the transfected cells
(Step 1624). After the basal signal is registered, it is saved
as a reference signal, (BS)rc (Step 1626) and the pump is
turned OFF (Step 1628). The computer 123 (FIGS. lA and
lB) triggers the incremental counter L (Step 1630).
The numerical content of the incremental counter L is
increased by one each time it is triggered, thus determining
the position of the nozzle of intake port 323 of autosampler
315 which samples the sets of agonists 319 to be tested.
Next, the numerical content, Lk, of the incremental counter
L is compared with the maximum number of the standard
agonists to be tested, Lmax (Step 1632).
If Lk does not exceed Lmax' autosampler 315 positions
nozzle 323 into the agonist reservoir located in the rack 319
at the position determined by the numerical value Lk (Step
1634). The pump 357 is turned ON in step 1636 and
proportioning valve 327 is switched ON to open its "nor-
mally closed" intake port 323 to the common outlet port 328
(Step 1638) thus allowing the particular agonist flows
through the priming valve 329, mixing zone 331 intake port
332 and outlet port 342 of the diverting valve 333, priming
valve 341, mixing zone 343, where it is mixed with the
suspension of transfected cells coming from outlet port 346
of multichannel diverting valve 347 and priming valve 345.
After being mixed with the transfected cells in mixing zone
343, the liquid flow goes through reaction developing lines
353, into detector 355 and further into waste reservoir 359.
After a delay provided by Step 1640, which is needed for the
US 6,558,916 B2
57
mixing process to stabilize, detector 355 registers an agonist
induced signal, (SLk)ro produced by the particular
orphaned receptor transfected cells in the presence of the
given agonist, Lk (Step 1642). After the signal is registered,
58
315 is moved to the zero position (step 1686), proportioning
valve 327 is turned ON (step 1688) and, after a washing
delay, determined in step 1690, is turned OFF again (step
1692) and the pump 357 is turned OFF (Step 1694). After the
5 step 1694 has been performed, the flow goes back to the
incremental counter L (step 1630) through the line 0.
its value, (SLk)ro is saved in step 1643 and the apparatus
proceeds to step 1644 where proportioning valve 327 is
turned OFF, thus connecting normally opened intake port
325 with buffer 305 to outlet port 328. In step 1646,
autosampler 315 moves its no=le 323 to position zero
occupied with reservoir filled with washing buffer. Propor-
tioning valve 327 is turned ON again (Step 1648) connecting
inlet 323 with outlet 328. A delay, determined in step 1650,
allows for the flow path, composed of intake port 323 and
outlet port 328 of the proportioning valve 327, priming valve
329, mixing zone 331, intake port 332 and outlet port 342 of
15
the diverting valve 333, priming valve 341, mixing zone
343, reaction developing lines 353, and detector 355 to be
washed out of the remains of the agonist. After the delay
(Step 1650), the proportioning valve 327 is turned OFF in
step 1652 and the pump 357 is turned OFF in step 1654, thus
bringing the system into its original state.
If in step 1632 (FIG. 17c), Lk does exceed L,,,ax' then the
process moves to step 1700 (FIG. 17g) where the counter L
is set to zero. Next, the autosampler 315 is moved to the zero
10 position in step 1702, the proportioning valve 327 is turned
ON in step 1704 and after a washing delay determined in
step 1706, both proportioning valve 327 and pump 357 are
turned OFF in steps 1708 and 1710 respectively, after which
the program stops in step 1712.
The above devices and procedures may be used to moni-
tor cellular responses to test compounds in living cells. For
example, living cells may be used to study signal transduc-
tion pathways and global cellular responses using reagents
such as fluorescent dyes to generate signals indicative of the
Next, the value of the registered signal for transfected
cells, (SLk)ro is compared with the value of the correspond-
ing reference basal signal, (BS)rc (Step 1658). If the agonist
induced signal, (SLk)ro is not greater than the basal value,
(BS)ro the computer 123 (FIGS. lA and lB) will "flag" the
corresponding agonist, Lk as "negative" (Step 1660), which
means that the cell does not contain a receptor which can be
stimulated by the particular agonist, and the flow returns
through line M back to the incremental counter L (Step
1630). If the (SLk)rc signal is greater than the (BS)rc signal,
the computer 123 will "flag" the corresponding agonist as
"POSITIVErc" (Step 1656), which means that the trans-
fected cells do express a receptor which can be stimulated by
the particular agonist.
20 activation of the signal transduction pathways or the trig-
gering of the global cellular responses. Preferably, the
fluorescent dyes are sensitive to the level or state of bio-
chemical mediators or biophysical parameters that indicate
changes in the activation state of the cell. In particular, to
25 measure changes in the concentration of unbound Ca2 + ions
in the cell, the indicator may be the dye indo I, Other
indicators may measure intracellular pH (e.g., SNARF-1),
intracellular K+ (e.g., PBFI), intracellular N+ (e.g., SBFI),
transmembrane potential (e.g., oxonols and DiBAC dyes),
30 lipid fluidity (e.g., merocyamine 540), lipid oxidation (e.g.,
cis-paranaric acid) and intracellular oxidation potential and
state (e.g., MC-H2DCFDA). The preceding indicators are
available from Molecular Probes (Eugene, OR) and other
vendors.
The next task is to determine if the host cells also express
this receptor to distinguish it from the orphaned receptor
which should be expressed only in transfected cells. To do
35 Example 7 below demonstrates the use of the present
devices and methods in which the detector is capable of
detecting a plurality of cellular responses simultaneously
and/or capable of measuring cellular responses in individual
cells, such as devices in which the detector is a flow so, after the standard agonist Lk has been flagged as positive
in the step 1656, the program flow enters loop K-0, where,
in step 1662, the autosampler 315 moves its nozzle 323 to
the position corresponding to the value obtained in counter
40 cytometer, to measure cellular responses to test compounds
in living cells.
L, Lk. Step 1664 switches diverting valve 347 to position 1,
which connects host cell suspension reservoir 349 with
mixing zone 343 through common output 346, through 45
intake port 348 of the multichannel diverting valve 347, and
through priming valve 345. Steps 1666 and 1668 turn valve
327 and pump 357 ON, respectively, allowing the buffer 305
coming from intake 309 of the autosampler 301, and stan-
dard agonist Lk, coming from the intake 323 and output port 50
328 of the proportioning valve 327, to be brought together
in mixing zone 331 with the subsequent mixing of this
solution with the host cells in the mixing zone 343. After a
delay determined by step 1670, which is needed for the
stream of the mixture of the cells and agonist to stabilize, 55
detector 355 registers the agonist induced signal in the host
cells, (SLk)Hc (Step 1672).
After the signal is registered, it is compared (Step 1674)
with the corresponding basal signal, (BS)Ho measured in
step 1614. Depending on the results of the comparison, the 60
agonist Lk is marked either as "POSITIVErc1POSITIVEHc"
(Step 1676) or as "POSITIVErclNEGATIVEHc" (Step
1678). In either case, the data are saved in step 1680 for
further evaluation by the researcher.
After the data are saved, the multichannel diverting valve 65
347 is switched over to position 2 in step 1682, the propor-
tioning valve 327 is turned OFF (step 1684), autosampler
EXAMPLE 7
Target cells are harvested from the culture vessel using
dissociation reagents such as 0.25%Trypsin/1 mM EDTA if
necessary, washed in buffered salts solution and counted. As
discussed above, the target cells may be obtained from
cultures containing a single cell type for from cultures
containing a plurality of cell types. If the cultures contain a
mixed population of cells, cell type identification reagents
will be used as described below.
After harvesting, the cells are loaded or stained with an
appropriate cellular response indicator, such as a dye. Load-
ing conditions will vary with the dye used. Conditions to be
considered include but are not limited to cell density,
temperature, duration and concentration of the dye probe.
A variety of dyes suitable for detecting cellular responses
and methods for introducing them into cells are known to
those skilled in the art. For example, indo-1, SNARF-1,
PBFL, SBFI and MC-H2DCFDA are loaded into cells using
the membrane permeant, esterase-cleaveable forms at 1-10
µM and room temperature. Oxonols and DiBAC dyes,
merocyanine 540, and cis-paranaric acid are loaded and
maintained under equilibrium conditions at 1-50 µM and
room temperature. To extend viability of the cells, loading
and staining arc performed in Hybridoma Medium (HM
Sigma) whenever possible.
US 6,558,916 B2
59
After loading with an appropriate indicator dye the cells
are washed in HM and resuspended in HM at 2E5 to 2E6
cells/ml. Cells are maintained at room temperature in a
vessel with constant stirring prior to mixing with the test
compound(s) and analysis.
Cells are drawn into the apparatus and exposed to test
compound(s) as described above. For example, the cells may
5
60
response is occurring. In this way, the cell type or cell types
which exhibit the cellular response when exposed to the test
compound or compounds may be identified.
Likewise in embodiments in which multiple cellular
responses are evaluated simultaneously, the cells are con-
tacted with a plurality of response indicating reagents and a
test compound or compounds. Alternatively, the cells may
be exposed to mixtures of test compound(s) and standard
compound(s) as described above. Each of the response
be drawn into the apparatus using negative pressure gener-
ated via a peristaltic pump. In some embodiments, the cells
may be exposed to mixtures of test compound(s) and stan-
dard compound(s) as described above.
In the preferred embodiment having a coupling device,
sample slugs comprising cells and test compound(s) or test
mixtures are then delivered to the flow cytometer. As dis-
cussed above, the coupling device preferably isolates the
slugs from the reaction developing lines coming from the
mixing chamber and delivers them to the flow cytometer
under positive air pressure. Alternatively, cells may also be
delivered directly to the flow cytometer without using the
coupling devices.
10
indicating reagents provides a distinct signal to the detector
to enable the determination of the cellular response which is
occurring. It will be appreciated that the cells may be a
single cell type or may comprise a mixed population of cell
types as described above. The cells which have been con-
tacted with the test compound or compounds and a plurality
15 of response indicating reagents are sent to the detector,
which detects signals indicative of the cellular responses
which are occurring. In addition, where a mixed population
of cell types is used, the detector detects signals indicative
of the cell type or cell types which are exhibiting each
20 response.
As the loaded cells enter the flow cytometer they are
excited by the laser(s). The excitation beam scatter proper-
ties and fluorescence emissions are optically selected using
steering dichroics and bandpass filters. Fluorescent and/or
non-fluorescent optical signals associated with each cell are 25
converted into electronic signals, which are processed and
represented graphically.
The response of the cells as indicated by the reporter dye
is detected from each cell at the point in time determined by
30
the configuration of the apparatus. The response can be
quantitated as the average of the entire population, or as the
percentage of cells exceeding a certain fluorescence value.
The data are stored in a database for future reference.
Examples 8A and 8B below provide examples of the
above methods.
EXAMPLE 8A
If multiple cell types are to be analyzed, the different cell
types are labeled with commercially available fluorescent
lipid intercalating dyes which enable the different cell types
to be distinguished from one another, such as Di0Cg18(3) or
DiIC18(3). Loading procedures vary according to the cell
type and dye used, but incubation with 1-5 ,uM dye for
several hours to overnight is usually sufficient. Other fluo-
rescent dyes may also be used to label the cells. Examples
are not limited to, but include, dyes that are trapped v-rithin
the cytoplasm due to ionic charges (e.g., carboxyfluorescein,
Molecular Probes) or dyes that become conjugated to intra-FIG. 18 illustrates the above procedure.
Embodiments of the present devices and methods in
which in which the detector is capable of detecting a
plurality of cellular responses simultaneously and/or capable
35 cellular proteins or organelles (e.g., Mitotracker Red,
Molecular Probes). Such dyes are typically loaded into the
cells using 1-50 µM incubation concentrations. Also, fluo-
rochrome conjugated antibodies specific for unique markers
of measuring cellular responses in individual cells, such as
devices in which the detector is a flow cytometer, may be 40
used to evaluate cellular responses in cell populations com-
prising more than one type of cell, to evaluate responses in
cell populations obtained from a single cell type or tissue in
which only a fraction of the population responds to a
stimulus, or to evaluate multiple cellular responses simul- 45
taneously.
In analyses in which the cell population contains more
than a single cell type, individual cells are tagged with a cell
type identification reagent specific for a particular cell type,
such as an antibody specific for that cell type. Each cell type 50
identification reagent provides a distinct signal to the detec-
tor to enable the determination of which cell type is respond-
ing to a test compound.
In addition, the cells are contacted with one or more test
compounds or test mixtures comprising one or more test 55
compounds and a standard compound as described above.
Alternatively, the cells may be exposed to mixtures of test
compound(s) and standard compound(s) as described above.
The cells are also contacted with a cellular response indi-
cating reagent, such as those described in Example 7 above, 60
which provides a signal indicative of a particular cellular
response.
The cells which have been contacted with the cell type
identification reagent, the response indicating reagent, and
the test compound(s) or test compound(s)/standard mixture 65
are sent to the detector, which detects signals indicative of
the cellular response and the cell type in which the cellular
on a cell population can be used to identify the populations.
Appropriate dyes and staining reagents are selected accord-
ing to the available excitation spectra in the FCM
configuration, and the desired spectral emissions that will
enable discrimination of one cell population from another.
Alternatively, the individual cell types may be labeled
with antibodies specific for each cell type. In such
procedures, the cells are harvested first and then labeled v-rith
the antibodies. To label the cells \vith antibodies, the cells
are reslL<>pended at high density in ice-cold staining solution.
Staining solution is a buffered salts solution containing a
sufficient concentration of antibody to completely saturate
all binding sites within 15 minutes on ice. Cells are washed
to remove unbound antibody.
The labeled cells are harvested and loaded with the
cellular response indicator, such as an indicator dye, of
choice as described above. Cells are exposed to one or more
test compound(s) or test compound(s)/standard mixtures
using the mixing system described above and sent to the
flow cytometer as described above.
Upon analysis of the cellular responses by the FCM,
electronic gating is performed to discriminate individual cell
types and to quantitate the frequency of responding cells
within a population.
FIG. 19 illustrates the above procedure.
EXAMPLE SB
Where multiple cellular responses are to be evaluated
simultaneously, a cell population is loaded with two or more
US 6,558,916 B2
61
different cellular response indicators, such as bioresponse
reporter dyes. For example, the dyes may be indo-1 for Ca2 +
responses and SNARF-1 to measure pH. Dyes are loaded
and cells are harvested as described above.
Alternatively, is some embodiments, fluorescent ligands 5
may be used to stimulate the cells. In this case the fluores-
cent ligands are used as normal test agonists or antagonists
62
ing a test compound(s) and a standard, and analyzed by the
FCM as described in Example 7 to determine the effects of
the test compound(s) or test compound(s)/standard mixture
on receptor activity.
The FCM is configured to detect each fluorescent param-
eter associated with the individual bioresponse reporter dyes
and/or with the each labeled population. Using electronic
gating, each bioresponse is determined for each individual
cell population simultaneously. Electronic gating is also
as described above. Thi<> enables the simultaneous analysis
and quantification of the cellular bioresponse and ligand
binding simultaneously. rn used to quantitate the frequency of responding cells within
a each population simultaneously. Cells are exposed to test compound(s) or test compound
(s)/standard mixtures comprising one or more test com-
pounds and a standard and analyzed as described above. The
FCM is configured to detect each fluorescent parameter
associated with the individual reporter dyes. Upon analysis 15
of the cellular responses by the FCM electronic gating will
be performed to determine the distinct bioresponses within
the cells and to quantitate the frequency of responding cells
within a population.
FIG. 21 illustrates the above procedure.
The above devices and methods in which the detector is
capable of detecting a plurality of cellular responses simul-
taneously and/or capable of measuring cellular responses in
individual cells, such as devices in which the detector is a
flow cytometer, may be used to analyze one or more test
compounds or test compound(s)/standard mixtures compris-
It will be appreciated that if one desires to simultaneously 20
evaluate a plurality of cellular responses in populations
comprising more than one cell type, the procedures
described in Examples 8A and 8B may be combined.
ing one or more test compounds and a standard for their
influence on the activity of ion channels or non-selective
pores.
In this embodiment, the reagents indicative of cellular
responses are reagents which indicate activation of ion
FIG. 20 illustrates the above procedure.
The above devices and methods in which the detector is
capable of detecting a plurality of cellular responses simul-
taneously and/or capable of measuring cellular responses in
individual cells, such as devices in which the detector is a
flow cytometer, may be used to evaluate the effects of test
compounds on receptor activity and to correlate the extent of
receptor occupancy by the ligand with the functional
response triggered by the receptor.
Ligand-specific receptors on cells activate specific second
messenger pathways and associated downstream events that
result in changes in cell activity or state. Compounds which
directly or indirectly block natural ligand binding or that
mimic the natural ligand to activate second messenger
pathways can be identified. Such compounds have a variety
25 channels or non-selective pores. In addition, where mixed
cell populations are used, the cells are also contacted with
cell type identification reagents. Different properties of
indicator dyes can be exploited to study ion channel and pore
function. For example, certain channels are permeant to
30 specific ions, and these ions can alter the fluorescent prop-
erties of intracellular dyes. For example, the activation of a
Ca2 + channel can be visualized directly using a Ca2 + sensing
dye, or indirectly using techniques based on the ability of
Mn2 + to pass through the channel and quench fluorescence
35 of a Ca
2
+ indicator dye.
of applications, including use as drugs or therapeutic agents. 40
The formation of non-selective pores may be monitored
using pore-permeant, fluorescent nucleic acid binding dyes
to detect permeability changes. These dyes increase their
fluorescence dramatically when bound to nucleic acids, and
hence would yield fluorescence: signals only when the
opening of pores permitted them to enter the cells. As discussed above, the present devices and methods may
be used to analyze receptor-mediated cellular responses in
mixed cell populations and/or to evaluate multiple receptor-
mediated responses simultaneously. In such embodiments,
the reagents indicative of cellular responses are reagents 45
which indicate receptor binding or activity. In addition,
where mixed cell populations are used, the cells are also
contacted with cell type identification reagents.
Example 9 describes the analysis of receptor-mediated
responses. SD
EXAMPLE 9
If populations of different cell types are being evaluated,
the cell types are labeled with cell type identification
reagents, such as distinct fluorescent dyes or antibodies, as 55
described above to enable discrimination by the FCM.
Thus, the present invention permits multiple channel- and
pore-mediated cellular responses to test compounds to be
monitored in distinct cell populations in an automated
fashion.
Example 10 describes the analysis of ion channels or
non-selective pores.
EXAMPLE 10
If populations of different cell types are being evaluated,
the cell types are labeled with cell type identification
reagents, such as distinct fluorescent dyes or antibodies, as
described above to enable discrimination by the FCM.
Labeling may be performed as described in Examples 8A
and 8B. Cells are harvested as described in Example 7. If a
plurality of cellular responses are being evaluated
simultaneously, after harvesting, the cells are loaded with
two or more cellular response indicating agents, such as
Labeling may be performed as described in Examples 8A
and 8B. Cells are harvested as described in Example 7. If a
plurality of cellular responses are being evaluated
simultaneously, after harvesting, the cells are loaded with
two or more cellular response indicating agents, such as
bioresponse indicator dyes, as described in Example 8B. If
it is desired to measure a plurality of cellular responses in a
population compri<>ing more than one cell type, the proce-
dures of Example 8A and 8B are combined.
60 bioresponse indicator dyes, as described in Example 8B. If
it is desired to measure a plurality of cellular responses in a
population comprising more than one cell type, the proce-
dures of Example 8A and 8B are combined.
After loading the cells are mixed with one or more test
compounds or test compound(s)/standard mixtures compris-
Cells are harvested as described in Example 7. The cells
65 are loaded with a Ca2 + sensing dye such as indo-1 as
described in Example 7. Such cells may be used to evaluate
the ability of test compound(s) to block or enhance the influx
US 6,558,916 B2
63 64
of Ca2 + through Ca2 + channels, or to block or enhance the
flow through the channel of other competitive channel
permeant ions, such as Mn2 + that quench dye fluorescence.
Other dyes that sense fluxes in other ions such as SBFI for
Na+ may also be used to detect flow through a Na+ channel 5
of competitive ions. In situations where the target cellular
activity is an activatable, non-selective pore then the cells
will not be loaded with a bioresponse indicator, unless more
than one bioresponse is to be monitored simultaneously.
single-transmembrane receptors. Upon activation the
enzyme catalyzes the formation of inositol trisphosphate
(IP3 ) from membrane lipids. 1 P 3 binds and activates an IP3 ,
receptor on the endoplasmic reticule (ER) that functions as
a Ca2 + channel. This results in release of stored Ca2 + from
the ER. This sudden release of Ca2 + can raise intracellular
free Ca2 + levels from 100 nM to 1 ,uM within seconds.
The apparatus of the present invention may be used to
study direct activators or inhibitors of phospholipase C, or of
Cells are mixed with one or more test compounds or test
compound(s)/standard mixtures comprising one or more test
compounds and a standard and analyzed by FCM as
described in above with the exception that competitive ions
such as Mn2 + (1-100 µM final concentration) or nucleic acid
binding dyes such as 7-AAD (1-100 ,uM) are introduced
with the test compounds or test compound(s)/standard mix-
tures. Sufficient time for the ions or nucleic acid sensing dye
is allowed by adjusting the reaction time of the system prior
to FCM analysis.
In embodiments in which the test compound(s) is mixed
with a standard(s), the standard may be an agonist or
antagonist of the channel being evaluated. For example, if
the channel being evaluated is an L-type voltage-gated Ca2 +
channel, the agonist Bay K 8644 or the antagonists
Nimodipine, Verapamil, or Diltiazem may be used as stan-
dard compounds.
Likewise if the channel being evaluated is an N-type
voltage-gated CA2 + channel, antagonists such as
w-conotoxin GVIA, w-conotoxin MVIIC may be used as the
standard compounds.
If the channel being evaluated is a voltage-gated Na+
channel, modulators such as batrachotoxin, a-scorpion
toxin, class2 13-scorpion toxin or antagonists such as
tetrodotoxin, or saxitoxin may be used as standard com-
pounds.
The FCM will be configured to detect the appropriate
optical signals in a multiparametric and multicellular format
suitable to the labeling and bioresponse indicator dyes
utilized.
FIG. 22 illustrates the above procedure.
rn the G-protein subunits associated with the receptor that
activate the phospholipase C. In such procedures, candidate
compounds are introduced to cells containing the desired
enzyme using the apparatus of the present invention. The
Ca2 + mobilization response is monitored with a fluorescent
15 Ca2 + indicator such as indol.
FIG. 23 illustrates the above procedure.
The present devices and methods in which the detector is
capable of detecting a plurality of cellular responses simul-
20 taneously and/or capable of measuring cellular responses in
individual cells, such as devices in which the detector is a
flow cytometer, may be used to analyze a test compound or
test compounds for their influence on cellular apoptosis or
cellular toxicity. As discussed above, the present devices and
25
methods may be used to analyze the effects of a test
compound or test compounds on cellular apoptosis in mixed
cell populations and/or to evaluate a plurality of cellular
responses including apoptosis simultaneously. In such
embodiments, the reagents indicative of cellular responses
30
are reagents which indicate activity of agents involved in
cellular apoptosis or cellular toxicity. In addition, where
mixed cell populations are used, the cells are also contacted
with cell type identification reagents.
Example 12 describes the analysis of the activity of agents
35
involved in cellular apoptosis or cellular toxicity.
EXAMPLE 12
If populations of different cell types are being evaluated,
the cell types are labeled with cell type identification
40
reagents, such as distinct fluorescent dyes or antibodies, as
described above to enable discrimination by the FCM.
Labeling may be performed as described in Examples 8A
and 8B. Cells are harvested as described in Example 7. If a
plurality of cellular responses are being evaluated
45
simultaneously, after harvesting, the cells are loaded with
two or more cellular response indicating agents, such as
bioresponse indicator dyes, as described in Example 8B. If
it is desired to measure a plurality of cellular responses in a
population comprising more than one cell type, the proce-
The present devices and methods in which the detector is
capable of detecting a plurality of cellular responses simul-
taneously and/or capable of measuring cellular responses in
individual cells, such as devices in which the detector is a
flow cytometer, may be used to analyze a test compound or
test compounds for their influence on the activity of second
messengers at points downstream of a receptor or channel
involved in the second messenger pathway. The above
devices and methods may be used to evaluate the effects of
test compounds on second messengers. As discussed above,
the present devices and methods may be used to analyze the
effects of a test compound or test compounds on second
messengers in mixed cell populations and/or to evaluate
multiple second messengers simultaneously. In such 55
embodiments, the reagents indicative of cellular responses
are reagents which indicate activity of second messengers
either directly or indirectly. In addition, where mixed cell
populations are used, the cells are also contacted with cell
50 dures of Example 8A and 8B are combined.
type identification reagents. 60
Certain of the fluorescent biosensors are loaded into the
cells prior to exposure to test compounds. Examples include,
but are not limited to, JC-1 loaded at 1-10 µM to measure
mitochondrial membrane potential collapse, dihydrofluores-
cin to measure accumulation of oxygen free radicals and
HOE 33342 loaded at 0.1 to 10 µg/ml to assess chromatin
condensation and denaturation.
Cell populations are treated with one or more test com-
pounds or with test compound(s)/standard mixtures com-
prising one or more test compounds and a standard prior to
harvest, and incubated for predetermined optimal time peri-
ods. The optimal incubation time will vary depending upon
the cell types, but will likely be from 1 to 4 hours under
normal culture conditions.
Example 11 describes the analysis of the activity of
second messengers.
EXAMPLEll
Phospholipase C is a 'second messenger' enzyme acti-
vated by certain 7-transmembrane receptors and certain
65 Cells are harvested as described in Example 7.
The cells are preferably input into the apparatus as
discrete, pre-treated populations.
US 6,558,916 B2
65
After input to the apparatus, the cells are exposed to extra
reagent<> in transit to the FCM. These reagents are additional
sensors and detectors of apoptosis. Such reagents may be
input through additional input lines in the system. Examples
of such reagents include, but are not limited to, FITC 5
conjugated Annexin V which binds phosphatidylserine ori-
ented to the external leaflet of the plasma membrane during
apoptosis. Time is allowed through adjustment of the lengths
of the reaction developing lines from the coupling device
and/or mixing chamber to permit interaction of the addi- rn
tional fluorescent reagents to their targets.
The FCM is configured to detect the appropriate optical
signals in a multiparametric and multicellular format suit-
able to the labeling and bioresponse indicator dyes utilized.
For example, cells are susceptible to the induction of 15
apoptosis by 1 µM of anti-cancer compounds such as
camptothecin or Taxol. Cells are loaded with the response
indicator dyes such as indol or Fura Red (Ca2 +), or JC-1
(mitochondrial potential), all at 1-10 ,uM. Next, the loaded
cells are aliquoted to multiwell plates and treated with test 20
compounds. The cells are incubated for 1-4 hours to permit
early apoptotic events to occur. The cells are then input into
the apparatus of the present invention through the
autosampler, and exposed to an additional apoptosis detec-
tion reagent, such as a fluorochrome-conjugated Annexin V 25
(0.1-100 ug/ml), through a sample input line.
Sufficient time (1-5 minutes) for the Annexin V to bind to
phosphatidylserine on the external leaflet of the plasma
membrane is provided to the sample mixture. For example,
30 the length of the reaction chamber may be extended to
ensure a sufficient amount of time is provided. Samples are
then delivered to the detector via the coupling device (PFC).
FIG. 24 illustrates the above procedure.
The above devices and methods in which the detector is 35
capable of detecting a plurality of cellular responses simul-
taneously and/or capable of measuring cellular responses in
individual cells, such as devices in which the detector is a
flow cytometer, may be used to analyze one or more test
compounds or test compound(s)/standard mixtures compris- 40
ing one or more test compounds and a standard for their
influence on the activity of agents involved in cellular
necrosis. As discussed above, the present devices and meth-
ods may be used to analyze the effects of a test compound
or test compounds on agents involved in cellular necrosis in 45
mixed cell populations and/or to evaluate multiple cellular
responses simultaneously. In such embodiments, the
reagents indicative of cellular responses are reagents which
indicate activity of agents involved in cellular necrosis. In
addition, where mixed cell populations are used, the cells are 50
also contacted with cell type identification reagents.
Example 13 describes the analysis of the activity of agents
involved in cellular necrosis.
66
The cells are exposed to additional fluorescent indicators of
membrane permeability, if appropriate, through use of addi-
tional input lines as described in Example 12. An example
is the red fluorescent, plasma membrane impermeant DNA
stain propidium iodide.
The FCM is configured to detect the appropriate optical
signals in a multiparametric and multicellular format suit-
able to the labeling and necrosis indicator dyes utilized.
FIG. 25 illustrates the above procedure.
The above devices and methods in which the detector is
capable of detecting a plurality of cellular responses simul-
taneously and/or capable of measuring cellular responses in
individual cells, such as devices in which the detector is a
flow cytometer, may be used to analyze one or more test
compounds or mixtures comprising one or more test com-
pound<> and a standard for their effects on both necrosis and
apoptosis. As discussed above, the present devices and
methods may be used to analyze the effects of a test
compound or test compounds on both necrosis and apoptosis
in mixed cell populations and/or to evaluate multiple cellular
responses simultaneously. In such embodiments, the
reagents indicative of cellular responses are reagents which
indicate activity of agents involved in cellular necrosis and
apoptosis. In addition, where mixed cell populations are
used, the cells are also contacted with cell type identification
reagents.
Example 14 describes the analysis of the activity of agents
involved in cellular necrosis and apoptosis.
EXAMPLE 14
Cells are labeled and treated as described in Examples 12
and 13 to detect both apoptosis and necrosis. Appropriate
combinations of labels and response sensing dyes are
selected according to the experimental needs to assess both
necrotic and apoptotic cells. Pre-stained cells are input to the
system through an autosampler port as described above. If
appropriate, additional dyes are added to the cell population
in transit as described in Examples 12 and 13.
The FCM is configured to detect the appropriate optical
signals in a multi parametric and multicellular format suit-
able to tile labeling and necrosis and apoptosi<> indicator
dyes utilized.
FIG. 26 illustrates the above procedure.
The above devices and methods in which the detector is
capable of detecting a plurality of cellular responses simul-
taneously and/or capable of measuring cellular responses in
individual cells, such as devices in which the detector is a
flow cytometer, may be used to obtain or clone populations
of cells having a desired phenotype or cellular response
profile. In such embodiments, the flow cytometer is
equipped to sort cells exhibiting a desired phenotype. The
system may be configured to enable fluorescence activated
cell sorting of cells having a desired phenotype or cellular
EXAMPLE 13
Cells are labeled and treated as described in Example 12,
except that the preloaded biosensors are appropriate for
detecting necrosis rather than apoptosis. An example would
be to load the cells with Calcein using Calcein AM at 1-10
µM. Unlike apoptotic conditions, under necrotic conditions
the plasma membrane will become permeant to large mol-
ecules like calcein early in the process. Normally well
retained green fluorescent calcein will leak out under
necrotic conditions and this will be apparent as decreased
green fluorescence.
55 response profile. Preferable, the reagents for detecting one or
more cellular responses used in such procedures provide a
fluorescent signal indicative of the cellular responses. Addi-
tional phenotypic markers, such as fluorescent antibodies
which detect the presence of particular cell surface markers
Pre-labeled cells, or naive cells if appropriate, are input to
the system through the autosampler port as in Example 12.
60 may also be used to separate cells having the desired
characteristics. This enables the separation or cloning of
cells having the desired phenotype or cellular responses
from other cells in the population which do not exhibit the
desired characteristics. In this way, a uniform population of
65 cells exhibiting the desired characteristics may be obtained
for use in assays.
Example 15 below describes such embodiments.
US 6,558,916 B2
67
EXAMPLE 15
If populations of different cell types are being evaluated,
the cell types are labeled with cell type identification
reagents, such as distinct fluorescent dyes or antibodies, as
described above to enable discrimination by the FCM.
Labeling may be performed as described in Examples SA
and SB. Cells are harvested as described in Example 7. If a
plurality of cellular responses are being evaluated
simultaneously, after harvesting, the cells are loaded with
two or more cellular response indicating agents, such as
bioresponse indicator dyes, as described in Example SB. The
cellular response indicating reagents may be any of those
listed herein. For example, if the cellular response to be
detected is calcium influx, the cellular response indicator
mav be indo-1. If it is desired to measure a plurality of
celiular responses in a population comprising more than one
cell type, the procedures of Example SA and SB are com-
bined.
Cells are mixed with one or more molecules to be
evaluated for inducing one or more cellular responses and
the mixture is directed through a detector capable of detect-
ing a plurality of cellular responses simultaneously or
detecting cellular responses in individual cells as described
above. The detector is also configured to sort or clone cells
exhibiting the desired characteristics from the population of
cells. Sample plugs comprising cells and the one or more test
compounds are continuously delivered to the flow cytom-
eter. As the plugs are analyzed by the flow cytometer, cells
having the desired characteristics are delivered into a receiv-
ing vessel. The delivered cells may be expanded in culture
for future storage or use.
FIG. 27 illustrates the above procedure.
The above devices and methods in which the detector is
capable of evaluating interactions with a plurality of par-
ticles simultaneously and/or capable of evaluating interac-
tions with individual particles, such as devices in which the
detector is a flow cytometer may be used to perform bio-
chemical analvses to determine whether one or more bio-
chemical molecules are present in a sample. In such
embodiments, the sample is contacted with a detection
reagent for identifying the presence of a biochemical mol-
ecule in a sample. In some embodiments, the presence of
several biochemical molecules in the sample is simulta-
neously evaluated using detection reagents which provide
distinct signals for each biochemical molecule being evalu-
ated. In some embodiments, the evaluation of multiple
biochemical molecules is accomplished using to beads or
particles which provide distinct signals.
For example, the present devices and methods may be
used to perform Western analyses to simultaneously evalu-
ate samples for the presence of multiple polypeptides.
Example 16 describes one embodiment of such Western
analyses in which multiple polypeptides are simultaneously
evaluated for modifications resulting from the activities of
signal transduction pathways. However, it will be appreci-
ated that the present devices and methods may be used to
conduct Western analyses on any types of polypeptides
present in a sample.
EXAMPLE 16
Microsphere populations bearing antibodies specific for a
plurality of cellular proteins are prepared by conjugating the
antibodies directly to the spheres. Optimal conjugation
conditions will vary for each antibody but examples include
the use of streptavidin coated microspheres to permit coating
68
with biotinylated antibody. The microspheres may be fluo-
rescent or of different sizes to render each bead population
identifiable by FCM. Based on size or fluorescence
parameters, several unique and addressable bead and anti-
s body combinations can be constructed. This allows multiple
assays to be performed in a single experiment when the
populations are analyzed by FCM.
For example, if the polypeptides to be assayed are
polypeptides which are modified through the activities of
rn signal transduction pathways, cell populations are treated
with test compounds prior to harvest, and incubated for
predetermined optimal time periods to allow appropriate
downstream signal transduction events to occur. The cells
are lvsed with a detergent, such as 0.1-1.0% NP-40, to
15 liber~te intracellular molecules. Micropsheres bearing spe-
cific antibody are added to the lysate to adsorb specific
cellular proteins.
Samples of beads are then drawn into the system fluidics
from the microtiter plate using an autosampler port. Bead
20 populations are exposed to a second antibody (10 µM)
within the fluidics system by introducing the antibody to the
sample slug through a second input system. The second
antibody is conjugated with a fluorescent molecule to permit
identification bv FCM. The second antibody is able to detect
25 modifications ~f the captured cellular protein on each unique
bead population. An example is the use of an anti-
phosphotyrosine antibody to detect phosphotyrosine labeled
proteins. The length of the reaction developing lines from
the coupling device and/or the mixing chamber is sufficient
30 to permit binding of the second antibody to the captured
protein (2 to 5 minutes).
The addressable, antibody covered beads with captured
cellular protein then enter into the FCM. The FCM is
configured with the correct excitation lasers and beam
35 scatter and emissions collecting optics to permit simulta-
neous analyses of multiple captured cellular proteins. Using
multiple unique bead populations, it is possible to simulta-
neously analyze the modification state of multiple cellular
proteins.
40
FIG. 28 illustrates the above procedure.
The above devices and methods in which the detector is
capable of evaluating interactions with a plurality of par-
ticles simultaneously and/or capable of evaluating interac-
45 tions with individual particles, such as devices in which the
detector is a flow cytometer, may be used to perform
Northern or Southern analyses to simultaneously evaluate
samples for the presence of multiple mRNAs or DNA5.
Example 17 describes one embodiment of such Northern
so analyses in which Northern analyses are performed on
mRNAs which may be induced following treatment with test
compounds. However, it will be appreciated that the North-
ern analyses may be performed to detect the presence of any
desired mRNAs in a sample. It will also be appreciated that
55 the method may be performed to detect the presence of any
desired DNAs in the sample.
EXAMPLE 17
Microsphere populations bearing oligonucleotide probes
60 complementary for portions of specific cellular mRNA are
prepared by conjugating the oligonucleotide directly to the
spheres. Optimal conjugation conditions will vary for each
oligonucleotide. An example is to use streptavidin coated
microspheres to permit coating with biotinylated oligonucle-
65 otide probe. The microspheres may be fluorescent or of
different sizes to render each bead population identifiable by
FCM. Based on size or fluorescence parameters, several
US 6,558,916 B2
69
unique and addressable bead and oligonucleotide combina-
tions can be constructed. Thi<> allows multiple assays to be
performed in a single experiment when the populations are
analyzed by FCM'
70
and fluorescent dideoxynucleotide triphosphates. Each reac-
tion will target a unique SNP site.
The last step is the addition of a slurry of streptavidin-
coated microspheres to adsorb the fluorescent labeled prim-
ers. This step is performed by the mixing system. If the mRNAs to be analyzed may be induced by test 5
compounds, cell populations are treated with test com-
pounds prior to harvest and incubated for predetermined
optimal time periods to allow appropriate cellular events to
occur that result in synthesis of new MRNA species. Cells
are lysed with a reagent such as RNAzol to liberate and
preserve intracellular mRNA. Micropsheres bearing specific
oligonucleotide probes are added to the lysate to permit
hybridization of the oligonucleotide probes on the beads to
complementary mRNA.
Extended, labeled primers enter into the system from the
microtiter plates using an autosampler port. The primers are
mixed with streptavidin coated beads taken from another
port. The reaction time is adjusted to permit the beads to
adsorb the extended primers while in transit through the
10 tubing from the coupling device or mixing chamber. The
FCM is configured to identify the bead populations and the
primer-associated fluorescence on each bead set.
FIG. 30 illustrates the above procedure.
The above devices and methods in which the detector is
Oligonucleotide probes containing biotinylated UTP spe-
cific for the remaining, unhybridized portions of the mRNA
are also added to the hybridization mix. Hybridization
between the bead bound probes, the mRNA species and the
biotinylated probes will proceed under optimized condi-
tions.
15 capable of evaluating interactions with a plurality of par-
ticles simultaneously and/or capable of evaluating interac-
tions with individual particles, such as devices in which the
detector is a flow cytometer, may be used for multiparamet-
ric analyses of enzymatic activities.
20
Bead populations are exposed to an anti-biotin antibody
(1-5 ,ug/ml) within the fluidics system by introducing the
antibody to the sample slug through a second input system.
The antibody is conjugated with a fluorescent molecule to
25 permit identification by FCM. The length of the reaction
developing lines from the coupling device and/or the mixing
chamber is sufficient to permit binding of the antibody to the
second oligonucleotide probe (2 to 5 minutes). Tile
addressable, antibody covered beads with captured mRNA
30 then enter the FCM.
Example 19 describes such analyses.
EXAMPLE 19
Microsphere populations bearing enzyme substrates are
prepared by conjugating the substrates directly to the
spheres. Optimal conjugation conditions will vary for each
substrate. An example is to use streptavidin coated micro-
spheres to permit coating with biotinylated substrate. The
microspheres may be fluorescent or of different sizes to
render each bead population identifiable by FCM. Based on
size or fluorescence parameters, several unique and addres-
sable bead and substrate combinations can be constructed.
This allows multiple assays to be performed in a single
experiment when the populations are analyzed by FCM'
Multiple identifiable bead populations, each bearing a
unique substrate, are entered into the mixing system. The
beads are mixed with enzyme(s) and candidate enzyme
The FCM is configured with the correct excitation lasers
and beam scatter and emissions collecting optics to permit
simultaneous analyses of multiple captured cellular mRNAs.
Using multiple unique bead populations, it will be possible
to simultaneously detect multiple mRNA species.
FIG. 29 illustrates the above procedure.
The above devices and methods in which the detector is
capable of evaluating interactions with a plurality of par-
ticles simultaneously and/or capable of evaluating interac-
tions with individual particles, such as devices in which the
detector is a flow cytometer, may be used to simultaneously
evaluate samples for the presence of multiple single nucle-
otide polymorphisms (SNPs).
35 antagonists or agonists. In some embodiments, the beads
may be mixed with enzyme(s), candidate antagonists or
agonists, and standards. The delay time of the reaction
developing lines from the coupling device and/or the mixing
chamber is determined a=ording to the reaction kinetics of
40 the enzyme activity towards the substrate and the desired
information.
Example 18 describes one embodiment of such analyses. 45
EXAMPLE 18
In this example, the ability of the enzyme to cleave
fluorescent substrate from the bead in the presence of a
compound test compound(s)/standard mixture is detected as
a decrease in fluorescence of the bead population. If the test
compound(s) or test compound(s)/standard mixture inhibits
catalytic activity of the enzyme, then less of the substrate
will be cleaved. The fluorescence intensity of the bead
population will remain nearly as bright as the control
50
population. If the test compound(s) or mixture increases the
catalytic activity of the enzyme then more of the substrate
will be cleaved.
There are multiple methodologies for determining the
identities of polymorphic bases in SNPs in genomic DNA
samples. One approach is to prepare a short minisequencing
primer that hybridizes to PCR-amplified genomic DNA
containing the SNP such that the 3' end of the primer is
immediately adjacent to the polymorphic base. DNA poly-
merase is used to extend the primer by one base to the
polymorphic site. If fluorescent bases are used, the identity 55
of the inserted base, which is dictated by the sequence of the
amplified DNA, can be identified by the fluorescent signa-
ture of the extended primer. Such fluorescent bases are
commonly used in high speed DNA sequencing efforts and
are commercially available. By constructing the oligonucle- 60
otide probes so that they can be affixed to PCM-addressable
microspheres, the products of the DNA polymerase reaction
can be detected by FCM. Multiple SNPs can be detected in
a single sample of DNA by linking primers for each SNP to
different addressable beads.
In a preferred embodiment, the extension reaction is
performed in microtiter wells using a biotinylated primer
65
The FCM is configured with the correct excitation lasers
and beam scatter and emissions collecting optics to permit
simultaneous analyses of modifications of multiple enzyme
substrates by the test compound(s) or mixtures.
FIG. 31 illustrates the above procedure.
The above devices and methods in which the detector is
capable of evaluating interactions with a plurality of par-
ticles simultaneously and/or capable of evaluating interac-
tions with individual particles, such as devices in which the
detector is a flow cytometer, may be used for multiparamet-
ric analyses of molecular assembly assays.
Example 20 describes such analyses.
EXAMPLE 20
Microsphere populations bearing first molecules to be
evaluated for interaction with second molecules are prepared
US 6,558,916 B2
71 72
by conjugating the first molecules directly to microspheres.
In some embodiments, both the first molecules and the
second molecules are polypeptides. For example, the first
molecules may be polypeptides involved in signal transduc-
tion. Thus, the devices and methods of the present invention 5
may be used to assess the ability of cognate molecules
involved in signal transduction to bind with their target
molecule in the presence of one or more test compounds.
test compound(s) on the association of the fluorescent ligand
with the receptors are assessed by FCM measurements.
The FCM is configured with the correct excitation lasers
and beam scatter and emissions collecting optics to permit
simultaneous analyses of binding of ligands to multiple
receptors, and the effect of the test compounds.
FIG. 33 illustrates the above methods.
It is not uncommon to find that therapeutic agents are only
partially effective in achieving the desired effect in a sub-
stantial proportion of patients. This is likely due to patient
heterogeneity and/or heterogeneity of the cell populations at
the disease site. Accordingly, there is a need for devices and
methods which evaluate heterogeneity in the response of a
Optimal conjugation conditions will vary for each mol-
ecule. An example is to use streptavidin coated microspheres rn
to permit coating with biotinylated substrate. The micro-
spheres may be fluorescent or of different sizes to render
each bead population identifiable by FCM. Based on size or
fluorescence parameters, several unique and addressable
bead and molecule combinations can be constructed. This 15
allows multiple assays to be performed in a single experi-
ment when the populations are analyzed by FCM.
subject's cells to therapeutic agents before initiation of a
treatment regimen. In this way, the therapeutic agents which
are most likely to be effective for that particular subject may
be administered at the initiation of treatment.
The beads bearing the first molecule, such as a molecular
target are entered into the mixing system and exposed to test
mixtures and a fluorescent cognate molecule in fluidics
format. In this type of assay, inhibition of the molecular
interaction by the test compound(s) will be visible as a
reduction in fluorescent signal of the cognate molecule due
to reduced binding to the target.
The FCM is configured with the correct excitation lasers
and beam scatter and emissions collecting optics to permit
simultaneous analyses of binding of cognate molecules to
multiple molecular targets, and the effect of the test mix-
tures.
The purpose of the assay is to assess the ability of cognate
signal transduction molecules that bind the target molecule
as part of a normal in vivo signaling cascade to bind with the
target molecule in the presence of test mixtures, may be used
for multiparametric analysis of receptor targets.
FIG. 32 illustrates the above methods.
Accordingly, one embodiment of the present invention is
20
a method for determining the effect of each of a plurality of
test agents on cells from a subject. The subject may be any
desired organism. For example, in some embodiments, the
subject is a mammal, such as a mouse, rat, rabbit, dog, cat,
sheep, goat, cattle, pig, monkey, human or other mammal for
25
which it is desired to identify effective agents prior to the
initiation of treatment.
In the method of the present invention, cells are obtained
from the subject. The cells may be any type of cells for
which it is desired to evaluate the response to each of the test
30 agents. A plurality of samples comprising the cells are
combined with one or more of the test agents to form a
plurality of test mixtures. For example, the test agents may
be sequentially combined with the cells using the automated
and computer controlled devices described above.
35 For example, the cells may be combined with the test
agents in wells of a multiwell tissue culture plate or in liquid
cultures. After contacting the cells with the test agent for a
period of time sufficient for the test agent to produce a
response in the cells, the test mixtures are directed through
40 a detection zone capable of detecting the effect of the test
agents on the cells. For example, the detection zone may be
the detection zone in one of the devices described above. In
some embodiments, the response of the cells to the test
The above devices and methods in which the detector is
capable of evaluating interactions with a plurality of par-
ticles simultaneously and/or capable of evaluating interac-
tions with individual particles, such as devices in which the
detector is a flow cytometer, may be used to simultaneously
evaluate the interactions of multiple receptors with ligands.
Example 21 describes one embodiment of such analyses in
which the interactions of multiple membrane spanning
receptors with their ligands are simultaneously evaluated. 45
However, it will be appreciated that the present devices and
methods may be used to simultaneously evaluate the inter-
actions of any type of molecule with potential binding
agents is measured using a detector which is capable of
detecting a plurality of cellular responses simultaneously
and/or a detector which is capable of detecting a cellular
response as a single cell is flowing through the detection
zone. For example, the detector may be a flow cytometer as
described above. partners.
EXAMPLE 21
Membrane spanning receptors are i-;olated from cells
using techniques familiar to those skilled in the art. For
example membrane spanning receptors may be isolated by
performing an imrnunoprecipitation from detergent solubi-
lized cells using a. microsphere-coupled antibody specific
for the receptor of interest. Following harvest of the beads
with bound receptor, the beads with coupled receptor can be
used in the mixing system.
Alternatively the receptor can be engineered to contain a
molecular tag such as histidine and expressed in a recom-
binant form. The tag is then used to isolate the receptor from
solubilized cells by mixing the lysate with a slurry of Ni+
coated microspheres. The receptor-coated microspheres can
be used in the mixing system.
Beads with attached receptors are entered into the mixing
system and mixed with a fluorescent ligand. The effects of
50 The test agents may be any agent or combinations of
agents for which it is desired to evaluate a cellular response.
In particular, the test agents may be any of the test agents
described above and the cellular response may be any of the
cellular responses described above. In some embodiments,
55 the test agents may be chemical compounds, biological
molecules or test cells. For example, the test agents may be
agents for treating cancer, immunosuppressive drugs,
antibiotics, anti-inflammatory drugs, neurotransmitters,
growth hormones, or analgesics. If desired, the test agents
60 may be mixed with the cells over a range of concentrations
in order to estimate the relative proportions of drugs to be
used in the patient.
In one particular embodiment of the present invention, the
test agent may be an anticancer agent, such as an agent used
65 in chemotherapy. Cells are obtained from a tumor or can-
cerous growth in the subject to be evaluated. The cells are
combined with one or more anticancer agents to evaluate
US 6,558,916 B2
73
their ability to reduce or eliminate the proliferation or
viability of the cells. The agent or combination of agents
which has the greatest effect on the proliferation or viability
of the cells is administered to the subject.
74
multiparametric cell viability measurements provide an
index of the relative susceptibility of the cells to the anti-
cancer agent or agents to which they were exposed.
Example 22 below describes the evaluation of the effect 5
of one or more anticancer agents on the viability or prolif-
eration of cells from the subject.
If desired, the devices described above may be used to
determine whether the effects of the test agent or combina-
tion of test agents resulted from activation of the apoptotic
pathway or through a necrotic mechanism. In such
embodiments, the cells are contacted with response indica-
tors indicative of one or the other of these pathways. EXAMPLE 22
Biopsied cells or blood samples containing tumor cells are
obtained from the subject. The subject's cells are obtained
by a clinically accepted method such as needle biopsy or
phlebotomy. A plurality of samples containing the cells are
each cultured in the presence of one or more anticancer
agents for a sufficient period of time to allow them to
produce a cellular response. The period of time may be
seconds, minutes, hours, days, weeks or any desired time
period.
The cells may be cultured in multiwell tissue culture
plates or in liquid cultures. The cells are combined with the
anticancer agents using an automated and computer con-
trolled device such as those described above to form test
mixtures comprising the cells and anticancer agents.
The cells are contacted with one or more response indi-
cating reagents which generate signals indicative of particu-
lar cellular responses. The response indicating reagent may
be added to the cultures of cells prior to directing the cells
to the detection zone or while the cells are in transit to the
detection zone in a device such as those described above.
The cells may be contacted with the response indicating
reagent<> using the devices described above. The response
indicating reagents may be any of those described above. In
some embodiments, the response indicating reagent may be
a fluorescent indicator such as those described above.
In some embodiments, the response indicating reagents
indicate one or more response, such as cellular toxicity,
necrosis, or apoptosis. For example, the response indicating
reagent may be any of those described in Examples 12-14
above.
10 During the apoptotic cascade, a series of biochemical
events and consequences can be measured that are typical of
the pathway. After exposure to any one compound not all
cells will clearly exhibit all features common to the apop-
totic cascade. However, it is typical for several of the
features to be detectable. Similarly, at a given concentration
15 of compound, not all cells will clearly exhibit all features of
apoptosis. And finally, not all features of apoptosis develop
at the same time following exposure to an array of pro-
apoptotic molecules. These variances arise from heteroge-
neity in a variety cellular properties, different mechanisms of
20 induction of apoptosis by the drugs and different concen-
trations and activities of the drugs. Thus, the ability to
simultaneously measure several parameters of cells exposed
to multiple drugs or to drugs in varying dosages would
permit detection of apoptotic hallmarks regardless of such
25 variables. The ability to develop a chemotherapeutic sensi-
tivity profile would improve the design of patient therapies
by optimizing the dosage regimen and combination therapy
before the patient is actually treated.
The cellular parameters to be measured may include, but
30 are not limited to, 1) forward and side light scatter
properties, 2) activation of the caspase family of proteases,
3) perturbations in mitochondrial function and integrity such
as the membrane potential, 4) inversion of phosphati-
dylserine from the inner leaflet of the plasma membrane to
35 the outer leaflet, 5) cellular acidification, 6) intracellular
generation of oxygen radicals and the effect of those species
on cellular molecules, 7) increases in the permeability of the
plasma membrane and 8) evidence of DNA damage and
chromatin condensation. Fluorescent probes or other
40 reagents for evaluating these parameters are known to those
skilled in the art. The test mixtures containing the cells, test agent, and
response indicating reagent are sequentially delivered to the
detector, which may be a flow cytometer (FCM) in one of
the devices described above. In some embodiments, the test
mixtures pass through a plug flow coupler before being 45
delivered to the detector. The detector, such as a flow
cytometer, may analyze individual cells in the test mixtures
If desired, the relative susceptibility of the tumor cells to
the test agents may be tested by comparing the effect of the
test agent or combination of test agents on the tumor cells to
their effects on normal, non-cancerous cells, such as periph-
eral lymphocytes. Also, if desired, the relative potency of
various agents towards tumor and normal cells can be
derived by testing the drugs across a broad dose range. The
dose of the drugs that causes 50% of the cells to die (LD50)
to assess the toxicity index of each agent combination on
those cells in order to provide an estimate of the frequency
of cells that respond to or are resistant to the therapy. 50 can be determined for each drug or drug combination. By
combining assays of drug activity towards tumor and control
cells, an index of the relative activity of the drugs towards
tumor and control cells can be developed. Comparison of
Alternatively, the detector may simultaneously evaluate
two or more parameters indicative of cellular toxicity,
necrosis, apoptosis, or other indicators decreased cell pro-
liferation or viability. Such parameters may include glu-
tathione or free calcium, dissolution of membranes, inter- 55
ference with mitochondrial energy generation, activities
against molecules with specific functions that are critical to
normal function of the organ where they reside, and inter-
ference with detoxification enzymes such as the cytochrome
P450 family. Cellular necrosismay be evaluated using 60
response indicating reagents which detect dissolution of the
cell plasma membrane or the random release of cellular
contents to the surrounding milieu. In addition to detecting
signals from response indicators such as fluorescent probes,
changes in the light scatter properties of the excitatory light 65
source used in the FCM may also be used to indicate
changes in the status of the cells. The results of these
this index for the various drugs or drug combinations will
indicate which drug or drug combination will most likely be
successful for the patient.
The results of the above analysis are used to identify the
anticancer agent or combination of anticancer agents which
is most effective on the subject's tumor. The most effective
agent or combination of agents is then administered to the
subject.
In vitro experiments using the HL-60 tumor cell line as a
model are described in Example 23 below.
EXAMPLE 23
The human monocvtic cell line HL-60 was used for the
toxicity testing. Sto~k cultures of cells were grmvn in
US 6,558,916 B2
75
spinner flasks in Iscove's modified Dulbecco 's medium with
4 mM L-glutamine adjusted to contain 1.5 g/L sodium
bicarbonate, 80%; fetal bovine serum, 20% Cultures were
incubated at 37 C. in 5% C02. Cells were maintained at
lx105/ml to 5xl05 cells/ml.
Stock solutions of Actinomycin D, ouabain, FCCP,
etoposide, acetaminophen and ibuprofen were prepared.
Stock solutions were prepared in appropriate solvents and
maintained at temperatures appropriate to the compounds.
5
76
input and analyzed in one minute, or an entire 96-well plate
could be analyzed in less than nine minutes. FIG. 34
illustrates multiparametric assessment of single dose toxic-
ity. The data was obtained from human monocytic cells
treated overnight in 24 wells of a 96-well plate with rela-
tively low or high concentrations of toxic molecules (FCCP,
actinomycin D, oubain, etoposide, acetaminophen,
ibuprofen), or buffer control. The cells were stained in the
wells with fluorescent probes that measure the following
10
parameters: intracellular calcium, cellular glutathione and
plasma membrane integrity propidium iodide and ToPro3).
Forward angle and 90 degree (side) scatter signals of the
incident laser beam were also determined as indicators of
HL-60 cells were incubated at 5x105 cells/ml in 200 ,ul of
media in 96-well flat bottom plates. Compounds were either
diluted into culture media for further dilution into wells or
diluted directly into wells from the stocks. Cells were
incubated with the compounds for 20 hours. To prepare for
loading the plates of cells were centrifuged (1000 g/5
15
minutes), and the supernatants were removed. The culture
media was replaced with 200 pl of a loading buffer. Cells
were stained directly in the wells by addition of fluorescent
probes with a multichannel pipettor. To measure intracellular
calcium levels the cells were incubated with indo-lAM (2
20
,uM). Glutathione levels were measured with CM-FDA (2
µM). The two probes were added simultaneously and the
cells were incubated for 40 minutes. After loading the cells
were analyzed directly from the wells without removing the
supernatant. TOPR0-3 and propidium iodide, PI, were
25
added to the wells at least 10 minutes before the analyses.
All stains were obtained from Molecular Probes except for
PI which was obtained from Sigma.
Rapid sample input from the 96 well plate into the FCM
was a=omplished using the embodiment of the device 30
described above comprising a plug flow coupler, an
autosampler, peristaltic pump and eight port switching valve
cell shape change or relative size. The well contents were
aspirated into the FCM at 10 samples per minute for the
simultaneous analysis of all the parameters. The results of
the 18 control well<> were pooled and the mean is shown. For
each parameter a threshold was set so that 10% to 20% of the
control cells exceeded the threshold level. The frequency of
cells that exceeded the threshold in the treated wells was also
determined. The results show that the toxins used at high
concentrations (FCCP, ouabain, actinomycin D) gave signs
of toxicity when measured by all six parameters. Etoposide
which was used at a relatively low concentration was not
detected by propidium iodide, the commonly used plasma
membrane indicator of toxicity. Acetaminophen and ibupro-
fen were only flagged by the light scatter parameters. This
figure is consistent with multiparametric assessment of
toxicity yielding more information than single parameter
measurement systems, and that information greatly
improves the ability to detect toxic molecules.
FIG. 35 illustrates the dose response curves for actino-
mycin D. These curves were generated from the same cell
line as shown in FIG. 34. Here the cells were exposed to a
range of concentrations of the toxic compound actinomycin
D. Four parameters, plasma membrane (probe 1), plasma
membrane (probe 2), intracellular calcium, and light scatter,
were measured. The results indicate that side scatter detec-
tion and intracellular calcium levels are more sensitive
to draw samples from the plate wells using the peristaltic
pump. Separate samples of cells were loaded with the
individual probes and introduced into a device of the present 35
invention as described above. These singly stained samples
were used to set the PMT voltages and amplifier gains on the
instrument for each stain. Once the instrument settings were
established, the wells containing the treated cells were input
via the autosampler into the flow cytometer. 40 detectors of toxicity than the plasma membrane integrity
probes. Furthermore these parameters give insight as to the
mechanism of the toxicity. These dose-response curves can
be used to determine LD50 values of 125 to 250 nM.
The system was controlled by software with functions
appropriate to flow cytometry applications such as control-
ling the switching valve. The autosampler was set to draw
from each well for three seconds, and to progress from well
to well with continuous aspiration driven by the pump. The 45
system can be directed to a washing station if desired but this
slows the sampling rate. Separate experiments with stained
or unstained cells in alternating wells indicated that well to
well contamination was less than 3% under these conditions.
Depending on the cell density in the wells a two second 50
aspiration delivers up to several hundred cells for analysis.
The switching valve serves to isolate the samples from the
oscillatory pressures of the peristaltic pump, and place them
under the positive air pressure normally used to drive
samples through a conventional FCM. This results in smooth 55
particle delivery to the laser interrogation point \Vith mini-
mal loss of data due to pressure surges. Sample uptake and
delivery into the system were synchronized.
The detector utilized in the device was a Coulter Epics
Elite flow cytometer (FCM). For cell analysis the instrument 60
was equipped with a Coherent Innova 90 argon laser using
UV optics to generate 350-355 nm UV excitation lines, a air
cooled argon laser to provide 488 nm excitation and a air
cooled HeNe laser to provide 633 nm excitation. Data files
were acquired and analyzed using Expo2 software (Coulter). 65
Data files were collected as one row of cells per file. For
the well aspiration times used one row of 12 wells could be
In some instances, cells are resistant to therapeutic agents
because cellular pumps, such as multidrug resistance pumps,
prevent the therapeutic agent from accumulating inside the
cell in an effective amount. Accordingly, devices such as
those described above may be utilized to measure the
activity of such pumps in cells obtained from the subject.
Multidrug resistance pumps (MDR) exist in the plasma
membranes of all cells. The MDR serve to extrude mol-
ecules from the interior of the cell. Overactivity or overex-
pression of this family of pumps accounts for some ability
of tumor cells to escape the toxic activities of chemothera-
pies by virtue of their ability to extrude tumor killing drugs
from the cytoplasm where they need to be present in
sufficiently high concentrations to have toxic activity
towards the tumor cells. Inhibitors of these pumps have been
sought as adjunct therapies to enhance the ability of cancer
chemotherapeutic drugs to accumulate in cancer cells,
thereby enhancing the drug therapeutic efficacy.
Cells from a subject may be pretreated with MDR inhibi-
tor drugs and their effectiveness against the cells from the
subject may be evaluated as described above. If desired, the
cells may be cultured in a multiwell plate or, alternatively,
the cells may be cultured in liquid culture. Some cultures of
the cells are contacted with the candidate MDR inhibitor
US 6,558,916 B2
77
drugs while control cultures are not contacted with the MDR
inhibitor drugs. If desired, the cells may be treated with
bioresponse probes to measure, for example, plasma mem-
brane integrity as an indicator of cell viability. The treated
cells (with or without bioresponse probes) are then drawn 5
from the wells of the multiwell plate into fluidics system of
a device such as those described above. A second line is used
78
integrity has been damaged. Alternatively, the test cells may
be loaded with the response indicating reagent as they are en
route to the detector.
The test mixtures are sequentially delivered to the detec-
tor. In some embodiments, the test mixtures pass through a
plug flow coupler before being delivered to the detector.
In some embodiments, the test cells may also be evaluated
to determine whether they express one or more molecules on
their surface which will cause a desirable or undesirable
to input a response indicating reagent that assesses the
relative activity of the MDR pumps in the cells. For
example, the fluorescent probe Calcein AM(Molecular 10 response. For example, the test cells may be contacted with
Probes, OR) is excluded by the MDR pump family of
detectably labeled antibodies against MHC molecules and
transporters. The slug of patient cells is thus exposed to the
analyzed using devices such as those described above to
MDR probe for a defined period of time and some amount
determine whether they have a pattern of MHC molecules
of the probe will leak into the cells, while most will be
which indicates that the subject will tolerate the tissue or
excluded by the pump. In the presence of active MDR 15 organ transplant or whether they have a pattern of MHC
inhibitors, less of the compound will enter the cell. The
molecules which indicates that the subject will reject the
decrease in fluorescence measured by the detector, such as
tissue or organ transplant.
a flow cytometer, indicates that the patient's cells are sus-
ceptible to the particular MDR inhibitor or combination of If desired, genetic analyses may also be performed as
MDR inhibitor. As an alternative mode of operation, the
20
described above to determine whether the subject is likely to
candidate MDR inhibitor compounds could be added to the a=ept or reject the transplant.
patient cells as a concentration gradient while the test probe It will be appreciated that the test cells need not be
would be added at a single concentration. In this way an candidate tissue or organ transplants, but may also be any
EC
50
value for the relative activity of each MDR inhibitor other cell type for which it is desired to evaluate the
drug could be derived. These single drug values could be 25 subject's response.
compared to values for drug combinations to determine if Although the invention has been described in detail with
there is synergy between the combined drugs. In this reference to certain particular embodiments thereof, it will
manner, drug therapies may be accurately tailored to each be understood that any variations and modifications apparent
patient to increase efficacy, and reduce treatment times and to those of skill in the art will still fall within the spirit and
overall costs. 30 scope of the invention. Other embodiments not specifically
In another embodiment of the present invention, the described herein may fall within the spirit and scope of the
methods for identifying the most effective test agent or present invention as provided by the following claims.
combination of test agents described above may be com- What is claimed is:
bined with genetic analysis techniques which indicate 1. A method for determining the effect of each of a
whether the subject is likely to respond positively to the test 35 plurality of test agents on cells from a subject comprising:
agent or combination of test agents. For example, a nucleic (a) obtaining cells from said subject;
acid sample may be obtained from a subject and analyzed to (b) combining each of a plurality of samples comprising
determine which test agent or combinations of test agents said cells with one or more of said test agents to form
are likely to be effective or not to be effective in treating the each of a plurality of test mixtures; and
subject. For example, the nucleic acid sample may be 40 (c) sequentially directing each of said plurality of test
evaluated to determine whether the subject has an allele of mixtures through a detection zone in an apparatus, said
one or more single nucleotide polymorphisms which is detection zone being capable of detecting the effect of
indicative of a positive or adverse response to a particular said agents on said cells.
test agent or combination of test agents. The identities of 2. The method of claim 1, wherein said method further
such single nucleotide polymorphisms in the sample may be 45 comprises determining whether said one or more test agents
determined using the methods described above or other has a desired effect on said cells from said subject.
methods familiar to those skilled in the art, including the 3. The method of claim 1, wherein said agents are selected
methods described in U.S. Pat. No. 5,952,174, U.S. Pat. No. from the group consisting of chemical compounds, biologi-
5,945,283, and U.S. Pat. No. 5,885,775, the disclosures of cal molecules, and test cells.
which are incorporated herein by reference in their entire- 50 4. The method of claim 3, wherein said agents are
ties. chemical compounds.
In another embodiment of the present invention, devices 5. The method of claim 4, wherein said subject is a
such as those described above may be used to determine mammal.
whether a subject will reject or accept a candidate organ or 6. The method of claim 5, wherein said mammal is
tissue for use in a transplant. The methods are similar to 55 selected from the group consisting of mouse, rat, rabbit, dog,
those described above for anticancer agents except that the cat, sheep, goat, cattle, pig, monkey, and human.
test agents are cells from the candidate organ or tissue. A 7. The method of claim 6, wherein said mammal is a
plurality of samples of immune cells from the subject are human.
each combined with test cells from the candidate organ or 8. The method of claim 1, wherein said cells are normal
tissue to generate test mixtures. The cells from the subject 60 cells.
and the test cells are placed in contact with one another for 9. The method of claim 8, wherein said normal cells are
a sufficient time to allow the immune cells to lyse or damage cells involved in generating or modulating an immune
the integrity of the test cells. The cells from the subject may response.
be combined with the test cells in a multiwell tissue culture 10. The method of claim 1, wherein said cells are abnor-
plate or in liquid culture. The test cells may have been 65 mal cells.
previously loaded with a response indicating agent which 11. The method of claim 10, wherein said abnormal cells
indicates whether they have been lysed and/or whether their are cancer cells.
US 6,558,916 B2
79
12. The method of claim 1, wherein said test agents are
selected from the group consisting of agents for treating
cancer, immunosuppressive drugs, antibiotics, anti-
inflammatory drugs, neurotransmitters, growth hormones,
and analgesics.
13. The method of claim 1, wherein said test mixtures
further comprise one or more response indicating agents.
14. The method of claim 13, wherein said response
indicating agents indicate a decrease or cessation of repli-
cation.
15. The method of claim 13, wherein said response
indicating agents indicate cell death.
5
1iJ
16. The method of claim 1, wherein said plurality of test
mixtures vary according to a condition selected from the
group consisting of incubation condition, type of cell, type 15
of test agent, and number of test agents, or a combination
thereof.
80
32. The method of claim 1, wherein said cells are abnor-
mal cells.
33. The method of claim 32, wherein said abnormal cells
are cancer cells.
34. The method of claim 1, wherein said test agents are
selected from the group consisting of agents for treating
cancer, immunosuppressive drugs, antibiotics, anti-
inflammatory drugs, neurotransmitters, growth hormones,
and analgesics.
35. The method of claim 1, wherein said test mixtures
further comprise one or more response indicating agents.
36. The method of claim 35, wherein said response
indicating agents indicate a decrease or cessation of repli-
cation.
37. The method of claim 35, wherein said response
indicating agents indicate cell death.
17. The method of claim 3, wherein said detection zone is
capable of detecting a plurality of cellular, responses simul-
taneously.
18. The method of claim 3, wherein said detecting step
comprises simultaneously measuring a plurality of an extent
38. The method of claim 1, wherein said plurality of test
mixtures vary a=ording to a condition selected from the
20 group consisting of the incubation condition, type of cell,
type of test agent, and number of test agents, or a combi-
nation thereof.
of response of said cells to said one or more test compounds
as each of said plurality of test mb,1:ures are flowing through
said detection zone.
39. The method of claim 1, wherein said detection zone is
capable of detecting a plurality of cellular responses simul-
25 taneously.
19. The method of claim 3, wherein said detection zone is
capable of detecting a cellular response as a single cell is
flowing through said detection zone.
40. The method of claim 1, wherein said detecting step
comprises simultaneously measuring a plurality of an extent
of response of said cells to said one or more test compounds
as each of said plurality of test mixtures are flowing through 20. The method of claim 3, wherein said detection zone
comprises a flow cytometer.
21. The method of claim 1, further comprising conducting
a genetic analysis to determine whether said subject is likely
30 said detection zone.
to have a desirable or adverse response to said one or more
test agents.
41. The method of claim 1, wherein said detection zone is
capable of detecting a cellular response as a single cell is
flowing through said detection zone.
42. The method of claim 1, wherein said detection zone
comprises a flow cytometer.
22. The method of claim 21, wherein said genetic analysis 35
comprises determining whether said subject has an allele of
one or more single nucleotide polymorphisms which indi-
cates that said subject will have said desirable or adverse
response.
43. The method of claim 1, further comprising conducting
a genetic analysis to determine whether said subject is likely
to have a desirable or adverse response to said one or more
40 test agents. 23. The method of claim 1, further comprising conducting
a cellular analysis to determine whether said subject is likely
to have a desirable or adverse response to said one or more
test compounds.
24. The method of claim 23, wherein said cellular analysis
comprising determining whether said one or more test 45
compounds will be maintained at effective concentrations in
said cells.
25. The method of claim 24, wherein said cellular analysis
comprises measuring the level of activity of one or more
multidrug resistance transporters in said cells.
26. The method of claim 1 wherein said test agent
comprises test cells.
SD
44. The method of claim 26, further comprising conduct-
ing a genetic analysis to determine whether said subject is
likely to have a desirable or adverse response to said test
cells.
45. The method of claim 44, wherein said genetic analysis
comprises determining whether said subject has an allele of
one or more single nucleotide polymorphisms which indi-
cates that said subject will have said desirable or adverse
response.
46. The method of claim 45, further comprising conduct-
ing a cellular analysis to determine whether said subject is
likely to have a desirable or undesirable response to said test
cells. 27. The method of claim 26, wherein said subject is a
mammal.
28. The method of claim 27, wherein said mammal is
selected from the group consisting of mouse, rat, rabbit, dog,
cat, sheep, goat, cattle, pig, monkey, and human.
47. The method of claim 26, wherein said cellular analysis
55 comprises determining whether said test cells express one or
molecules on their surface which will cause a desirable or
29. The method of claim 28, wherein said mammal is a
human.
30. The method of claim 1, wherein said cells are normal 60
cells.
31. The method of claim 30, wherein said normal cells are
cells involved in generating or modulating an immune
response.
undesirable response.
48. The method of claim 47, wherein said one or mol-
ecules comprise major histocompatibility molecules.
49. The method of claim 1, where in said test agents are
test cells from a candidate organ or tissue to be tested for use
in an organ or tissue transplant.
* * * * *