Ex Parte Palecek et al

Board of Patent Appeals and Interferences

Jul 16, 2010

10993468 (B.P.A.I. Jul. 16, 2010)

UNITED STATES PATENT AND TRADEMARK OFFICE
____________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
____________

Ex parte SEAN P. PALECEK, JUAN J. DE PABLO,
LIN JI, and JAMES A. THOMSON
____________

Appeal 2010-001157
Application 10/993,468
Technology Center 1600
____________

Before KIMBERLY R. JORDAN, Division 1 Support Administrator.

ORDER RE-MAILING BOARD DECISION1
This appeal was originally decided on July 8, 2010 (see attached copy
of the decision) and communicated via electronic delivery. There is no
record, however, in the electronic file that e-mail notification was sent to
Appellants notifying them of the decision. Accordingly, the decision is
being re-mailed.

1 The two-month time period for filing an appeal or commencing a civil
action, as recited in 37 C.F.R. § 1.304, or for filing a request for rehearing,
as recited in 37 C.F.R. § 41.52, begins to run from the “MAIL DATE”
(paper delivery mode) or the “NOTIFICATION DATE” (electronic delivery
mode) shown on the PTOL-90A cover letter attached to this order.
Appeal 2010-001157
Application 10/993,468

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If there are any questions pertaining to this Order, please contact the
Board of Patent Appeals and Interferences at 571-272-9797.

KRJ/clj

QUARLES & BRADY LLP
33 E. MAIN ST, SUITE 900
P.O. BOX 2113
MADISON, WI 53701-2113

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte SEAN P. PALECEK, JUAN J. DE PABLO,
LIN JI, and JAMES A. THOMSON
__________

Appeal 2010-001157
Application 10/993,468
Technology Center 1600
__________

Before RICHARD M. LEBOVITZ, MELANIE L. McCOLLUM, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

FREDMAN, Administrative Patent Judge.

DECISION ON APPEAL1

1 The two-month time period for filing an appeal or commencing a
civil action, as recited in 37 C.F.R.§ 1.304, or for filing a request
for rehearing, as recited in 37 CF.R.§ 41.52, begins to run from
July 8, 2010 shown on the PTOL-90A cover letter attached to this
decision.

Appeal 2010-001157
Application 10/993,468

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This is an appeal under 35 U.S.C. § 134 involving claims to methods
of cryopreserving human embryonic stem cells. We have jurisdiction under
35 U.S.C. § 6(b). We affirm.

Statement of the Case
The Claims
Claims 1, 2, 5-10, 12-22, 24-38, and 40-45 are on appeal. The claims
subject to each rejection have not been separately argued and therefore stand
or fall together. See 37 C.F.R. § 41.37(c)(1)(vii). We selected Claim 17 as
representative and Claim 17 reads as follows:
17. A method of cryopreserving stem cells, comprising
the steps of:
a) growing undifferentiated human embryonic stem
cells on a bottom layer of solid support matrix, such that the
cells adhere to the matrix;
b) adding an effective amount of a carbohydrate-based
cryopreservation medium over the matrix adherent cells;
c) adding an effective amount of a freezing medium to
the cells of step b); and
d) cooling the matrix adherent cells of step c) to a
temperature sufficient to cryopreserve the cells.

The prior art

The Examiner relies on the following prior art references to
show unpatentability:
Reid et al. US 2002/0182188 A1 Dec. 5, 2002
Funk et al. US 6,667,176 B1 Dec. 23, 2003

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Ure et al., A rapid and efficient method for freezing
and recovering clones of embryonic stem cells, 8 TRENDS
GENETICS 6 (1992).

Reubinoff et al., Effective cryopreservation of human
embryonic stem cells by the open pulled straw vitrification
method, 16 HUMAN REPRODUCTION 2187-2194 (2001).

Wolkers et al., Human platelets loaded with trehalose
survive freeze-drying, 42 CRYOBIOLOGY 79-87 (2001).

Voight et al., Cultured epidermal keratinocytes on a
microspherical transport system are feasible to reconstitute
the epidermis in full-thickness wounds, 5 TISSUE
ENGINEERING 563-572 (1999).

Limaye et al., Cryopreservation of human
hematopoietic cells with membrane stabilizers and
bioantioxidants as additives in the conventional freezing
medium, 10 J. HEMATOTHERAPY STEM CELL RES. 709-718
(2001).

The issues
A. The Examiner rejected claims 17-20, 22, 24-27, 32, 33, 36, 38, 42, 43,
and 45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, and
Wolkers (Ans. 4-7).
B. The Examiner rejected claims 17-22, 24-27, 32-34, 36, 38, 42, 43, and
45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers,
and Voigt (Ans. 7-8).
C. The Examiner rejected claims 1, 2, 5-9, 12-14, 16-22, 24-33, 35-38,
and 40-45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk,
Wolkers, and Reid (Ans. 8-9).
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D. The Examiner rejected claims 1, 2, 5-10, 12-14, 16-22, 24-38, and 40-
45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers,
Reid, and Voigt (Ans. 9-10).
E. The Examiner rejected claims 1, 2, 5-9, 12-22, 24-33, 35-38, and 40-
45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers,
Reid, and Limaye(Ans. 10-11).
A. 35 U.S.C. § 103(a) over Ure, Reubinoff, Funk, and Wolkers
The Examiner finds that “the mere fact that nobody combined these
well known techniques before, does not mean that the instant invention was
not obvious over the prior art. It is noted that all cited references are
pertinent art, i.e., they all teach cryopreservation” (Ans. 17).
Appellants argue that:
Although the Examiner alleged that one of skill in the art
would have been motivated to use the Ure method with
human ESCs [embryonic stem cells] (office action dated
October 30, 2007, page 3, lines 17-18), Reubinoff explicitly
discourages such combination by explaining that while
“slow-rate freezing and rapid thawing methods ... are
efficient for the cryopreservation of mouse ES cells, it has
been observed that the survival of undifferentiated human
ES cells following slow-rate freezing is very poor, and most
often the cells either differentiate or die” (Reubinoff, page
2187, right column, second paragraph (emphasis added))

(App. Br. 9). Appellants argue that “Funk teaches away from freezing ESCs
on a Matrigel matrix by separating the cells from the matrix before
cryopreservation” (App. Br. 10).
Appellants argue that “Wolkers teaches . . . that human cell lines
require genetic engineering to express a bacterial pore protein to permit
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trehalose penetration into the cells . . . Therefore, a skilled artisan would . . .
have no reasonable expectation of success using trehalose on cells not
provided with such structures” (App. Br. 10). Appellants argue that “there is
no absolute Matrigel requirement in human ESC cultures. Rather, the cells
can be cultured on feeder layers or other matrices, which is explicitly stated
by Funk . . .Therefore, the Examiner failed to show a motivation to combine
Ure with Reubinoff and Funk” (App. Br. 11).
Appellants argue that even “if the prior art could be combined as
suggested by the Examiner, and even if such combination of four or more
documents would teach the elements of the claims, which it does not, the
claimed invention would still be nonobvious due to the well-documented
high level of unpredictability in the art as of the filing date” (App. Br. 12-
13).
In view of these conflicting positions, we frame the obviousness issue
before us as follows:
Does the evidence of record support the Examiner’s conclusion that
the prior art rendered obvious the method of cryopreservation of human
embryonic cells of Claim 17?
Findings of Fact (FF)
1. Ure teaches “freezing ES cells in situ on 24-well plates. We
have developed a simplified method in which the ES cells are exposed to the
minimum volume of freezing mix, which is frozen uniformly and thawed
very rapidly” (Ure 6).
2. Ure teaches that “clones were picked and expanded in gelatine-
coated 24-well plates. Individual clones were trypsinized when near
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confluent and one-third of the cell suspension was passaged into a fresh
well” (Ure 6).
3. Ure teaches that the “minimum volume of freezing mix
required to coat the wells completely (0.25 ml of complete media containing
10% dimethyl sulphoxide, DMSO) was then added” (Ure 6).
4. Ure teaches that “[p]lates were placed in polystyrene boxes
packed with sytrofoam and immediately transferred to a -80°C freezer” (Ure
6).
5. The Examiner acknowledges that “Ure et al. do not teach
cryopreserving human embryonic stem cells” (Ans. 4). The Examiner also
acknowledges that “Ure et al. taken with Reubinoff, and Funk et al. do not
teach adding a carbohydrate-based cryopreservation medium over the matrix
adherent cells” (Ans. 5).
6. Reubinoff teaches that “to exploit this remarkable potential of
human ES cells . . . an efficient cryopreservation method would be highly
valuable for the development and widespread use of these cell lines”
(Reubinoff 2187, col. 1-2).
7. Reubinoff teaches that:
Slow-rate freezing and rapid thawing methods are most
commonly used for the cryopreservation of cell lines
(Freshney 1994). While these standard methods are efficient
for the cryopreservation of mouse ES cells (Robertson,
1987), it has been observed that the survival of
undifferentiated human ES cells following slow-rate
freezing is very poor, and most of the cells either
differentiate or die.

(Reubinoff 2187, col. 2).
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8. Reubinoff teaches that “ES cells were cryopreserved in clumps
of about 100-200 cells by using either the conventional slow-freezing
method . . . or the open pulled straw (OPS) vitrification method” (Reubinoff
2188, col. 10).
9. Reubinoff teaches that “[h]uman pluripotent ES cells from both
cell lines could be successfully recovered and propagated after
cryopreservation with conventional slow-rate freezing and rapid thawing
methods . . . and the cells retained their fundamental characteristics.
Nevertheless, the efficiency of these standard methods with human ES cells
was relatively low” (Reubinoff 2190, col. 1-2).
10. Funk teaches “a system for obtaining expression libraries from
primate pluripotent stem (pPS) cells. pPS cells can be maintained in vitro
without requiring a layer of feeder cells to inhibit differentiation” (Funk, col.
5, ll. 39-42).
11. Funk teaches that “the role of feeder cells can be replaced by a
combination of one or more features in the culture environment that support
proliferation of the cells without differentiation. One such feature is a
suitable substrate on the culture surface, such as extracellular matrix
exemplified by Matrigel® and laminin” (Funk, col. 5, ll. 43-48).
12. Wolkers teaches “[s]mall carbohydrates, such as trehalose . . .
have an exceptional ability to stabilize cells during freezing, freeze-drying,
and air-drying” (Wolkers 79, col. 1).
13. Wolkers teaches that “[t]rehalose . . . is particularly effective at
stabilizing dry cells. . . . Its remarkable effectiveness is apparently due to its
ability to replace the water shell of macromolecules, thus preventing
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damaging effects during drying, and to its high glass transition temperature,
resulting in the formation of stable glasses at room temperature that preserve
cells in the dried state” (Wolkers 79, col. 1-2).
14. The Specification teaches that “trehalose is believed to maintain
thermodynamic stability of membranes by preserving phospholipid head
group spacing and inhibiting lipid phase transitions and separation during
freezing and drying” (Spec. 1-2 ¶ 0004).
15. The Specification teaches that “[i]t has been proposed that
freezing destruction from extracellular ice is essentially a plasma membrane
injury resulting from osmotic dehydration of the cell” (Spec. 9 ¶ 0052).
Principles of Law
“Often, it will be necessary . . . to look to interrelated teachings of
multiple [references] . . . and the background knowledge possessed by a
person having ordinary skill in the art, all in order to determine whether
there was an apparent reason to combine the known elements in the fashion
claimed.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 418 (2007). “[T]his
analysis should be made explicit,” and it “can be important to identify a
reason that would have prompted a person of ordinary skill in the relevant
field to combine the elements in the way the claimed new invention does.”
Id.
An obviousness “analysis need not seek out precise teachings directed
to the specific subject matter of the challenged claim, for a court can take
account of the inferences and creative steps that a person of ordinary skill in
the art would employ.” Id.
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In assessing a claim’s obviousness, “a court must ask whether the
improvement is more than the predictable use of prior art elements according
to their established functions.” Id. at 417. “The combination of familiar
elements according to known methods is likely to be obvious when it does
no more than yield predictable results.” Id. at 416.
A reference may be said to teach away when a person of
ordinary skill, upon reading the reference, would be
discouraged from following the path set out in the reference,
or would be led in a direction divergent from the path that
was taken by the applicant. The degree of teaching away
will of course depend on the particular facts; in general, a
reference will teach away if it suggests that the line of
development flowing from the reference’s disclosure is
unlikely to be productive of the result sought by the
applicant.

In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994).
“[A] reference may teach away from a use when that use would render
the result inoperable.” In re ICON Health and Fitness Inc., 496 F.3d 1374,
1381 (Fed. Cir. 2007).
Analysis
Ure teaches a method of cryopreserving embryonic stem cells by
growing the stem cells on gelatine coated plates, adding a freezing medium
to the cells, and cooling the matrix to cryopreserve the cells (FF 1-4). There
is no dispute that Ure does not teach freezing of human embryonic stem cells
or adding trehalose to the cells (FF 5).
Applying the KSR standard of obviousness to the findings of fact, we
conclude that the person of ordinary creativity would have predictably
applied the cryopreservation method of Ure to human embryonic stem cells
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for the reasons of Reubinoff that cryopreservation is “highly valuable” and
expected to work to at least some degree (FF 6-7) and incorporated trehalose
since Wolker teaches that trehalose has “an exceptional ability to stabilize
cells during freezing” (Wolkers 79, col. 1; FF 12). Such a combination is
merely a “predictable use of prior art elements according to their established
functions.” KSR, 550 U.S. at 417.
Appellants argue that:
Although the Examiner alleged that one of skill in the art
would have been motivated to use the Ure method with
human ESCs (office action dated October 30, 2007, page 3,
lines 17-18), Reubinoff explicitly discourages such
combination by explaining that while “slow-rate freezing
and rapid thawing methods ... are efficient for the
cryopreservation of mouse ES cells, it has been observed
that the survival of undifferentiated human ES cells
following slow-rate freezing is very poor, and most often the
cells either differentiate or die” (Reubinoff, page 2187, right
column, second paragraph (emphasis added))

(App. Br. 9). Appellants argue that “Funk teaches away from freezing ESCs
on a Matrigel matrix by separating the cells from the matrix before
cryopreservation” (App. Br. 10).
We are not persuaded. While Reubinoff teaches that “the efficiency
of these standard methods with human ES cells was relatively low,”
Reubinoff also teaches that “[h]uman pluripotent ES cells from both cell
lines could be successfully recovered and propagated after cryopreservation
with conventional slow-rate freezing and rapid thawing methods . . . and the
cells retained their fundamental characteristics” (Reubinoff 2190, col. 1-2;
FF 9). The claim does not require any degree of efficiency or survival rate.
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Reubinoff does not teach or suggest that cryopreservation of human
embryonic stem cells by the method of Ure would be unlikely to work (see
FF 6-9). Rather, Reubinoff simply does not prefer slow-rate freezing, but
Reubinoff clearly shows that the conventional methods are operable (FF 6-
9).
Regarding Funk, Appellants argue that Funk teaches to separate the
cells into solution prior to cryopreservation (App. Br. 10). However, Funk
teaches that “[c]ells maintained in Matrigel®/conditioned medium and
laminin/conditioned medium were cryopreserved as follows: The cells were
frozen in standard hES medium (not conditioned medium) supplemented
with 10% DMSO and additional 10% SR(total 30%). The cells were thawed
onto Matrigel® or laminin in conditioned medium” (Funk, col. 29, ll. 42-
47). This teaching of Funk does not indicate that the cells should be
separated and certainly does not teach away from freezing cells on the
matrix. Like our appellate reviewing court, “[w]e will not read into a
reference a teaching away from a process where no such language exists.”
DyStar Textilfarben GmbH v. C.H. Patrick Co., 464 F.3d 1356, 1364 (Fed.
Cir. 2006).
Appellants argue that “Wolkers teaches . . . that human cell lines
require genetic engineering to express a bacterial pore protein to permit
trehalose penetration into the cells . . . Therefore, a skilled artisan would . . .
have no reasonable expectation of success using trehalose on cells not
provided with such structures” (App. Br. 10).
We are not persuaded. In fact, Wolkers teaches that “[s]mall
carbohydrates, such as trehalose . . . have an exceptional ability to stabilize
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cells during freezing, freeze-drying, and air-drying” (Wolkers 79, col. 1; FF
12). While Wolkers gives one example where the use of genetically
engineered pores enhanced the freeze tolerance of human cell lines,
Wolkers’ own experiments do not use such cells with genetically engineered
pores, but rather use normal platelets (see Wolkers 79, col. 1 and 80, col. 1-
2). The Specification recognizes, consistent with Wolkers, that “trehalose is
believed to maintain thermodynamic stability of membranes by preserving
phospholipid head group spacing and inhibiting lipid phase transitions and
separation during freezing and drying” (Spec. 1-2 ¶ 0004). The
Specification teaches that “[i]t has been proposed that freezing destruction
from extracellular ice is essentially a plasma membrane injury resulting from
osmotic dehydration of the cell” (Spec. 9 ¶ 0052; FF 15). Thus, the ordinary
artisan would have expected that trehalose would enhance the stability of
cells during freezing, whether the trehalose penetrated the cells or not (FF
12-15).
Appellants argue that “there is no absolute Matrigel requirement in
human ESC cultures. Rather, the cells can be cultured on feeder layers or
other matrices, which is explicitly stated by Funk . . .Therefore, the
Examiner failed to show a motivation to combine Ure with Reubinoff and
Funk” (App. Br. 11).
We are not persuaded. First, the use of Matrigel is not a requirement
of Claim 17 (or indeed of any of independent claims 1, 16, 28, or 32).
“[L]imitations are not to be read into the claims from the specification.” In
re Van Geuns, 988 F.2d 1181, 1184 (Fed. Cir. 1993) (citing In re Zletz, 893
F.2d 319, 321 (Fed. Cir. 1989)).
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Second, Funk teaches that in growing embryonic stem cells “the role
of the feeder cells can be replaced by a combination of one or more features
in the culture environment that support proliferation of the cells without
differentiation. One such feature is a suitable substrate on the culture
surface, such as extracellular matrix exemplified by Matrigel® and laminin”
(Funk, col. 5, ll. 43-48; FF 11). The Supreme Court has clearly stated that
the “combination of familiar elements according to known methods is likely
to be obvious when it does no more than yield predictable results,” KSR, 550
U.S. at 401. This reasoning is applicable here, since Funk teaches an
alternative matrix for growing human embryonic stem cells which would
predictably support the cell growth (FF 10-11). Appellants have provided
no evidence that the use of Matrigel® would have been unpredictable.
Appellants argue that even “if the prior art could be combined as
suggested by the Examiner, and even if such combination of four or more
documents would teach the elements of the claims, which it does not, the
claimed invention would still be nonobvious due to the well-documented
high level of unpredictability in the art as of the filing date” (App. Br. 12-
13).
We are not persuaded. Appellants argument that there is a “well-
documented high level of unpredictability” requires evidentiary support.
Attorney argument is not evidence. In re Pearson, 494 F.2d 1399, 1405
(CCPA 1974). Nor can it take the place of evidence lacking in the record.
Meitzner v. Mindick, 549 F.2d 775, 782 (CCPA 1977). For example,
Appellants did not provide evidence that it would be unpredictable to apply
the method of Ure to human embryonic stem cells with the incorporation of
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trehalose which has “an exceptional ability to stabilize cells during freezing,
freeze-drying, and air-drying” (Wolkers 79, col. 1; FF 12).
Conclusion of Law
The evidence of record supports the Examiner’s conclusion that the
prior art rendered obvious the method of cryopreservation of human
embryonic cells of Claim 17.
C. 35 U.S.C. § 103(a) Rejection over Ure, Reubinoff, Funk, Wolkers and
Reid
Appellants argue that “Reid does not teach adding of a top layer of a
solid support matrix.” (App. Br. 16). Appellants argue that “it would not
have been obvious to the skilled artisan reading Reid to add a layer of matrix
over adherent human ESCs for cryopreservation because the Reid cells are
in suspension” (App. Br. 16). Appellants also argue that “Funk does not
teach growing cells to about 1000-10,000 cell colonies” (App. Br. 16).
The Examiner finds that “Reid et al. teach that cryopreservation of the
human liver stem cells embedded in extracellular matrix results in better cell
viability and function” (Ans. 29). The Examiner finds that the “skilled
Artisan would have known and would have been motivated to increase the
efficiency of the cryopreservation method . . . by further covering their
Matrigel-adherent cells with a layer of Matrigel (i.e., to embed the HES cell
in Matrigel)” (Ans. 29).
The Examiner also finds that “since Funk et al. teach that only
colonies comprising 10-2,000 cells are optimal for growth and propagation
. . . one of skill in the art would have known and been motivated to freeze
colonies of this size” (Ans. 30).
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We find that the Examiner’s position is supported by a preponderance
of the evidence. Reid teaches “optionally embedding the cells in forms of
extracellular matrix” (Reid 10 ¶ 0109). We agree with the Examiner that
Reid’s teaching to embed cells in the extracellular matrix reasonably
suggests coating Matrigel-adherent cells with a layer of Matrigel to the
ordinary artisan (see Ans. 29). We also agree with the Examiner that Funk’s
teaching of a particular size colony as optimal would suggest the use of these
sized colonies.
B., D., and E. 35 U.S.C. § 103(a) Rejections
Appellants do not separately argue the additional rejections under 35
U.S.C. § 103(a) which depend upon the primary rejection over Ure,
Reubinoff, Funk, and Wolkers. Instead, Appellants state with regard to each
of these rejections that for “the reasons discussed above, Ure, Reubinoff,
Funk, and Wolkers do not teach cryopreservation of adherent human ESCs
as recited by the claims” (see, e.g., App. Br. 14). The Examiner has found
specific evidence and reasoning to support the rejections (see Ans. 7-11).
Appellants have not provided substantial reasons to doubt the correctness of
the Examiner’s rejections. On appeal to this Board, Appellant must show
that the Examiner has not sustained the required burden. See Ex parte
Yamaguchi, 88 USPQ2d 1606, 1608 and 1614 (BPAI 2008) (precedential);
Ex parte Fu, 89 USPQ2d 1115, 1118 and 1123 (BPAI 2008) (precedential).
Therefore, we affirm the Examiner’s rejections.
SUMMARY
In summary, we affirm the rejections of:
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claims 17-20, 22, 24-27, 32, 33, 36, 38, 42, 43, and 45 under 35
U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, and Wolkers.
claims 17-22, 24-27, 32-34, 36, 38, 42, 43, and 45 under 35
U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, and Voigt.
claims 1, 2, 5-9, 12-14, 16-22, 24-33, 35-38, and 40-45 under
35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, and
Reid.
claims 1, 2, 5-10, 12-14, 16-22, 24-38, and 40-45 under 35
U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, Reid, and
Voigt.
claims 1, 2, 5-9, 12-22, 24-33, 35-38, and 40-45 under 35
U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, Reid, and
Limaye.

No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a)(1)(iv)(2006).

AFFIRMED

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