Ex Parte KleinsekDownload PDFBoard of Patent Appeals and InterferencesMay 6, 201011711921 (B.P.A.I. May. 6, 2010) Copy Citation UNITED STATES PATENT AND TRADEMARK OFFICE ____________________ BEFORE THE BOARD OF PATENT APPEALS AND INTERFERENCES ____________________ Ex parte DON A. KLEINSEK, Appellant1 ____________________ Appeal 2010-001935 Application 11/711,921 Technology Center 1600 ____________________ Decided: May 6, 2010 ____________________ Before CAROL A. SPIEGEL, FRANCISCO C. PRATS, and JEFFREY N. FREDMAN, Administrative Patent Judges. SPIEGEL, Administrative Patent Judge. DECISION ON APPEAL Appellant appeals under 35 U.S.C. § 134(a) from an Examiner's final rejection of all pending claims, claims 1-5 and 7-28. We have jurisdiction under 35 U.S.C. § 134. We REVERSE. 1 The real party in interest is GERIGENE, LLC (Corrected Brief for Appellant, filed 8 April 2009 ("Br."), 3). Appeal 2010-001935 Application 11/711,921 I. Statement of the Case The subject matter on appeal is directed to methods of treating a tissue defect in a patient, such as wrinkles, depressed scars, and lip hyperplasia, comprising introducing adipose (fat) cells at or near the site of the defect. The adipose cells may be obtained from the patient (autologous) or from another source of the same species (allogenic). Claims 1, 12, 16, 18, 19, 22, and 24 are illustrative (Br. 47-50, emphasis added): 1. A method of treating a tissue associated with a defect in a patient using autologous materials derived from the patient, the method comprising: introducing a plurality of autologous adipose cells to the tissue at, or proximal to, the defect of the patient after the plurality of autologous adipose cells have been cultured in vivo to expand the number of adipocyte cells in at least one medium that comprises autologous serum. 12. The method of claim 1 wherein the tissue comprises uscle tissue. m 16. A method of treating a tissue associated with a defect in a patient, the method comprising: introducing a plurality of isolated adipose cells to treat the defect of the patient after the lurality of adipose cells have been isolated in vitro. p 18. The method of claim 16 wherein the cultured [sic, isolated] adipose cells are expanded in vitro.[ ]2 19. The method of claim 16 wherein the cells are introduced into the patient in a mixture with a filler material, wherein the filler material, prior to mixing with the cells, comprises a member of the group consisting of collagen, fibronectin, fibrillin, laminin, elastin, glycosaminoglycan, exogenous extracellular matrix, whole extracellular matrix, and utologous extracellular matrix. a 2 Since claim 16 refers to "isolated" adipose cells and culturing is a known means for expanding a cell population in vitro, we interpret claim 18 as referring to "isolated" adipose cells. 2 Appeal 2010-001935 Application 11/711,921 22. The method of claim 16 wherein the adipose cells omprise undifferentiated mesenchymal cells. c 24. The method of claim 16 wherein the adipose cells are allogenic. The Examiner rejected claims 16-22 and 25-28 as unpatentable under 35 U.S.C. § 102(b) as anticipated by Billings3 or, in the alternative, under 35 U.S.C. § 103(a) as obvious over Billings in view of Oliver4 (Ans.5 5-7). The Examiner has also rejected claims 1-5, 7-15, 23, and 24 as unpatentable under 35 U.S.C. § 103(a) over the combined teachings of Billings, Smith,6 Gillis,7 Gruber,8 and Naughton9 (Ans. 7-11). The Examiner also relies on the following rebuttal references: Green,10 Nakajima,11 and Shigeno12 (Ans. 12, 17-19, 24, and 25). 3 Edmund Billings, Jr., and James W. May, Jr., Historical Review and Present Status of Free Fat Graft Autotransplantation in Plastic and Reconstructive Surgery, 83 PLASTIC AND RECONSTRUCTIVE SURGERY 368- 381 (1989), Br. Exh. E ("Billings"). 4 U.S. Patent 4,919,664, Stimulation of Hair Growth, issued 24 April 1990, to Oliver et al., Br. Exh. A (hereinafter "Oliver"). 5 Examiner's Answer mailed 27 July 2009 ("Ans."). 6 Ulf Smith, Morphologic Studies of Human Subcutaneous Adipose Tissue In Vitro, 169 ANATOMICAL RECORD 97-104 (1971), Br. Exh. P ("Ulf"). 7 U.S. Patent 5,199,942, Method for Improving Autologous Transplantation, issued 6 April 1993, to Steven Gillis, Br. Exh. B ("Gillis"). 8 Gruber et al., Study on In Vitro Cultivation of Human Chondrocytes Using Autologous Human Serum as a Medium Additive: Minimizing the Possible Risk of Infection with Prion Disease Pathogens, 75 LARYNGO-RHINO- OTOLOGY 105-108 (1996). This decision cites the English language translation by FLS, Inc. of record, Br. Exh. K ("Gruber"). 9 U.S. Patent 5,842,477, Method for Repairing Cartilage, issued 1 December 1998, to Naughton et al., Br. Exh. C ("Naughton"). 10 Howard Green and Olaniyi Kehinde, Formation of Normally Differentiated Subcutaneous Fat Pads by an Established Preadipose Cell Line, 101 JOURNAL OF CELLULAR PHYSIOLOGY 169-171 (1979) ("Green"). 3 Appeal 2010-001935 Application 11/711,921 A. Rejection of claims 16-22 and 25-28 As to claims 16-22 and 25-58, the Examiner found that Billings teaches the use of cultured adipose cells and preadipocytes (including undifferentiated mesenchymal cells) in human autologous transplantation to treat tissue defects, such as scars and wrinkles (Ans. 6). The Examiner found that cultured adipose cells inherently express an extracellular matrix (hereinafter "ECM") containing fibronectin, collagen, and hyaluronic acid and that some of these ECM materials would necessarily be introduced into a patient along with the cultured adipose cells (id.). Thus, the Examiner found the subject matter of claims 16-22 and 25-28 anticipated by Billings (Ans. 5 and 7). In the alternative, the Examiner concluded that the claimed method would have been an obvious matter of culture design choice since Oliver teaches removing cells from a culture while keeping the ECM intact and whole, thereby allowing flexibility in the manner of processing and administering the cellular composition (Ans. 7). Appellant argues that Billings does not teach the use of "isolated" adipose cells as required by claim 16, but rather that the use of preadipocytes in a single cell suspension (see e.g., claim 28) is a subject "ripe for investigation" (Br. 13-15). According to Appellant, Billings "teaches that 11 Nakajima et al., Adipose Tissue Extracellular Matrix: Newly Organized by Adipocytes during Differentiation, 63 DIFFERENTIATION 193-200 (1998) ("Nakajima"). 12 Yoichi Shigeno and Brian A. Ashton, Human Bone-Cell Proliferation In Vitro Decreases with Human Donor Age, 77-B THE JOURNAL OF BONE AND JOINT SURGERY 139-142 (1995) ("Shigeno"). 4 Appeal 2010-001935 Application 11/711,921 free fat transplantation is controversial, and unpredictable" (id. at 15, original emphasis), a position that is confirmed by Ersek13 (id. at 16). Appellant further argues that Billings does not teach in vitro expansion of adipose cells as required by claim 18 (Br. 17) or the "mixing" of isolated adipose cells with the filler material recited in claim 19 (id. at 18). Appellant contends that one of ordinary skill in the art understands an "undifferentiated mesenchymal cell" to refer to a cell at a stage where it can differentiate into fibroblasts, adipose tissue, cartilage, tendon, bone, or muscle cells, not to a pre-adipocyte per se and cites to Smith for support (Br. 19-20). Thus, Appellant argues that Billings does not teach using undifferentiated mesenchymal cells as required by claim 22 (id.). B. Rejection of claims 1-5, 7-15, 23, and 24 As to claims 1-5, 7-15, 23, and 24, the Examiner found that Billings differs from the claimed invention in failing to culture the adipose cells in the presence of autologous serum and in failing to use allogenic adipose cells (Ans. 9). The Examiner found that Smith, Gruber, and Gillis each teach culturing adipose tissue in the presence of human serum and that Gruber teaches that use of fetal calf serum in cell cultures of autologous cells present a potential risk of prion disease transmission (id. at 9-10). Therefore, the Examiner concluded that it would have been obvious to culture the human adipose cells of Billings in the presence of human (autologous) serum, rather than fetal calf serum, in order to eliminate the risk of transmission of prion disease to the human with a reasonable expectation of 13 Robert A. Ersek, Transplantation of Purified Autologous Fat: A 3-Year Follow-Up Is Disappointing, 87 PLASTIC AND RECONSTRUCTIVE SURGERY 219-227 (1991), Br. Exh. H ("Ersek"). 5 Appeal 2010-001935 Application 11/711,921 success based on the known use of human serum in adipose cell culture taught by Smith, Gruber, and Gillis (id. at 10). In addition, the Examiner found that Naughton teaches adding ECM proteins to cell preparations and using allogenic cells for transplantation (id. at 10-11). Thus, the Examiner concluded that "the combined teachings of Billings, Smith, Gillis, Gruber and Naughton render obvious the claimed invention" (id. at 11). Appellant argues that (i) adipocyte treatment of patients is an unpredictable art, (ii) Appellant proceeded against conventional wisdom and succeeded, (iii) successful use of autologous serum for culturing a particular cell type is unpredictable, (iv) the prior art has rejected the Examiner's proffered rationale of using autologous serum to control prion disease transmission, (v) none of Gruber, Gillis, Smith, and/or Naughton provide a reasonable expectation of success using autologous serum with autologous adipose cells because Gruber, Gillis, and Naughton are using different cell types and Smith does not use autologous serum, and (vi) assuming arguendo that Gruber's chondrocytes were predictive for adipocytes, Gruber teaches that the chondrocytes lose their phenotype and, therefore, their intended function (Br. 22-23). In essence, Appellant argues that there can be no prima facie case of obviousness because there is no reasonable expectation of success. Appellant relies on ten (10) references to support his position, i.e., an Associated Press Article,14 Freshney I,15 14 CNN.com, HEALTH Study: Stem Cell Lines Contaminated, downloaded 3 February 2005 from 6 Appeal 2010-001935 Application 11/711,921 Sogn,16 Oppenlander,17 Zhao,18 Koller,19 Choi,20 Brittberg,21 Shah,22 and Stute23 (Br. 24-33), as well as discussing the teachings of Gruber, Gillis, Naughton, and Smith (id. at 32-39). Appellant additionally relies on http://www.cnn.com/2005/HEALTH/01/23/stem_cells.ap/, Br. Exh. D ("AP Article"). 15 R. Ian Freshney, CULTURE OF ANIMAL CELLS: A Manual of Basic Technique, 3rd ed., 5, 100-101, and 253-255, Wiley-Liss, Inc., New York, NY (1999), Br. Exh. I ("Freshney I"). 16 John A. Sogn and Susan Jackson, Long Lived Nonadherent Rabbit Macrophages Obtained From Spleen Cell Cultures, 19 IN VITRO 90-98 (1983), Br. Exh. Q ("Sogn"). 17 Barbara K. Oppenlander and Douglas C. Dooley, Serum-Free Expansion of CD34NEG Cord Blood Cells Is Enhanced by Low Levels of Fetal Bovine Serum but Not Autologous Serum, 100 BLOOD Abstract No. 5249 (November 2002), Br. Exh. N ("Oppenlander"). 18 Zhao et al., Serum-free Culture of Dendritic Cells from Patients with Chronic Myeloid Leukemia In Vitro and Estimation of Their Cytotoxicity, 115 CHINESE MEDICAL JOURNAL 1296-1300, abstract only (2002), Br. Exh. S ("Zhao"). 19 Koller et al., Alternatives to Animal Sera for Human Bone Marrow Cell Expansion: Human Serum and Serum-Free Media, 7 JOURNAL OF HEMATOTHERAPY 413-423 (1998), Br. Exh. L ("Koller"). 20 Choi et al., The Effect of Serum on Monolayer Cell Culture of Mammalian Articular Chondrocytes, 7 CONNECTIVE TISSUE RESEARCH 105-112 (1980), Br. Exh. G ("Choi"). 21 Brittberg et al., Treatment of Deep Cartilage Defects in the Knee with Autologous Chondrocyte Transplantation, 331 THE NEW ENGLAND JOURNAL OF MEDICINE 889-895 (1994), Br. Exh. F ("Brittberg"). 22G. Shah, Why Do We Still Use Serum in the Production of Biopharmaceuticals? 99 DEVELOPMENTAL BIOLOGY STANDARDS 17-22 (1999), Br. Exh. O ("Shah"). 23 Stute et al., Autologous Serum for Isolation and Expansion of Human Mesenchymal Stem Cells for Clinical Use, 32 EXPERIMENTAL HEMATOLOGY 1212-1225 (2004), Br. Exh. R ("Stute"). 7 Appeal 2010-001935 Application 11/711,921 Malpeli24 and Freshney II25 to support his position that chondrocytes are unusual, atypical cells which are not suitable predictors for the behavior of other cell types (Br. 39-41). Appellant reiterates the patentability arguments made for claims 18, 19, and 22 vis-à -vis claims 2, 7, 8, and 15 (id. at 41-45). Finally, Appellant argues that none of Billings, Smith, Gillis, Gruber, or Naughton teach or suggest treating a defect in muscle tissue as required by claim 12 (Br. 44) or use of allogenic cells as required by claim 24 (id. at 45-46). Appellant has provided the same patentability arguments for (I) claims 1, 3-5, 9-11, 13, 14, and 23; (II) claims 2, 7, 8, 15, and 19; (III) claim 12; (IV) claims 16, 17, 20, 21, and 25-28; (V) claim 18; (VI) claim 22; and, (VII) claim 24. An argument which merely points out what a claim recites is not considered an argument for separate patentability of the claim.26 37 C.F.R. § 41.37(c)(1)(vii). Therefore, we decide this appeal on the basis of claims 1, 12, 16, 18, 19, 22, and 24. Id. Based upon the positions of Appellant and the Examiner, the dispositive issues before us are: 24 Malpeli et al., Serum-Free Growth Medium Sustains Commitment of Human Articular Chondrocyte through Maintenance of Sox9 Expression, 10 TISSUE ENGINEERING 145-155 (2004), Br. Exh. M ("Malpeli"). 25 R. Ian Freshney, CULTURE OF ANIMAL CELLS: A Manual of Basic Technique, 4th ed., 367-368, Wiley-Liss, Inc., New York, NY (2000), Br. Exh. J ("Freshney II"). 26 Appellant lists sixteen claim groupings (Br. 6-7). However, many of these separately grouped claims are not argued separately, e.g., "Group 17: Claim 28 (suspension)" (Br. 7) is argued as being patentable for the same reasons discussed with respect to claim 16, "bearing in mind that it differs in scope from claim 16 and has further reasons for patentability" (id. at 20). 8 Appeal 2010-001935 Application 11/711,921 Does the evidence of record establish that Billings is a nonenabling reference, i.e., does Billings describe the subject matter of claim 16 sufficiently to have placed it in the possession of a person of ordinary skill in the field of the invention? Does the evidence of record establish a reasonable expectation of success of treating tissue associated with a defect by introducing isolated adipose cells after the adipose cells have been isolated in vitro as recited in claim 16? Has the Examiner articulated a sound basis for culturing adipocytes in media supplemented with autologous serum in place of other animal serum as required by the method of claim 1? Does the evidence of record establish a reasonable expectation of successfully expanding autologous adipocytes in vitro in a culture medium supplemented with autologous serum and of successfully treating a tissue associated with a defect by introducing expanded autologous adipocytes at or near the tissue defect as required by claim 1? II. Findings of Fact The following findings of fact ("FF") are supported by a preponderance of the evidence of record. A. The 921 application [1] According to the 921 application ("Spec."), the practice of cosmetic and reconstructive surgery often uses various injectable materials to augment and/or repair defects of subcutaneous or dermal tissue to obtain an aesthetic result (Spec. 2:20-22). [2] Surgical correction of various defects have been accomplished by initial removal and subsequent re-implantation of excised adipose 9 Appeal 2010-001935 Application 11/711,921 tissue either by injection or by a larger scale surgical implantation (Spec 7:17-22). [3] However, large scale repairs, e.g., breast reconstruction, are said to be limited by the amount of adipose tissue which may be excised from the patient (Spec. 8:2-3). [4] According to the 921 application, in a preferred embodiment, the present invention utilizes the surgical engraftment of autologous adipocytes which have been cultured on a solid support typically derived from … collagen or isolated extracellular material. The culture may be established from a simple skin biopsy specimen and the amount of adipose tissue which can be subsequently cultured in vitro is not limited by the amount of adipose tissue initially excised from the patient. [Spec. 8:6-11.] [5] Typically, adipocytes are mechanically or enzymatically disaggregated (i.e., isolated) from adipose tissue derived from a biopsy specimen, seeded onto a collagen gel matrix or a cultured extracellular matrix, grown until near-confluence, gently scraped from the matrix surface, and then expanded using tissue culture techniques used for propagating undifferentiated mesenchymal cells to increase the number of adipose cells (Spec. 23:9-17; 18:1-2). [6] According to the 921 application, "[a]lthough adipocytes, prior to the storage of lipid, resemble fibroblasts, it is likely that they arise directly from undifferentiated mesenchymal tissue" (Spec. 15:21-24). B. Adipogenesis 10 Appeal 2010-001935 Application 11/711,921 [7] It is known in the art that adipogenesis is a sequential process, see e.g., Dani27 Figure 1 reproduced below. Figure 1 of Dani illustrates the process of adipogenesis. C. The applied prior art 1. Billings [8] Billings summarized the history of autologous free fat grafts (adipose transplants) as follows: Free fat graft autotransplantation for soft-tissue replacement has been a neglected subject in recent years. In a review of the literature, investigations of the various uses of free fat autotransplantation in animals and humans provide an understanding of the problems associated with the use of fat as a free graft. Results of free fat autotransplantation were found to be quite unpredictable, with wide variations in the resulting bulk of the graft. Microscopic studies of this behavior led to controversy as to whether the graft ultimately was made of surviving graft adipocytes (cell survival theory) or host adipocytes (host replacement theory). Studies revealed a "fibroblast-like" mesenchymal cell within adipose tissue that was believed to be an immature 27 C. Dani, Embryonic Stem Cell-Derived Adipogenesis, 165 CELLS TISSUES ORGANS 173-180 (1999) ("Dani") (copy enclosed). 11 Appeal 2010-001935 Application 11/711,921 adipocyte precursor or preadipocyte. Further characterization of the preadipocyte and its complete differentiation was accomplished using tissue-culture techniques. These investigations provide evidence of the dynamic nature of adipose tissue that strongly supports the cell survival theory and gives explanation to the unpredictable behavior of free fat autografts. Many conditions treated by plastic surgeons require soft-tissue augmentation. Autogenous [i.e., autologous] adipose tissue is the most appropriate and natural replacement material. With new culturing techniques, preadipocytes in a single cell suspension may provide an injectable soft-tissue replacement. This subject appears ripe for investigation. [Billings 376 SUMMARY, emphasis added.] [9] Billings concluded that [i]t is possible that preadipocytes grown in tissue culture could be a useful autotransplantable material in the treatment of a variety of soft-tissue defects. The preadipocyte is a "fibroblast-like" cell of mesenchymal origin that can be assumed to have greater resistance to trauma than mature adipose tissue laden with lipid. When reimplanted as an autogenous graft in pellet form or injected, the preadipocytes differentiate and mature to become normal-sized adipocytes and form a "fat pad" at the site. [Billings ¶ bridging 375-376, reference notes omitted.] [10] Billings further concluded that [a] single-cell suspension of preadipocytes from a patient grown in tissue culture might be used as a injectable autogenous transplant to treat soft-tissue deficiency in the same patient. This might provide an unlimited amount of soft-tissue replacement that could be tailored through multiple injections. It seems likely that further investigation into this area will prove fruitful. [Billings 376 ¶ 2, emphasis added.] 12 Appeal 2010-001935 Application 11/711,921 [11] Among the sites treated by free fat autografts noted by Billings is the temporalis muscle (Billings 368 ¶ 3). 2. Oliver [12] Oliver teaches a method for stimulating hair growth using cultured human scalp lower follicular dermal cells which are preferably removed from the culture system without using enzymes, thereby leaving the cell membranes and attached ECM in substantially non- degraded, relatively adhesive form (Oliver col. 2, ll. 35-47). 3. Smith [13] Smith teaches culturing a 3 mm x 3 mm x 1 mm piece of human adipose tissue (an explant) in Parker medium 199 with or without added human serum (Smith 98, col. 1, ¶¶ 3-4). [14] Outgrowths of fibroblast-like cells were noted in explants cultured in the presence of added serum, but not in explants cultured in the absence of serum (Smith 99, col. 2, ¶¶ 2-3). However, the serum fraction responsible for stimulating the growth of the fibroblast-like cells was not known (id. at 103, col. 2, ¶ 1). [15] According to Smith, samples of adipose tissue cultured for 30 weeks in Parker medium 199 supplemented with greater than 5% added serum maintained a morphology that compared well to that of freshly excised specimens (Smith abstract; 102, col. 1, ¶ 2). [16] Smith also teaches using collagenase to isolate adipose cells from adipose tissue in order to determine individual cell sizes (Smith 98, col. 2, ¶ 1). 4. Gillis 13 Appeal 2010-001935 Application 11/711,921 [17] Gillis teaches methods of improving hematopoietic progenitor blood cell transplants comprising (a) obtaining the progenitor cells from a patient, e.g., from bone marrow or peripheral blood, (b) expanding the progenitor cells ex vivo in the presence of selected growth factors, and (c) administering the expanded progenitor cells to the patient in conjunction with or following cytoreductive therapy (Gillis col. 3, ll. 12-28). [18] Preferred expansion media include minimal essential medium, Hanks medium, McCoys medium, and RPMI 1640 minimal essential medium, each supplemented with autologous serum and possibly antibiotics (Gillis col. 5, ll. 27-37). [19] Fetal bovine serum ("FBS") was substituted for autologous serum in experiments where the expanded cells were not readministered to their original donors (Gillis col. 8, ll. 11-15). 5. Gruber [20] According to Gruber, in vitro cultivation could become a significant source of autologous material increasingly needed for reconstructive surgery (Gruber 2, penultimate ¶). [21] Gruber teaches a method of cultivating cartilage tissue comprising isolating cells (chondrocytes) from donor tissue and amplifying them, first in a monolayer culture and then in a three-dimensional culture system (Gruber 2, last ¶). [22] Fetal calf serum ("FCS") was added to both the monolayer and three- dimensional cultures (Gruber 3, ¶ 1). 14 Appeal 2010-001935 Application 11/711,921 [23] Gruber teaches that FCS may be infected with prions which may transmit diseases, e.g., bovine spongiform encephalopathy, scrapie, and Creutzfeldt-Jakob disease (Gruber 3, ¶ 2). [24] According to Gruber, it was not only possible to substitute autologous human serum for the FCS in the culture system, but also about 80% of the cultures showed higher proliferation rates (Gruber 7, ¶ 2). [25] Gruber concluded that growing human chondrocytes in a monolayer culture using DMEM (Dulbecco's minimal essential medium) supplemented with 10% human serum was feasible and avoided the potential risk of infection with disease-transmitting prions (Gruber 10 ¶¶ 2-3). 6. Naughton [26] Naughton teaches methods of making and/or repairing cartilage in vivo comprising (a) implanting a biocompatible, nonliving three- dimensional scaffold structure, in combination with periosteal/perichondrial tissue, at the site of cartilage damage or loss, and (b) administering a preparation of chondrocytes and/or other stromal cells, e.g., chondrocyte progenitor cells, to the site of the implant (Naughton col. 1, ll. 5-15; col. 3, ll. 60-4:10). [27] Preferably, the periostial/perichondrial tissue and stromal cells are from the patient's own tissues or from cells of fetal origin ("universal donor") to minimize the risk of immunological rejection (Naughton col. 9, ll. 21-29; col. 10, ll. 62-67; col. 13, ll. 28-31). D. Appellant's rebuttal evidence28 28 The 921 application was filed February 28, 2007 and is a continuation of application 09/632,581, filed August 3, 2000, which is a continuation of 15 Appeal 2010-001935 Application 11/711,921 1. Ersek [28] According to Ersek, "[t]ransplantation of autologous fat is an old idea that has recently gained new interest" (Ersek 219, ¶ bridging cols. 1-2). [29] Ersek reports treating over a hundred patients by (a) obtaining autologous fat by blunt liposuction, (b) straining and rinsing the fat, and (c) injecting the "cleansed" fat into the patient (Ersek 219, col. 2, ¶ 2; 221, col. 1, ¶ 2 and col. 2, ¶ 2). [30] Ersek concluded that autologous fat transplantation "is very much an experimental technique, one that must be pursued with extreme caution" (Ersek 226, first full sentence). [31] According to Ersek, "[w]hile further efforts to define transplantation methods should be encouraged, my discouraging results with viable autologous fat transplant methods are leading me in other directions," i.e., implanting biocompatible, non-degradable inert substances (Ersek 226, last ¶). 2. Shah [32] According to Shah, growing cells in vitro requires culture conditions that mimic in vivo conditions with respect to temperature, pH, oxygen and carbon dioxide concentration, osmolarity, and nutrition (Shah 17, ¶ 1). application 09/003,378, filed January 6, 1998, which claims benefit under 35 U.S.C. § 119 of the February 20, 1997 filing date of provisional application 60/037,961. Accordingly, Ersek, Sogn, Choi, and Brittberg were published prior to the earliest possible filing date for the 921 application, while the AP Article, Freshney I and II, Oppenlander, Zhao, Koller, Shah, Stute, and Malpeli were published thereafter. 16 Appeal 2010-001935 Application 11/711,921 [33] Eukaryotic cell cultures are generally supplemented with animal serum, e.g., 10% FBS, horse serum, and even human serum (for some fastidious human cell lines), because serum provides unidentified, but essential factors needed for efficient cell growth (Shah abstract; 17, ¶ 2 – 18, ¶ 3). [34] Shah discusses known advantages and disadvantages of using serum, e.g., while it is an almost universal growth supplement effective with most types of cells, it also raises safety, reproducibility, and cost concerns (Shah 18, ¶¶ 1-2). [35] According to Shah, attempts to replace serum with serum free substitutes have been disappointing for a number of reasons, including the difficulty of finding non-animal derived replacement components, the cost of optimizing a serum-free medium for a cell line, and the need for formulations that are cell-line specific (Shah 20, last ¶; 22, ¶ 1). 3. Freshney I [36] According to Freshney I, there is evidence of cellular phenotypic and genotypic instability as a result of variations in culture conditions, selective overgrowth of constituents of the cell population, and genomic variation during evolution of a cell line from a primary culture and during subsequent maintenance as a cell line or purified cell strain (Freshney I 253, col. 1, ¶ 2). [37] As a result, a finite cell line should be grown to between the 5th and 10th population doubling to create sufficient bulk for freezing, while continuous cell lines should be cloned and the appropriate clone selected and grown up for freezing (Freshney I 254, col. 2, ¶ 2). 17 Appeal 2010-001935 Application 11/711,921 [38] Furthermore, in view of the expected variation between batches of serum, resulting from different methods of preparation, sterilization, storing, age, and animal stocks, it is important to select a batch of serum, use it for as long as possible, and replace it with one as similar as possible (Freshney I 255, col. 1, ¶ 3). [39] Alternatively, the best method of eliminating serum variation is to convert to a serum-free medium, which may be costly and time- consuming (Freshney I 255, col. 1, ¶ 4). 4. Choi [40] Choi reports that its findings reinforce the statement that optimal conditions for culturing mammalian chondrocytes must be determined for each species individually and underscore the importance of the particular sera employed in studies of the growth and phenotypic expression of cultured chondrocytes and in interpreting the findings based on them (Choi 111, last ¶). [41] Specifically, according to Choi, "there is much variation in the response of three species of cultured articular chondrocytes – rabbit, human, and dog – to a range of types and concentrations of sera" (Choi 105, ¶ 1). [42] For example, human chondrocyte cultures have been improved by using explants (small pieces of tissues) rather than dissociation methods (which provide isolated cells) for initiating cultures and by using human sera following priming or initial supplementation with FCS (Choi 111, ¶ bridging cols. 1-2). 5. Malpeli 18 Appeal 2010-001935 Application 11/711,921 [43] Malpeli also teaches that serum is a common supplement for in vitro cell cultures (Malpeli 152, col. 2, ¶ 1). [44] Malpeli reports that serum interferes with the maintenance of chondrogenic phenotype commitment and differentiation potential, likely through the loss of Sox9 expression (which specifies the chondrogenic lineage) (Malpeli abstract; ¶ bridging 152-153). [45] "More important[ly], serum is a 'nonphysiological' medium for chondrocytes" (Malpeli 153, col. 1, ¶ 2). [46] Malpeli discloses a serum-free growth medium that sustains commitment of human articular chondrocytes through maintenance of Sox9 expression (Malpeli abstract; 146, ¶ bridging cols. 1-2; 150, col. 2, ¶ 2 – 151, col. 2, ¶ 2; 153, col. 1, ¶ 2 – col. 2, ¶ 3). 6. Brittberg [47] Brittberg performed autologous chondrocyte transplants using cartilage that was arthroscopically obtained, minced, digested with collagenase, washed, resuspended in culture medium supplemented with 15% autologous serum, and transplanted (Brittberg abstract; 890 "Isolation and Culture of Chondrocytes"). [48] According to Brittberg, autologous chondrocytes and serum were used to minimize potential side effects and the likelihood of transmitting infectious disease (Brittberg 894, col. 1, ¶ 3). [49] Further according to Brittberg, the culturing procedure increased the number of initially isolated chondrocytes 10 to 20 times; and, a fraction of the culture cells were able to reexpress their phenotype after using procedures known to facilitate the production of a cartilage matrix (Brittberg 894, col. 1, ¶ 4). 19 Appeal 2010-001935 Application 11/711,921 7. Freshney II [50] Freshney II states that chondrocytes are "highly specialized cells of mesenchymal origin that are responsible for synthesis, maintenance, and degradation of the cartilage matrix" (Freshney II ¶ bridging 367- 368). [51] According to Freshney II, articular chondrocytes cultured in a monolayer divide rapidly, become fibroblastic, and lose their biochemical characteristics (Freshney II ¶ bridging 367-368). [52] Freshney II also exemplifies a growth medium supplemented with 10% FCS (Freshney II 368, col. 1, ¶ 4). 8. Stute [53] According to Stute, one of the major hurdles to clinical use of human mesenchymal stem cells is the biosafety of the FCS used for their culture (Stute abstract; 1213, col. 1, ¶ 2). [54] Stute investigated the feasibility of replacing FCS in media with autologous human serum based on the theory that autologous serum contains all of the growth factors and substances necessary for the isolation and expansion of human mesenchymal stem cells (Stute abstract; 1213, col. 1, ¶ 3). [55] Stute concluded that 10% autologous serum appeared as good as 10% FCS in terms of isolation and expansion, while 1% and 3% autologous serum appeared inferior (Stute abstract; 1223, col. 1, last ¶). [56] Stute further concluded that 10% autologous serum appeared superior to the other serum conditions with respect to osteogenic (bone growth) differentiation (Stute abstract; 1223, col. 1, last ¶). 9. the AP Article 20 Appeal 2010-001935 Application 11/711,921 [57] According to the AP article, "people have always been concerned about the possibility that something deleterious might be transferred from feeder cells to stem cells" (AP Article 1, last full sentence). [58] Researchers reported that human embryonic stem cells available for research are contaminated with nonhuman molecules from the culture medium used to grow the cells (AP Article title). [59] Specifically, the stem cells are contaminated by a sialic acid, Neu5Gc, which human cells are unable to make (AP Article 2, ¶¶ 5-6). [60] According to the researchers, growing stem cells in human serum selected for low amounts of anti-Neu5Gc antibodies reduced, but did not eliminate, the immune response (AP Article 2, ¶ 9). 10. Oppenlander [61] According to Oppenlander, the proliferation of peripheral blood CD45NEG cells, but not umbilical cord blood CD45NEG cells, in serum-free medium suggests that the cells have different requirements for survival and/or growth (Oppenlander abstract). [62] Oppenlander added various combinations of growth-modulating factors to serum-free medium to assess their ability to enhance proliferation (Oppenlander abstract). [63] In some cases, media were further supplemented with 10% autologous cord blood or FBS (Oppenlander abstract). [64] Oppenlander found that adding FBS enhanced the ability of cord blood CD34NEG cells to form colonies in methylcellulose and to generate uncommitted CD34POS cells in liquid culture for up to two weeks (Oppenlander abstract, last two sentences). 11. Koller 21 Appeal 2010-001935 Application 11/711,921 [65] According to Koller, human hematopoietic cell cultures are traditionally carried out in media supplemented with both 10% FBS and 10% horse serum (Koller 416, col. 1, last ¶). [66] Specifically, serum contains cofactors that potentiate the effect of growth factors (Koller 420, col. 2, ¶ 2). [67] Koller found that the horse serum could be eliminated from the medium without loss of performance if the FBS was increased to 20% (Koller 416, col. 2, ¶ 1). [68] Unfortunately, FBS may contain adventitious contaminants, e.g., infectious agents (Koller 421, col. 1, ¶ 2). [69] Koller also found that human plasma and serum, from both autologous and allogeneic sources, supported less than 50% of the culture output as compared with animal sera-containing media (Koller 421, col. 1, ¶ 3). [70] Although use of autologous human serum eliminates concerns regarding the transmission of known and unknown human infectious agents, "generation of autologous serum for cell expansion protocols is undesirable and has significant limitations from a manufacturing, regulatory, and quality assurance perspective, particularly as the scope of cell expansion therapy increases" (Koller 421, col. 1, ¶ 3). 12. Sogn [71] According to Sogn, rabbit splenocytes proliferate in media containing high levels (10%) of FBS, but not in media supplemented with either horse serum or autologous serum (Sogn abstract; 96, col. 1, ¶ 1). 13. Zhao 22 Appeal 2010-001935 Application 11/711,921 [72] According to Zhao, dendritic cells from patients with chronic myeloid leukemia expand better in serum-free medium containing the cytokines (1) stem cell factor, (2) granulocyte colony-stimulating factor, (3) tumor necrosis factor alpha, and (4) interleukin-4, than in serum-free medium containing only cytokines (2)-(4) (Zhao Methods & Results). [73] Results with serum-free medium were not significantly different from results of medium containing FCS, but results from medium containing autologous serum were inferior to medium containing FCS or serum-free medium containing cytokines (Zhao Results). E. Examiner's rebuttal evidence29 1. Green [74] According to Green, cells of an established mouse preadipocyte cell line, 3T3, differentiated into adipose cells with a rather low frequency when their growth in culture slowed or arrested (Green 169, ¶ 1). [75] Green selected from sublines that underwent differentiation into adipose cells with a much higher frequency (Green 169, ¶ 1). [76] Cells of one of these sublines, 3T3-F442A, were (a) cultured and harvested by trypsinization when they were nearly confluent or confluent, but before appreciable adipose conversion had begun, and (b) injected into mice in a suspension of medium, giving rise to typical fat pads (Green 169, ¶ 2). 2. Nakajima 29 Green and Shigeno were published prior to the earliest possible filing date for the 921 application, while Nakajima was published thereafter. 23 Appeal 2010-001935 Application 11/711,921 [77] According to Nakajima, an exponentially growing cloned preadipocyte cell line (BIP) derived from Japanese Black cattle produced type I, II, III, IV, V, and VI collagens, laminin, and fibronectin in an in vitro culture system (Nakajima abstract; 198, col. 1, ¶ 1). [78] The cell culture system contained Dulbecco's modified Eagle medium supplemented with 10% FBS (Nakajima 194, col. 1, ¶ 4). 3. Shigeno [79] Shigeno cultured marrow-free, washed bone fragments in media supplemented with either 10% autologous serum or 10% FCS (Shigeno 139, col. 2, ¶¶ 2-3). [80] According to Shigeno, [f]etal calf serum, a standard supplement for in vitro culture media, is rich in growth factors. It is therefore interesting that autologous serum supported greater cell division than did fetal calf serum and that the relative efficiencies of the two sera did not change with age. This suggests that changes in the systemic or local availability of growth factor are not the cause of age-related changes in bone-cell proliferation. [Shigeno 142, last ¶.] III. Discussion A. Legal principles 1. claim construction A claim is given its broadest reasonable construction consistent with the specification. In re Prater, 415 F.2d 1393, 1404-05 (CCPA 1969). 2. anticipation 24 Appeal 2010-001935 Application 11/711,921 Anticipation requires that each and every element as set forth in the claim is found, either expressly or inherently described, in a single prior art reference. In re Robertson, 169 F.3d 743, 745 (Fed. Cir. 1999). If a prior art reference does not expressly set forth a particular element of the claim, that reference still may anticipate if that element is "inherent" in the disclosure of the reference. Id. Inherency, however, may not be established by probabilities. Id., citing In re Oelrich, 666 F.2d 578, 581 (CCPA 1981). It is also well settled that "the reference must be enabling and describe the applicant's claimed invention sufficiently to have placed it in the possession of a person of ordinary skill in the field of the invention." In re Paulsen, 30 F.3d 1475, 1478-79 (Fed. Cir. 1994). 3. obviousness An invention is obvious if "the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious … to a person having ordinary skill in the art to which said subject matter pertains." 35 U.S.C. § 103. The factual inquiries underlying obviousness include (1) the scope and content of the prior art, (2) the differences between the prior art and the claims at issue, (3) the level of ordinary skill in the art at the time the invention was made, and (4) any objective evidence of non-obviousness. Graham v. John Deere Co., 383 U.S. 1, 17-18 (1966). A rejection for obviousness must include “articulated reasoning with some rational underpinning to support the legal conclusion.†KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 418 (2007), quoting In re Kahn, 441 F.3d 977, 988 (Fed. Cir. 2006). "For obviousness under § 103, all that is required is a reasonable expectation of success." In re O’Farrell, 853 F.2d 894, 903-04 25 Appeal 2010-001935 Application 11/711,921 (Fed. Cir. 1988). Expectation of success is assessed from the perspective of a person of ordinary skill in the art, at the time the invention was made. Life Techs., Inc. v. Clontech Labs., Inc., 224 F.3d 1320, 1326 (Fed. Cir. 2000). B. Claim construction We begin by construing the terms adipose cells, adipocyte cells, adipocytes, preadipocytes, isolated adipose cells, and mesenchymal cells. Mesenchymal cells are multipotent stem cells that can differentiate into a variety of cell types, first as precursor "blast" cells and ultimately as mature (terminal) cells, including cartilage cells (chondrocytes), fat cells (adipocytes), muscle cells (from myoblasts), and bone cells (from ostoblasts) (see e.g., FF 7). Adipoblasts are fibroblast-like cells for which no specific genetic markers have been identified so far (id.). Differentiation of an adipoblast into a preadipocyte (preadipose cell) is associated with genes such as A2Col6 and lipoprotein lipase LPL (id.). Differentiation continues from the preadipocyte to the terminally differentiated adipocyte (adipose cell or fat cell) and is associated with genes such as α-FABP, GPDH, and leptin (id.; see also Spec. 15:17). The adipocyte synthesizes and accumulates lipid (fat) (Spec. 16:7-11). An isolated adipocyte is an adipocyte which has been mechanically or enzymatically dissociated from adipose tissue as a single cell (see e.g., FF 5 and 16). Thus, a suspension of isolated adipocytes cannot be an excised piece of adipose tissue per se (explant). B. Analysis 1a. Rejection of claims 16-22 and 25-28 under § 102 over Billings 26 Appeal 2010-001935 Application 11/711,921 Appellant argues that Billings does not teach the use of isolated adipose cells as required by claim 16 (Br. 13-14). In particular, Appellant argues that Billings is not an enabling reference (id.). In response, the Examiner points to Billings' "use of a single-cell suspension of preadipocytes … as an injectable autologous transplant to treat soft-tissue deficiency … (page 376, first full paragraph)" (Ans. 12). According to the Examiner, "Billings provides references to specific culture studies … (page 374), thus demonstrating that all the necessary tools are available to those of skill in the art to carry out the suggested method" (id.). The Examiner relies on Green for support (id.). The method of claim 16 requires, in relevant part, introducing a plurality of isolated adipose cells into a patient to treat a tissue defect after the plurality of adipose cells have been isolated in vitro. Billings teaches that a single-cell suspension of preadipocytes might provide an injectable soft-tissue replacement, a subject ripe for investigation which will likely prove fruitful with further investigation (FF 8 and 10). The Examiner has not explained how the "tools" taught by Billings would have put one of ordinary skill in the art in possession of the method of claim 16. For example, the Examiner has not explained how (and which) culture conditions disclosed by Billings would have allowed isolated preadipocytes to differentiate into functional adipocytes (adipose cells) or how and when to isolate the adipocytes from the culture for use as claimed. "One shows that one is 'in possession' of the invention by describing the invention, with all its claimed limitations, not that which makes it obvious." Lockwood v. American Airlines Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997). 27 Appeal 2010-001935 Application 11/711,921 Further, Green is directed to use of a selected subclone of a cloned preadipocyte cell line (FF 74-76). Freshney I teaches that cellular phenotypic and genotypic instability occurs during evolution of a cell line from a primary culture and during subsequent maintenance as a cell or purified cell strain (FF 36). Again, the Examiner has not explained how Green relates to Billings, e.g., does Green use the same culture conditions disclosed by Billings and, if so, would Green's results reasonably have been expected to be comparable with those obtained from a primary culture. Absent a fully articulated explanation, we must reverse the rejection of claim 16, as well as claims 17-22 and 25-28 dependent thereon, under § 102(b) as anticipated by Billings because the evidence of record fails to establish that Billings is an enabling reference. 1b. Rejection of claims 16-22 and 25-28 under § 103 over Billings in view of Oliver Alternatively, the Examiner concluded that the claimed method would have been "an obvious design choice based on the fact that there are methods known in the art of cell therapy to remove cells from a culture while keeping the extracellular matrix intact and whole as described by Oliver et al (column 2 lines 35-47) thus allowing flexibility in the manner of processing and administering the cellular composition" (Ans. 7). Appellant argues that the combined teachings of Billings and Oliver "do not amount to the required evidence of a reasonable expectation of success" (Br. 15). Appellant relies on Billings and Ersek as evidence of unpredictability (Br. 15-17). 28 Appeal 2010-001935 Application 11/711,921 In response, the Examiner offers Green as a rebuttal reference to support her position "that the art of transplanting suspensions of adipocytes is not as unpredictable as Appellant asserts" (Ans. 12). Here, Billings expressly teaches that free fat graft autotransplantation for soft-tissue replacement is problematic and "results … were found to be quite unpredictable" (FF 8). Although Billings suggests that new culturing techniques might allow single cell suspensions of preadipocytes to be used as an injectable autologous transplant to treat soft-tissue defects (FF 8 and 10), Billings did not provide insight on what such new culture techniques might entail. Oliver does not provide any further insight because Oliver involves culturing hair cells (FF 12) and the evidence of record establishes that growing cells in vitro requires culture conditions that mimic in vivo conditions for those particular cells (see e.g., FF 32, 40, and 61). Rather than explaining the relevance of Oliver, the Examiner directed our attention to Green (Ans. 12). However, while Green is directed to use of a selected subclone of a cloned murine preadipocyte cell line (FF 74-76), Green is problematic for at least the reasons discussed above regarding enablement for anticipation. Furthermore, since the rejection is based on the combined teachings of Billings and Oliver, the Examiner cannot now rely on Green to overcome the deficiencies of Billings and/or Oliver. Turning to Appellant's rebuttal evidence, Ersek mechanically isolated at least some adipocytes and injected them into a patient (FF 29) and essentially found the method ineffective (FF 31). Thus, Ersek's characterization of and experience with autologous fat transplantation as 29 Appeal 2010-001935 Application 11/711,921 "very much an experimental technique" is consistent with Billings’ findings of unpredictability (FF 30). A conclusion of obviousness which is not supported by sufficient factual findings and/or clearly articulated reasoning cannot be sustained. Therefore, we reverse the rejection of claim 16, and its dependent claims 17- 22 and 25-28, under § 103 over the combined teachings of Billings and Oliver because the evidence of record fails to establish a reasonable expectation of success. For completeness, we note the following: As to claim 18, for the reasons given above, the Examiner has similarly failed to explain how Billings' invitation to further experimentation and/or variety of disclosed culture techniques on page 374 would have provided one of ordinary skill in the art with a reasonable expectation of success. As to claim 19, its plain language requires introducing the isolated adipose cells in essentially a carrier matrix or "filler material" of extracellular matrix. To the extent that Oliver teaches removing cells from a culture system without disassociating them, i.e., using enzymes (FF 12), Oliver teaches away from using "isolated" cells as required by claims 16 and 19. Further, the Examiner reasons that since adipocytes require a surface to attach to during their culture whereupon they deposit ECM proteins and Billings does not teach removing the matrix, it is reasonable to conclude that ECM proteins would be in the suspensions collected from cultures and used for injection (Ans. 14). However, the Examiner appears to be reading limitations from the Specification into the claims (see e.g., FF 5). The adipocytes could just as easily be grown on feeder cells (see e.g., Spec. 23:9) 30 Appeal 2010-001935 Application 11/711,921 and the evidence of record suggests that cultured cells are often washed and resuspended in clean culture medium before injection (see e.g., FF 47 and 76; accord FF 29 and 57). As to claim 22, we agree with Appellant that a preadipocyte is not an undifferentiated mesenchymal cell (see claim construction supra and FF 7). To summarize, we reverse the rejection of claims 16-22 and 25-28 under § 103 over the combined teachings of Billings and Oliver. 2. Rejection of claims 1-5, 7-15, 23, and 24 under § 103 over the combined teachings of Billings, Smith, Gillis, Gruber, and Naughton The method of claim 1 comprises treating a tissue defect by obtaining adipocytes, expanding the number of adipocytes in vitro in at least one culture medium containing autologous serum, and then introducing the expanded adipocytes into the tissue at or near the defect. The Examiner found that Billings teaches the subject matter of claim 1, but for teaching culturing the autologous adipocytes in medium containing autologous serum (Ans. 9). The Examiner found that Smith, Gruber, and Gillis each teach culturing adipose tissue in the presence of human serum and that Gruber teaches that supplementing cell cultures of autologous cells with FCS runs the risk of prion disease transmission (id. at 9-10). Therefore, the Examiner concluded that it would have been obvious to culture human adipocytes in the presence of autologous human serum, instead of FCS, to eliminate the risk of transmission of prion disease to the human with a reasonable expectation of success based on the known use of human serum in adipose cell culture taught by Smith, Gruber, and Gillis (id. at 10). The Examiner also found that Billings failed to disclose use of allogenic (not autologous) adipose cells as required by claim 24 (Ans. 9). 31 Appeal 2010-001935 Application 11/711,921 The Examiner found that Naughton teaches adding ECM proteins to cell preparations and using allogenic cells for transplantation (id. at 10-11). The Examiner concluded that the combined teachings of Billings, Smith, Gillis, Gruber and Naughton rendered the claimed invention obvious (id. at 11). We find the Examiner's articulated reason for culturing adipocytes in media supplemented with autologous serum in place of other animal serum to have a sufficient factual basis and Appellant's argument to the contrary unpersuasive. Appellant's own rebuttal evidence teaches or suggests using autologous serum for the same reason asserted by the Examiner. To wit, Brittberg expressly teaches using autologous chondrocytes and serum to minimize potential side effects and the likelihood of transmitting infectious disease (FF 48) and Koller teaches that use of autologous human serum eliminates concerns about transmitting infection (FF 70). As to Appellant's argument that the applied prior art fails to teach or suggest treating a defect in muscle tissue (Br. 44), we agree with the Examiner (Ans. 26) that Billings teaches treating defects in muscle tissue, e.g., in the temporalis muscle (FF 11). Therefore, this argument is not persuasive of patentability because it is factually inaccurate. We also note that both claims 23 and 24 depend from independent claim 16, not from independent claim 1 -- a fact not overlooked by the Examiner (Ans. 8). A dependent claim includes all of the limitations of the independent claim from which it depends. By failing to address independent claim 16 in this rejection, the Examiner has failed to address all of the limitations of dependent claims 23 and 24. Obviousness requires a suggestion of all limitations in a claim. In re Royka, 490 F.2d 981, 985 (CCPA 1974). Moreover, claim 16 has not been shown to be unpatentable 32 Appeal 2010-001935 Application 11/711,921 over Billings for the reasons given above. Therefore, we summarily reverse the rejections of claims 23 and 24 under § 103 over Billings, Smith, Gillis, Gruber, and/or Naughton. Thus, the dispositive issue before us is whether a reasonable expectation of success exists in the Examiner's prima facie conclusion of obviousness, i.e., whether one of ordinary skill in the art would have had a reasonable expectation of successfully expanding autologous adipocytes in vitro in a culture medium supplemented with autologous serum and of successfully treating a tissue associated with a defect by introducing expanded autologous adipocytes at or near the tissue defect. According to Appellant, no reasonable expectation of success exists. Appellant relies on Billings, Ersek, the AP Article, Freshney I and II, Sogn, Oppenlander, Zhao, Koller, Choi, Brittberg, Shah, and Stute to support its position, as well as arguing asserted defects of the applied prior art (Br. 21-41). We find Appellant's arguments persuasive. First, as to expanding autologous adipocytes in culture media containing autologous serum, the evidence of record establishes that growing cells in vitro requires culture conditions that mimic in vivo conditions for those particular cells (see e.g., FF 32, 40, and 61). The evidence also establishes that culture media are typically supplemented with serum, such as 10% FCS or 10% FBS, to provide unidentified, but essential factors needed for cell growth (see e.g., FF 33, 34, and 43). However, serum may also be "nonphysiological" for certain cell types and may contain factors which interfere with the cell's phenotype and differentiation (see e.g., FF 44-45). In some cases, only a fraction of cultured cells are able to reexpress their phenotype and then only after taking additional corrective 33 Appeal 2010-001935 Application 11/711,921 steps (see e.g., FF 49 and 42). In other cases, autologous serum supplementation may be more effective in culturing some cell types (see e.g., FF 55-56 and 69). For example, Sogn disclosed that rabbit splenocytes proliferate in media containing 10% FCS but not in media supplemented with either horse serum or autologous serum (FF 71). Thus, based on the evidence of record, one of ordinary skill in the art would reasonably have concluded that not all cell types and serum-supplemented media are mutually interchangeable for expanding cells. Hence, the successful expansion of cells in culture supplemented with autologous serum is evidently both cell type and serum species specific. Consequently, we agree with Appellant that the teachings of Gillis (hematopoietic progenitor cells (FF 17)) and Gruber (chondrocytes (FF 21)) have not been shown to be predictive of the behavior of adipocytes expanded under various culture conditions. The Examiner points out that Smith successfully cultured adipocytes in media containing human serum (Ans. 17). The Examiner also maintains that "Naughton clearly indicates that connective tissue cells such as chondrocytes and adipocytes [from stromal cells] are seeded together onto a scaffold for implantation … [implying] that the two cell types are capable of growth under the same conditions" (Ans. 21). However, as pointed out by Appellant (Br. 23), Smith does not use autologous human serum and, analogous to use of other animal serum (see e.g., FF 38), one of ordinary skill in the art would have had a reasonable basis for expecting variation between batches of human serum, e.g., concentration and/or type of unidentified growth factors found therein. Furthermore, it is unclear what effect such variation would have had on the 34 Appeal 2010-001935 Application 11/711,921 claimed method. Moreover, the Examiner did not substantively challenge Appellant's contention that "Smith … did not report any increase in adipose cell number" (Br. 37). At best, the Examiner responded that "Billings … suggest[s] that … Smith is predictive of success as far as expanding adipocytes" (Ans. 20) without explaining why or how. Thus, Smith is of little, if any, probative value. Regarding Naughton, Appellant argues that co-culture conditions are not predictive of other culture conditions because one cell type or feeder cells may provide important factors vital to the culture of the other cell type (Br. 38-39). Notably, Naughton discloses culturing in the presence of, preferably autologous, periosteal/perichondrial tissue (FF 26-27). The Examiner did not substantively respond to Appellant's argument, e.g., by explaining whether autologous periosteal/perichondrial tissue and autologous serum would have reasonably been expected to provide the same growth factors. Therefore, we accord Naughton little, if any, probative value. As to a reasonable expectation of successfully treating tissue defects with autologous adipocyte transplants, on balance the evidence of record also weighs in favor of unpredictability. Billings expressly characterizes free fat autotransplantation as "quite unpredictable" (FF 8) and invites further experimentation in the area (FF 8 and 10). Ersek characterizes autologous fat transplantation "very much an experimental technique" (FF 30) and has chosen to pursue "other directions" (FF 31). Green uses specially selected sublines of an established mouse preadipocyte line for transplantation (FF 74-76) and Freshney I reasonably suggests that cloning and maintaining cell lines produce genomic variation from the parent (primary) culture (FF 36). 35 Appeal 2010-001935 Application 11/711,921 Therefore, based on the foregoing, we reverse the rejection of claims 1-5, 7-15, 23, and 24 under § 103 over the combined teachings of Billings, Smith, Gillis, Gruber, and Naughton. C. Conclusion We reverse the rejection of claims 16-22 and 25-28 under § 102(b) as anticipated by Billings. On balance, the evidence of record establishes that Billings is a non-enabling reference because Billings does not describe the subject matter of claim 16 sufficiently to have placed it in the possession of a person of ordinary skill in the field of the invention. We reverse the rejection of claims 16-22 and 25-28, under § 103 over the combined teachings of Billings and Oliver. On balance, the evidence of record, while establishing a reason to combine the teachings of the references, also establishes that fat cell transplantation of is an unpredictable technique which lacks a reasonable expectation of success. In particular, the evidence of record fails to establish a reasonable expectation of success of treating tissue associated with a defect by introducing isolated adipose cells after the adipose cells have been isolated in vitro. Finally, we reverse the rejection of claims 1-5, 7-15, 23, and 24 under § 103 over the combined teachings of Billings, Smith, Gillis, Gruber, and Naughton. The Examiner has articulated a sound basis for culturing adipocytes in media supplemented with autologous serum in place of other animal serum as required by the method of claim 1. However, the evidence of record fails to establish a reasonable expectation of successfully expanding autologous adipocytes in vitro in a culture medium supplemented with autologous serum and of successfully treating a tissue associated with a 36 Appeal 2010-001935 Application 11/711,921 defect by introducing expanded autologous adipocytes at or near the tissue defect as required by claim 1. IV. Order Upon consideration of the record, and for the reasons given, it is ORDERED that the decision of the Examiner to reject claims 16-22 and 25-28 as unpatentable under 35 U.S.C. § 102(b) as anticipated by Billings or, in the alternative, under 35 U.S.C. § 103(a) as obvious over Billings in view of Oliver is REVERSED; and, FURTHER ORDERED that the decision of the Examiner to reject claims 1-5, 7-15, 23, and 24 as unpatentable under 35 U.S.C. § 103(a) over the combined teachings of Billings, Smith, Gillis, Gruber, and Naughton is REVERSED. REVERSED enc: C. Dani, Embryonic Stem Cell-Derived Adipogenesis, 165 CELLS TISSUES ORGANS 173-180 (1999) cdc DON A. KLEINSEK, PH.D. 5582 BANTRY LANE FITCHBURG, WI 53711 37 Application/Control No. 11/711,921 Applicant(s)/Patent Under Reexamination Kleinsek, Don A. Notice of References Cited Examiner Laura Schuberg Art Unit 1600 Page 1 of 1 U.S. PATENT DOCUMENTS DOCUMENT SOURCE ** * DOCUMENT NO. DATE NAME CLASS SUBCLASS APS OTHER A B C D E F G H I J K L M FOREIGN PATENT DOCUMENTS DOCUMENT SOURCE ** * DOCUMENT NO. DATE COUNTRY NAME CLASS SUBCLASS APS OTHER N O P Q R S T NON-PATENT DOCUMENTS DOCUMENT SOURCE ** * DOCUMENT (Including Author, Title Date, Source, and Pertinent Pages) APS OTHER U Christian Dani, Embryonic Stem Cell-Derived Adipogenesis, 165 Cells Tissues Organs 173-180 (1999). V W X *A copy of this reference is not being furnished with this Office action. (See Manual of Patent Examining Procedure, Section 707.05(a).) **APS encompasses any electronic search i.e. text, image, and Commercial Databases. U.S. Patent and Trademark Office PTO-892 (Rev. 03-98) Notice of References Cited Part of Paper No. 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Embryonic Stem Cell-Derived Adipogenesis Christian Dani Cells Tissues Organs; 1999; 165, 3-4; ProQuest Health and Medical Complete pg. 173 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. 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