DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of Application/Amendments/Claims
Applicant’s response filed on 6/26/2026 has been considered. Claim 1 has been amended. Claims 1, 7 and 9 are pending and examined. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
Applicant’s claim for the benefit of a prior-filed application KR10-2015-0190629 and PCT/KR2016/008003 filed on 12/31/2015 and 7/22/2016, respectively, under 35 U.S.C 119(e) or under 35 U.S.C 120, 121 or 365(c) is acknowledged.
Accordingly, the effective priority date of the instant application is granted as 12/31/2015.
Withdrawn Rejections
The 35 U.S.C. 103 rejection of claims 1, 7 and 9 have been withdrawn and reapplied in modified form to address applicants claim amendments which further describe the limbal tissue-derived epithelial cell sheet as comprising JC-1 low stem cells and ABCG2 positive as well as modifications to the method step (a).
Reply to Applicants Arguments
Although the rejections are newly applied, some of applicant’s arguments are relevant and are addressed below.
Applicant points to newly amended claim 1 and argues that the cited prior art does not describe tightly affixing the epithelial cell-removed amniotic membrane onto the slide with an epithelial sheet size of 250 to 300 mm. Applicant points to additional amendments describing the epithelial cell sheet comprising at least 30% limbal stem cells, which are defined as JC-1 low population and are positive for ABCG2. Applicant argues that a person skilled in the art would recognize that Mahadeorao teaches away from the claimed method, pointing to para 10 and 15 for support showing that the method of culturing limbal biopsies on amniotic membranes fails to achieve satisfactory ocular surface repair.
These arguments have been fully considered, but are not found persuasive since teaching away requires the prior art to criticize, discredit, or otherwise discourage the claimed solution, see MPEP § 2141.02(VI): “the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed.” Since the prior art clearly does not do this, this argument is unconvincing since Mahadeorao describes scraping off the epithelial layer of the amniotic membrane using blunt forceps and placing the epithelial cell-removed membrane on a sterile glass slide in a 100mm petri plate (Mahadeorao, para 78). Mahadeorao describes fastening the epithelial cell-removed amniotic membrane to a PET inset using medical grade silicon O-rings so that the membrane is spread uniformly and the denuded epithelial side of the membrane faces up (Mahadeorao, para 79). Mahadeorao describes seeding USCs on the epithelial cell-removed amniotic membrane using a culture medium containing DMEM/F12 (1:1) supplemented with 10% human serum (Mahadeorao, para 81).
Applicant points to MPEP 2143.02 arguing that a prior art reference must be considered in its entirety, including portions that would lead away from the claimed invention. Applicant points to amendments to claim 1 which describe a culture medium containing DMEM/F12(1:1), wherein the medium does not contain cholera toxin and fetal bovine serum.
Applicant further argues that Francis indicates a major limitation in that the proportion of stem cells is very low in amniotic membranes and would find it difficult to use for transplantation therapy, pointing to pg 343 for support. Applicant argues that a person skilled in the art would recognize that a cell sheet produced from limbal tissue simply cultured on an amniotic membrane without stem cell isolation would contain a low proportion of stem cells and would be unstable and not suitable for transplantation therapy. Applicant further points to the technical limitations, including the size of the cell sheet and proportion of stem cells. Applicant points to the specification for support and references Fig 2A, 3A-3C and 5 which shows various culturing conditions and the resulting structural effects on the limbal tissue-derived epithelial cell sheet. Applicant argues that the method of Mahadeorao cannot produce a sheet of the same size claimed and cannot be used for direct transplantation. Applicant further argues that Mahadeorao does not teach the claimed method describing tightly fixed amniotic membrane on a slide glass of a certain size. Applicant submits that Mahadeorao only discloses a step to fix the amniotic membrane using a PET insert, which is different from the present invention that isolates single cells from limbal tissue rather than using the limbal tissue itself. Applicant submits that fixing the amniotic membrane using a PET ring is not the same as fixing the amniotic membrane using the slide class of the present invention. Applicant states that the amniotic membrane collapses when cultured for a long period of time in the case of Mahadeorao. Applicant references Fig 3C of the specification which compares fixing the amniotic membrane using a PET ring and glass slide which shows an increased proportion of stem cells. Applicant argues that these are unexpected results arising from the increased tension of the amniotic membrane fixed on the glass slide which results in an increased ratio of limbal stem cells, leading to an increased ration of p63 positive limbal stem cells. Applicant references example 5 from the specification for support.
These arguments have been fully considered, but are not found persuasive. It is argued that these limitations are taught by the prior art since Mahadeorao describes fastening the epithelial cell-removed amniotic membrane to a PET inset using medical grade silicon O-rings so that the membrane is spread uniformly (Mahadeorao, para 79). Mahadeorao presents immunocytochemical data showing p63 expression among the cultured limbal stem cells (Mahadeorao, para 16, 22, 34). Thus, it is argued that Mahadeorao’s description of fastening the epithelial cell-removed amniotic membrane to a PET inset using medical grade silicon O-rings so that the membrane is spread uniformly is functionally equivalent to the claimed method for affixing (Mahadeorao, para 79).
Claim Objections
Claim 1 is objected to because of the following informalities: the claim contains the acronyms JC-1 and ABCG2. While acronyms are permissible shorthand in the claims, the first use should include the full recitation followed by the acronym in parentheses.
Claim Interpretation
Claim 1 describes a method for culturing limbal stem cells and further describes placing limbal tissue on the slide glass scaffold. Taking the broadest reasonable interpretation in light of the specification, the limbal tissue contains limbal stem cells which are then cultured further within the limbal tissue-derived epithelial cell sheet.
New Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1 and 7 are newly rejected under 35 U.S.C. 103 as being unpatentable over Mahadeorao et al. US 2005/0186672, published 8/25/2005 (hereinafter Mahadeorao, reference of record) as evidenced by TGP Catalogue, 2015 (hereinafter TGP, reference of record) in view of Francis et al. "Myogel supports the ex-vivo amplification of corneal epithelial cells." Experimental eye research 88.3 (2009): 339-346 (hereinafter Francis, reference of record) and Shaharuddin et al. "Limbal side population cells: a future treatment for limbal stem cell deficiency." Regenerative Medicine 8.3 (2013): 319-331 (hereinafter Shaharuddin). This is a new rejection necessitated by amendment of the claims in the responses filed 6/26/2026.
Claim 1: Mahadeorao describes culturing undifferentiated stem cells (USC) derived from corneal limbus tissue on epithelial cell-removed amniotic membranes (Mahadeorao, para 16-21, 24-25 and 41). Mahadeorao provides a description of USCs in para 41 and categorizes USCs as a species of limbal stem cells (Mahadeorao, para 41 and claim 5). Mahadeorao confirms the presence of stem cell-specific markers such as p63α using immunofluorescence and immunoperoxidase assays (Mahadeorao, para 62 and 90). Mahadeorao describes amniotic membrane preparation in para 77-78. Mahadeorao describes cutting a 5cm x 5cm section of amniotic membrane (Mahadeorao, para 77). Mahadeorao describes scraping off the epithelial layer of the amniotic membrane using blunt forceps and placing the epithelial cell-removed membrane on a sterile glass slide in a 100mm petri plate (Mahadeorao, para 78). Mahadeorao describes fastening the epithelial cell-removed amniotic membrane to a PET inset using medical grade silicon O-rings so that the membrane is spread uniformly and the denuded epithelial side of the membrane faces up (Mahadeorao, para 79). The fastening of the epithelial cell-removed amniotic membrane to a PET inset using medical grade silicon O-rings will result in no empty space between them and ensures the amniotic membrane is neither wrinkled nor torn (Mahadeorao, para 79). Applicants claim amendments describe tightly affixing the epithelial cell-removed amniotic membrane on a “slide glass” by covering the slide glass with the amniotic membrane, which is considered an equivalent structure to the one generated by Mahadeorao.
Mahadeorao describes seeding USCs on the epithelial cell-removed amniotic membrane using a culture medium containing DMEM/F12 (1:1) supplemented with 10% human serum (Mahadeorao, para 81). Mahadeorao lists other media factors including epithelial cell growth factor (EGF), DMSO, insulin, sodium selenite, human transferrin (Mahadeorao, para 57, 81 and example 1). The culturing media described in para 81 excludes the use of cholera toxin and fetal bovine serum, corresponding to the exclusionary provision of claim 1 e). Mahadeorao describes culturing the limbal tissue-derived epithelial cell sheet to 95% confluency, which reads on growing to 95% of the area of the slide glass scaffold as described in claim 1 d) (Mahadeorao, para 51). Mahadeorao describes transplanting the limbal tissue-derived epithelial cell sheet into patients (Mahadeorao, para 65, 81, and claims 9, 18).
Mahadeorao does not disclose the same dimensions of the limbal tissue, amniotic membrane, glass slide and limbal tissue-derived epithelial cell sheet as described in newly amended claim 1. Mahadeorao does not describe the inclusion of O-phosphoethanolamine into the culture medium. Mahadeorao describes the use of DMEM/F12 media supplemented with 10% human serum rather than 4-6% as described in claim 1. Mahadeorao does not mention that the limbal tissue-derived epithelial sheet comprises at least 30% limbal stem cells as a JC-1 low population which is characterized by a positive expression of ABCG2.
Claim 7: Mahadeorao describes culturing times ranging from 10-15 days at 37oC until confluent, with specific embodiments reaching 95% confluent (Mahadeorao, para 51).
Mahadeorao does not mention that the limbal tissue-derived epithelial sheet comprises at least 30% limbal stem cells as a JC-1 low population which is characterized by a positive expression of ABCG2 as described in newly amended claim 1.
Claim 1: Shaharuddin characterizes limbal stem cell side populations and shows that ABCG2 transport proteins are highly expressed and can be identified using JC1, which binds to the mitochondria instead of the nuclei and carries lesser cytotoxicity (Shaharuddin, pg 324 col 1). Shaharuddin shows that these cells are present in both isolated and cultivated limbal epithelial sheets using flow cytometry and other techniques.
It would have been prima facie obvious to one of ordinary skill in the art to generate limbal tissue-derived epithelial sheets that comprises at least 30% limbal stem cells as a JC-1 low population and are ABCG2 positive since Shaharuddin shows that these are inherent features to a cultivated limbal epithelial sheet. Inherent features need not be recognized at the time of the invention. There is no requirement that a person of ordinary skill in the art would have recognized the inherent disclosure at the time of invention, but that the subject matter is in fact inherent in the prior art reference, see MPEP 2112.
Claim 1: Similar to Mahadeorao, Francis describes an ex vivo culturing method for limbal stem cell expansion using de-epithelialized human amniotic membrane explants (Francis, abstract and intro para 2). Francis describes preparing a 50 mm amniotic membrane disk by thawing on a glass microscope slide and removing the amniotic membrane epithelium using 2 mL of trypsin for 20 min combined with mechanical scraping. Francis then cut a 15 x 15 mm section of the denuded amniotic membrane to serve as an explant culture substrate for the limbal tissue (Francis, 2.1 Cell carrier preparation). Francis describes culturing the limbal tissue containing limbal stem cells on the denuded amniotic membranes using a corneal media comprising 1:1 DMEM/F12 and 15% human serum supplemented with epidermal growth factor and insulin transferrin and selenium supplement (Francis, 2.2 Explant culture technique). The culturing media described in section 2.2 excludes the use of cholera toxin and fetal bovine serum, corresponding to the limitations described in claim 1. Francis found that culturing on de-epithelialized human amniotic membranes provided the most reliable medium for confluent growth when compared to other supports such as matrigel, myogel and stromagel (Francis, 3. Results para 2 and Fig 2). Francis describes methods for transplanting the ex vivo cultured limbal stem cells into patients (Francis, Discussion second to last paragraph). Francis describes the use of 15% human serum supplemented medium rather than 4-6% as described in claim 1. Francis does not describe the inclusion of O-phosphoethanolamine into the culture medium. Francis does not disclose the same dimensions of the limbal tissue, amniotic membrane, glass slide and limbal tissue-derived epithelial cell sheet as described in newly amended claim 1.
Claim 7: Francis describes monitoring cellular growth over a 15-day period and/or until confluence was reached (i.e. 100% of the area of the amniotic membrane explant was covered with limbal tissue) (Francis, 2.3 Growth rate and Fig 1, Fig 2).
It would have been prima facie obvious to one of ordinary skill in the art to use an amniotic membrane with dimensions 28-35 mm, slide glass with dimensions 18-28 mm, limbal tissue with dimensions 2x3 mm and limbal tissue-derived epithelial cell sheet with dimensions 2550 to 300 mm2 as described in claim 1 given the disclosures of Mahadeorao and Francis. It would have been a matter of combining prior art elements according to known methods to yield predictable results since both Mahadeorao and Francis disclose similar dimensions for each of the limbal tissue, amniotic membrane, glass slide and limbal tissue-derived epithelial cell sheet component and there exists motivation to experiment with different dimensions. Furthermore, a product catalogue from Technical Glass products (TGP) is provided as further evidence showing polished plates, disks and microscope slides with dimensions corresponding to the limitations described in claims 1 (TGP, pg 12, polished plates with dimensions 1-1/4” x 1-1/4”). One would have a reasonable expectation of success given that both Mahadeorao and Francis observed timbal tissue expansion on varying substrate dimensions and both achieved confluency (limbal tissue growth greater than 95% of the area of the slide glass scaffold).
Furthermore, it is argued that the percentage (v/v) of human serum and standard culture media components like O-phosphoethanolamine would have resulted from routine experimentation using standard laboratory techniques available at the time of filing. For instance, Mahadeorao describes the use of 10% human serum supplemented medium rather than 4-6% and Francis describes the use of 15% human serum supplemented medium. Thus, it would have been a matter of routine experimentation for one of ordinary skill to adjust the percentage (v/v) of human serum to arrive at 4-6% given that both methods share similar goals of culturing limbal stem cells on de-epithelialized human amniotic membranes. Furthermore, O-phosphoethanolamine is a well-known culture media supplement and is inherently present in adult human serum. "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454,456, 105 USPQ 233,235 (CCPA 1955). Accordingly, in the absence of evidence to the contrary, one of ordinary skill in the art would have considered claims 1 and 7 to have been prima facie obvious to at the time the invention was made.
Claims 1, 7 and 9 are rejected in modified form under 35 U.S.C. 103 as being unpatentable over Mahadeorao (supra), TGP (supra), Shaharuddin (supra) and Francis (supra) as applied to claims 1 and 7 above in further view of Zhang et al. "The effect of amniotic membrane de-epithelialization method on its biological properties and ability to promote limbal epithelial cell culture." Investigative ophthalmology & visual science 54.4 (2013): 3072-3081 (hereinafter Zhang, reference of record). This is a new rejection necessitated by amendment of the claims in the responses filed 6/26/2026.
A description of Mahadeorao, TGP, Shaharuddin and Francis can be found above. The collection of cited art does not describe the removal of epithelial cells from the amniotic membrane using 4 to 6 M urea.
Claim 9: Zhang describes using 5 M urea to de-epithelized human amniotic membranes for limbal tissue culture (Zhang, Methods: HAM Denudation, pg 3073). Zhang concludes that urea treatment resulted in the complete amniotic epithelial cell removal with a higher level of basement membrane integrity and more favorable extracellular matrix growth factor composition when compared to similar conventional approaches using EDTA or trypsin (Zhang, Discussion, pg 3080).
It would have been prima facie obvious to one of ordinary skill in the art to incorporate the 5 M urea de-epithelization process described by Zhang into the ex vivo culturing method for limbal stem cell expansion described by Mahadeorao in view of Francis in order to achieve improved de-epithelialized human amniotic membrane integrity. It would have been a matter of applying a known technique to improve limbal stem cell expansion since Zhang reported superior membrane integrity and extracellular matrix growth factor composition when using urea treatment for epithelial cell removal when compared to trypsin treatment as reported by Mahadeorao and Francis. For these reasons, one would have been motivated to do so in order to improve membrane integrity and extracellular matrix growth factor composition. One would have a reasonable expectation of success given the favorable de-epithelialization results achieved by Zhang. Accordingly, in the absence of evidence to the contrary, one of ordinary skill in the art would have considered the claimed invention to have been prima facie obvious to at the time the invention was made.
Conclusion
No claims allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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Alexander Nicol
Patent Examiner
Art Unit 1634
/ALEXANDER W NICOL/Examiner, Art Unit 1634
