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 .
Claim Status
Claims 1 has been amended.
Claims 3,4, and 6 is canceled.
Claim 1 is under examination.
Rejections Withdrawn
Claim Rejections under 35 U.S.C. § 112
The rejection of claim 4 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second
paragraph, as being allegedly indefinite is withdrawn in view of claim cancelation.
Edited rejections necessitated by claim amendment
Claim Rejections - 35 USC § 103
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1 is rejected under 35 U.S.C. 103 as being unpatentable over Matsuura-Hachiya et al (Experimental dermatology,2018m, of record), hereafter Matsuura et al. in view of Matsuura (JP 2020- 202757A, of record) hereafter, JP 2020- 202757A , Riva et al (Tissue Engineering, 2007). Gibo’s product data sheet ( Life Technologies Corporation, 2014), and Ibidi’s product data sheet (Ibidi,2024) . JP 2020- 202757A is in the Japanese language. A machine translation is provided herewith. Citations are made to the machine translation.
Matsuura et al, teach a method for producing human skin equivalents (HSEs). In the method, the HSEs are reconstructed with dermal equivalents consisting of collagen type I and fibroblasts, as well as epidermal equivalents consisting of keratinocytes cultured on top of the dermal equivalents. The method of Matsuura, also includes coating the dermal equivalents with collagen IV at the dermal-epidermal junction to promote keratinocytes proliferation and terminal differentiation, thereby improving the epidermal layers stratification in HSEs.
The method of Matsuura et al reads on instant claim as follow:
Regarding claim 1- step (a), the method of Matsuura et al teaches forming dermal equivalents by pouring a medium containing collagen type I and fibroblasts into a culture dish to form gels (see e.g., page 444, section 2.4). The gels read on the “three-dimensional hydrogel scaffold”. According to Matsuura et al, for gelation the matrix solution comprising Collagen I and fibroblast is placed in an incubator at 37C̊. ( See page 444, section 2.4).This reads on claimed step (a).
Matsuura et al, do not teach collagen type I at a concentration of 6 mg/ml in a mixture consisting of phosphate-buffered saline, NaOH, and distilled deionized water, or the percentage of CO2 required for the gelation step.
While Matsuura et al do not teach the same concentration for collagen type I of 6 mg/ml as recited in instant step. However, this is an optimizable parameter and it is well recognized that it is prima facie obvious for one of ordinary skill in the art to use routine experimentation to discover an optimum value of a result effective variable. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum of workable ranges by routine experimentation. The "discovery of an optimum value of a result effective variable in a known process is ordinarily within the skill of the art." Application of Boesch, 617 F.2d 272, 276, 205 USPQ 215, 218-219 (C.C.P.A. 1980). See MPEP 2144.05.
It should also be mentioned that neutralizing collagen I with NaOH and the incubation in an incubator at 37C̊ and 5% CO2 is a common practice in the art since it facilitates self-assembly into cross-linked fibrils forming gel, as disclosed by Life TechnologiesCorporation and Ibidi. It is also a common practice in the art to dilute collagen I with phosphate-buffered saline and distilled deionized water to the optimum working concentration, as disclosed by Life Technologies Corporation and Ibidi. ( See Life Technologies Corporation product data sheet , section “ Use-gelling procedures” page 1, and Ibidi product data sheet, table 1- column 4-page 6). Therefore, neutralizing collagen type I with NaOH, diluting it with 10X PBS, and incubating in an incubator at 37C̊ and 5% are all claim limitations that are well-understood, routine, and conventional steps in the relevant art. Consequently, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to incorporate such common practices into the claimed invention.
Regarding step (b), Matsuura et al also teach pouring a layer of collagen type IV on top of the dermal equivalents (see page 445, section 2.5). Specifically, the method of Matsuura involves pouring 100 µg/ml DMEM-diluted collagen IV into a glass ring with 11 mm diameter that is directly placed on top of the dermal equivalent (see page 445, section 2.5, and for the size of the ring see page 445, section 2.4). It is noted that method of Matsuura et al involves diluting collagen IV in DMEM rather than PBS, and it doesn’t specify the final concentration after the dilution step. In addition, Matsuura et al teach that the solution is stored at 37 C̊ for 24 hours, which differs from the incubation step recited in instant step (b) e.g. at room temperature for 1 hour. The method of Matsuura also doesn’t teach the exact amount of collagen applied in the ring, nor does it teaches the ring height. Hence, we cannot calculate the concentration in µg /cm2 (which is dependent on the surface area within the glass ring and the amount of collagen IV applied). It is noted that Matsuura’s method involves diluting collagen IV in DMEM and does not teach the exact concentration of collagen IV in µg /cm2 , as well different incubation step. However, these are all optimizable parameters and it is well recognized in the art that it would have been prima facie obvious for one with ordinary skill in the art to rely on routine experimentation when determining the appropriate concentration, and the incubation temperature and time. When the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimal or workable ranges through routine experimentation. See MPEP 2144.05.
Regarding step (c) and (d), The method of Matsuura also involves culturing the keratinocytes on top of the coated dermal equivalents to form an epidermis (see page 445, section 2.5). Specifically, Matsuura et al teach culturing keratinocytes, suspended in a culturing medium at a concentration of (4×104 cells/0.4 mL- e.g.1X105 cells/ml), on top of coated dermal equivalents placed into a glass ring mounted on stainless steel meshes. It should be noted that the stainless steel meshes with the glass ring are placed in a 3.5-cm culture dish. The method of Matsuura et al also involves adding a culturing medium into the 3.5-cm culture dish (2.5 mL/dish). Also, it is important to highlight that, while this rejection is based on skin equivalents described in section 2.5, this section doesn’t explicitly give details about the culturing of the keratinocytes; however, Matsuura et al note that the seeding of keratinocyte in 2.5 was performed as described above (e.g. referring to section 2.4)”. Section 2.4 describes the details for culturing keratinocyte in the control example, thus it would be obvious that the two experiment were subjected to the same experimental conditions. (See section 2.4 and 2.5 on pages 444-445).
Matsuura’s method includes producing HSEs in conventional culture vessel. Matsuura et al do not teach constructing HSEs in a double-walled culture vessel comprising an outer chamber; and an inner chamber surrounded by an inner wall and a bottom surface made of porous material, as required by the amended claim 1, nor do they teach the keratinocyte seeding density, as recited in step (c).
JP 2020- 202757A, teaches a method for stably producing a skin-like tissue with a
structure similar to natural skin using a double-walled vessel. This culture vessel has an inner chamber with a porous bottom surface and an outer chamber surrounding said inner chamber, with a means for preventing water vapor derived from a medium filled in the outside of the cell culture vessel from mixing into the inside of the cell culture vessel. (See Fig.2-3). The method of JP 2020- 202757A for producing the skin-like tissue discloses the making of a hydrogel, preferably of collagen type I mixed with fibroblasts, which is then poured onto the inner chamber’s porous membrane. After the gel is formed, the inner and outer chambers are filled with a medium for cell culturing. Then, keratinocytes are seeded on top of the hydrogel in the inner chamber to form a keratinocyte layer, and the inside and outside of the cell culture vessel are filled with a culture medium. JP 2020- 202757A also teaches that the keratinocyte can be seeded at a density ranging from 100,000 to 1000,000 cells/cm2, which includes the seeding density of 250,000 cells/cm2 recited in instant step. JP 2020- 202757A further teach removing the medium from the inner chamber and exposing it to air, while the outer chamber is again filled with a medium for cell culturing. The medium is then replaced once every 2 to 3 days, and the cells are cultured for 2 weeks. (See paragraphs [0012] , [0031]), example 1; and Figures 1-2). JP 2020- 202757A discloses that using double-walled vessel allows for strict control the gas phase and relative humidity on the stratum corneum side of the skin-like tissue, which is important for improving keratinocyte’s stratification and terminal differentiation. (See paragraphs [0023] ; and Fig.5). It is noted that the frequency of changing medium once every 2 to 3 days, as taught by JP 2020- 202757A, differs from that recited by instant step e.g. every day. However, this is also an optimizable parameter that would depend on the seeding density of the cells, and it is well recognized in the art that it would have been prima facie obvious for one with ordinary skill in the art to rely on routine experimentation when determining the appropriate occurrence. When the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimal or workable ranges through routine experimentation. See MPEP 2144.05.
Both Matsuura et al and JP 2020- 202757A teach a dermal equivalents consisting of collagen type I and fibroblasts. This differs from instant application, which teaches “three-dimensional hydrogel scaffold matrix by pouring a matrix solution consisting of a type I collagen without fibroblast, phosphate-buffered saline, NaOH, and distilled deionized water”.
Riva et al teach a simple procedure for culturing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen type I layer alone, without fibroblast. The method of Riva, involves seeding keratinocytes at high density directly on collagen I-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. (See abstract). Riva et al demonstrate that keratinocytes grown on collagen type I without fibroblast formed epithelial colonies and stratified sheets that were typical of keratinocytes and comparable with those reported for keratinocyte grown on collagen I containing fibroblast. (See abstract, and Fig.6-page 2774). According to Riva et al, using human or murine feeder cells in epidermal constructs present a major risk of contaminating the tissues with allogenic molecules, pathogens, or macromolecules, which could be transmitted to human cells if the engineered tissues are intended for clinical use. (See Discussion page 2775). Furthermore, Riva et al teach that, while type I collagen alone may be sufficient to trigger and sustain keratinocytes differentiation and epidermal histogenesis; the number of the cell layers (4–6 layers, including basal cells) was lower than in natural skin and other models of bilayered skin equivalent. (See Discussion, 1st column, last paragraph, page 2776).
Taken together, it would have been prima facie obvious for one with ordinary skill in the art at the time the invention was filed to combine the teachings of Matsuura, JP 2020- 202757A, and Riva to use a double-walled vessel for culturing keratinocytes on an acellular dermal equivalent coated with collagen IV to make a skin-like tissue with an improved structure similar to the natural skin. Because Matsuura et al teach coating the dermal equivalents with collagen IV at the dermal-epidermal junction promotes keratinocytes proliferation and terminal differentiation, thereby improving the epidermal layers stratification in HSEs. JP 2020- 202757A teaches that utilizing a double-walled culture vessel to produce HSEs allows for strict control of the gas phase and relative humidity, which improves keratinocyte stratification and terminal differentiation. Riva et al, disclose that culturing keratinocytes on collagen I layer alone, without fibroblast, supports keratinocytes proliferation and terminal differentiation, resulting in epidermal 3D equivalents, and suggest that culturing keratinocytes on a acellular hydrogel reduces the risk of contaminating the tissues with allogenic molecules or pathogens that could be transmitted to human cells if the engineered tissues are intended for clinical use.
Thus one would have been motivated to employ the double-walled vessel, as disclosed by JP 2020- 202757A’s, and culture the keratinocyte on top of acellular dermal hydrogel coated with collagen type IV to produce HSEs in accordance with Matsuura’s et al and Riva’s directions. There would be a reasonable expectation of success in using a double-walled vessel for the culturing of keratinocyte on top of acellular hydrogel coated with collagen IV, because doing so would stimulate the proliferation of basal keratinocytes, improve the stratification of epidermal layers in HSEs, and reduce the risk of contaminating the tissues with allogenic molecules or pathogens. Some Teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. See MPEP 2143 (I)(G).
Response to Arguments
Applicant's arguments filed 07/10/2025 have been fully considered but they are not
persuasive.
Applicants argue that Matsuura et al, JP 2020- 202757A, and Riva are insufficient to establish a prima facie case of obviousness of claim 1 at least because claim 1 yields unexpected and superior method of producing reconstructed human skin over Matsuura et al, JP 2020- 202757A, and Riva,
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive. This is because the method of Matsuura et al clearly show that seeding keratinocyte on dermal equivalents coated with collagen IV increased keratinocyte proliferation and improved the stratification of the epidermal layers (See Fig.2). For example, Fig.2, on page 446 of Matsuura et al shows the effect of coating the dermal equivalents with collagen IV prior to keratinocyte seeding on epidermal stratification. As such, the thickness of epidermal layers was around twice than that of control culture (i.e. without the coating layer) ( See. Figure 2B). Furthermore, HSEs containing type IV collagen aggregates included more proliferative basal cells than control HSEs. (See Fig.3). Therefore, the argument of unexpected and superior method is not persuasive because the beneficial results (e.g. the limited expression of a loricrin to the stratum qranulosum and the enhancement of the structural integrity of the epidermis) would have been expected given the combined teachings of Matsuura, JP 2020- 202757A, and Riva. As per the MPEP "Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof." In re Gershon, 372 F.2d 535, 538, 152 USPQ 602,604 (CCPA 1967). Applicants also appear to misread section 2.4 and 2.5 of Matsuura et al. It should be noted that section 2.4 cited in Matsuura et al describes the control experiment, which includes the details of preparing dermal equivalents and the seeding of keratinocyte, whereas section 2.5 describes the details of the experimental example, which include preparing the dermal equivalents using the steps in section 2.4, coating with collagen IV, and then seeding with keratinocyte following the steps described in 2.4. This is evident from the statement of Matsuura et al recited in the last sentence of section 2.5 “ e.g. the keratinocytes in the culture medium were poured into a glass ring as described above (e.g. referring to section 2.4)”.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. 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 nonprovisional extension fee (37 CFR 1.17(a)) 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to FATIMAH KHALAF MATALKAH whose telephone number is (703)756-5652. The examiner can normally be reached Monday-Friday,7:30 am-4:30 pm EST.
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/FATIMAH KHALAF MATALKAH/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638