DETAILED ACTION
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 8/13/2026 has been entered.
Examiner’s Note
Please note that the case has been transferred and the Examiner of record has changed. All subsequent papers filed in response to actions received from the Office should be addressed to Examiner GILLIAN REGLAS, AU 1632.
Claim Status
As of the Final Office Action mailed 3/18/2026, claims 1-19 were pending.
In Applicant's Response filed on 8/13/2026, claims 1 was amended and claims 3 and 5 were canceled.
As such, claims 1-2, 4, and 6-19 are pending and have been examined herein.
Withdrawn Objections/Rejections
The objections and rejections presented herein represent the full set of objections and rejections currently pending in this application. Any objections or rejections not specifically reiterated are hereby withdrawn.
Claim Interpretation
For the purpose of applying prior art, “non-adherent substrate” is interpreted to comprise PDMS because Applicant’s specification discloses PDMS is a non-adherent substrate at para. 0006, 0054, 00155, and 0158 and as Applicant asserts in the remarks on page 10, last para., PDMS reads on “non-adherent”. The examiner is also interpreting that, absent evidence to the contrary, the PDMS substrate as described below are “substantially flat.”
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 (i.e., changing from AIA to pre-AIA ) 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.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-2, 4, 6-10, and 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Murray et al (Journal of biomedical materials research Part A 104.7 (2016): 1638-1645.) in view of Shahin-Shamsabadi et al (ACS Biomaterials Science & Engineering 6.9 (2020): 5346-5356; previously cited; in the IDS filed 03/24/2025) and Kikuchi (WO2019177146-A1; Filed 03/15/2019; Published 09/19/2019; previously cited) as evidenced by Selvaganapathy (The Good Food Institute, 01/28/2021, “Dr. Ravi Selvaganapathy: Cultivating whole muscle cuts without scaffolds”, YouTube, youtube.com/watch?v-hIMyzTKE_-o; of record) and Imashiro et al (Int J Mol Sci. 2021 Jan 3;22(1):425; of record).
Regarding claim 1, Murray teaches a method of forming a cell sheet comprising seeding C2C12 myoblasts with a culture medium onto a flat PDMS substrate and incubating the cells on the substrate for 5 days to attain confluence and an additional 6 days for differentiation at 37 °C (Abstract; page 1639, left col. para. 4 and 7 – 8 and right col. para. 1 – 3; page 1640, right col. para. 3; page 1643, left col. para. 1 and right col. para. 2; Figure 8; page 1645, left col. para. 2 and right col. para. 1).
Murray continues to teach plating 5 x 103 cells/cm2 on PDMS in a 6-well plate and 72 hours later the cells reached 90% confluence (page 1639, left col. para. 4 and 7; page 1641, left col. para. 4).
Murray continues to teach that the cells are monolayer culture that peeled off the PDMS (Fig. 8). Murray continues to teach that the method produces a C2C12 cell sheet that has interesting implications for patterned PDMS substrates as biomaterials for potential ex vivo tissue development models and regenerative wound healing applications which require an adhesive construct during growth but need to be easily separated from the substrate for patient application (p. 1645, left col. para. 2; p. 1643, left col. para. 1; Figure 8). Murray teaches in tissue repair and medical implants, understanding the role of substrate materials and topography is essential for better control over cell behavior and more effective cell integration with tissues in situ (Abstract; p. 1638, left col. para. 1). Murray teaches the sheets that peeled off the substrate indicates the intercell forces were stronger than forces of attachment between the cells and PDMS substrate (p. 1643, right col. last para.; p. 1645, right col. para. 1). Murray teaches on patterns of pillars, cells grew between an over the features on PDMS because of its hydrophobicity (p. 1644, left col. para. 2; p. 1645, left col. last para.; Figure 4 and 6).
Regarding claim 4, Murray teaches 37 °C (page 1639, left col. para. 7 and right col. para. 1).
Regarding claim 5, Murray teaches the sheet is a 3D sheet (page 1641, right col. para. 3; Fig. 3; Fig. 8).
Regarding claim 6, Murray teaches the cells are seeded on the PDMS surface in a 6-well plate (p. 1639, left col. para. 4 and 7).
Regarding claim 7, Murray teaches the PDMS substrate is patterned (p. 1640, left col. and right col. para. 1; p. 1641, right col. para. 4; p. 1642, left col. para. 2; Figure 4).
Regarding claims 8 and 9, Murray teaches the non-adherent substrate is a silicone polymer that is PDMS and that the surface is unmodified (p. 1639, left col. para. 4).
Regarding claim 10, Murray teaches the cells are C2C12 (i.e., “animal”) (p. 1638, right col. last para.; p. 1639, left col. para. 7).
Regarding claim 15, Murray teaches the cell culture medium is DMEM (page 1639, left col. para. 8).
Murray does not teach:
seeded on the substrate to a monolayer density of at least 65%
seeding to a density of 90% to 100% or
the incubation time ranges from 20 minutes to 10 hours.
Regarding (i) and (ii), Shahin-Shamsabadi teaches seeding C2C12 myoblasts at 90% confluence (0.235 x 106 cells per well in 24 well plates or at 0.48 x 106 cells per well in 12 well plates) on flat plates in DMEM medium results in the formation of cell sheets (p. 5347, right col. para. 2; p. 5349, left col. and right col. para. 1; Figure 2A).
Shahin-Shamsabadi continues to teach it is also possible to form sheets using 6 well plates and 1.15 x 106 cells and 10 cm dishes and 6.3 x 106 cells (p. 5349, right col. para. 1). Due to the high cell number and metabolic activity of the cells, the medium was refreshed to prevent the medium from becoming acidic (page 5349, left col.).
Shahin-Shamsabadi teaches the ability to form tissue-like constructs that have high cell density with proper cell-cell and cell-ECM interactions is critical for many applications including tissue models for drug discovery and tissue regeneration (Abstract). Shahin-Shamsabadi teaches methods using self-assembly can be used to create tissue-like constructs with high cellular density and well-defined microstructure in the form of cell sheets (Abstract). Shahin-Shamsabadi teaches cell sheets have a particularly interesting architecture in the context of tissue regeneration and repair as they can be applied as patches to integrate with surrounding tissues (Abstract; page 5346, left col.). Shahin-Shamsabadi teaches the cell sheets generated by the method can be stacked to form thick grafts that can be used for regeneration purposes or as in vitro models (Abstract).
Regarding (iii), Shahin-Shamsabadi teaches that the cell sheets can adhere to each other within a short incubation time (Abstract).
Shahin-Shamsabadi continues to teach incubating stacks of cell sheets for one hour followed by imaging (p. 5348, left col. para. 1 – 2; p. 5349, left col. last para.). When the layers were stacked on each other, the cells formed interlayer attachments attributed to cell-cell, cell-ECM or ECM-ECM adherence between the different sheets (p. 5349, right col. para. 1).
The teachings of Selvaganapathy are relied upon as providing evidence that skeletal muscle cells grown to confluence connect with each other, generate ECM by themselves and form a contiguous sheet that is connected with each other (minute 33:03 – 33:32; page 61 – 62 of the transcript).
The teachings of Imashiro are relied upon as providing evidence that when cells reach confluence, they adhere to each other and develop a confluent monolayer with tight adhesions between them and with trypsin treatment, the cells detach in a state of cell sheet if adhesions between the cells and culture surface are selectively lost (page 3, para. 1; Figure 2).
Regarding (iii), Kikuchi teaches a method of forming a cell sheet comprising seeding myoblasts and fibroblasts obtained from skeletal muscle with a culture medium onto a substantially flat substrate and incubating the cells at 37 °C for 1 hour, 2 hours, 4 hours, 5.5 hours, 6 hours, 11.5 hours, and 12 hours (p. 69 – 70, para. 0077; Figure 1). Kikuchi teaches that when the incubation time was 2 hours or more, a sheet was formed (p. 70, para. 0078; Figure 1; p. 8, para. 0010). Kikuchi teaches a sheet-shaped cell culture of sufficient strength can be formed by incubating for about 2 hours after seeding and that the sheet naturally detaches (p. 5, para. 0007; p. 7, para. 0009; p. 34, para. 0038). Kikuchi teaches the intercellular adhesive forces of the cells that make up the sheet increase as the sheet is formed, exceeding the adhesive forces between the cells and the substrate (p. 5, para. 0007). Kikuchi teaches the time required for sheet formation varies depending on the type of cells contained in the seeded population and the state of the cell (p. 34, para. 0037 – 0038). Kikuchi teaches the cell sheet can be used in the treatment of various disease including grafts and therapeutic applications of cell sheets have already reached the stage of clinical application (p. 2, para. 0001; p. 3, para. 0005; p. 4, para. 0006). Kikuchi teaches there is a growing demand for sheet-shaped cell cultures that are of higher quality, easier to handle and can be produced simply (p. 4, para. 0007).
Therefore, it would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to form a cell sheet on a PDMS surface as taught by Murray, where forming a cell sheet by plating cells at near confluence and forming multi-layer cell sheets and the cells can adhere to each other in one hour as taught by Shahin-Shamsabadi, to arrive at the instantly claimed invention. One would have been motivated to combine the teachings of Murray and Shahin-Shamsabadi because both teach methods of forming C2C12 cell sheets and both teach the cell sheets may be useful as tissue models and tissue engineering.
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to form a cell sheet on a PDMS surface as taught by Murray and Shahin-Shamsabadi in combination, where the cells are incubated for 2 hours as taught by Kikuchi, to arrive at the instantly claimed invention. One of ordinary skill in the art would have been motivated to combine the teachings of Murray, Shahin-Shamsabadi, and Kikuchi in a method of rapidly forming cell sheets for tissue engineering and regenerative therapies as Murray teaches the sheet has interesting implications for potential ex vivo tissue development models and regenerative wound healing applications which require an adhesive construct during growth but need to be easily separated from the substrate for patient application, Shahin-Shamsabadi teaches the cell sheets generated by the method can be stacked to form thick grafts that can be used for regeneration purposes or as in vitro models, and Kikuchi teaches there is a growing demand for sheet-shaped cell cultures that are of higher quality, easier to handle and can be produced simply. One would have a reasonable expectation of success in combining the teachings because Murray teaches the C2C12 cells reach confluence on PDMS and the sheets peel off the substrate naturally and Shahin-Shamsabadi teaches seeding C2C12 cells at ~90% confluence forms cell sheets and have a particularly interesting architecture in the context of tissue regeneration and repair, and Kikuchi teaches a sheet-shaped cell culture of sufficient strength can be formed by incubating for about 2 hours after seeding, the sheet naturally detaches from the cells and intercellular adhesive forces of the cells that make up the sheet increase as the sheet is formed, exceeding the adhesive forces between the cells and the substrate.
Response to Arguments
Applicant's arguments have been fully considered but they are not persuasive.
On p. 6 of Remarks, Applicant argues that the culture time as described in Murray (24 hours) purportedly allow non-specific adsorption of proteins, make the cells capable of attaching to the proteins, and to the substrate itself. Applicant argues that Murray uses low density seeding and that the cells remain attached to the PDMS surface. Applicant argues that Murray provides no teaching, suggestion, or motivation to increase seeding density and decrease incubation time to prevent substrate adhesion. Applicant generally argues that the cited references do not remedy the deficiencies of Murray.
While applicant’s arguments have been fully considered, they are not persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the “objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support” (see MPEP 716.02(d)). Applicant’s arguments regarding forming cell-cell connections rather than cell-substrate connections are not commensurate in scope with the claims in view of MPEP 716.02(d).
Moreover, Murray explicitly teaches that the cells cultured on the PDMS surface were a “monolayer culture that peeled off the PDMS” (see rejection above). Therefore, Murray teaches forming a cell sheet on a non-adherent substrate that does not adhere to the substrate (PDMS). Shahin-Shamsabadi teaches seeding C2C12 cells at 90% confluence (0.235 x 106 cells per well in 24 well plates or at 0.48 x 106 cells per well in 12 well plates) on flat plates in DMEM medium results in the formation of cell sheets (page 5347, right col. para. 2; page 5349, left col. and right col. para. 1; Figure 2A). Therefore, Shahin-Shamsabadi teaches plating C2C12 cells at 90% confluence produces C2C12 cell sheets and that C2C12 cells can adhere to each other in one hour. When cells reach confluence, they adhere to each other and develop a confluent monolayer with tight adhesions between them and with trypsin treatment, the cells detach in a state of cell sheet if adhesions between the cells and culture surface are selectively lost as evidenced by Imashiro (page 3, para. 1; Figure 2). Therefore, plating C2C12 at 90% confluence on the PDMS substrate of Murray would be expected to form a cell sheet that peels off the PDMS substrate as taught by the prior art. Further, the 103 rejection above has been amended to address the amended limitations. The teachings of Kikuchi in point (iii) above are particularly informative because it specifically states forming a cell sheet on a substantially flat substrate and incubating the cells at 37 °C for 1+ hours (p. 69 – 70, para. 0077; Figure 1). Notably, Kikuchi teaches that when the incubation time was 2 hours or more, a sheet was formed (p. 70, para. 0078; Figure 1; p. 8, para. 0010), a sheet-shaped cell culture of sufficient strength can be formed by incubating for about 2 hours after seeding and that the sheet naturally detaches (p. 5, para. 0007; p. 7, para. 0009; p. 34, para. 0038). Thus, Applicant’s arguments are not persuasive.
Claim(s) 11-14 is/are rejected under 35 U.S.C. 103 as being unpatentable Murray in view of Shahin-Shamsabadi and Kikuchi as applied to claims 1-2, 4, 6-10, and 15 above, and further in view of Javaherian et. al. (PLoS One. 2011;6(6):e20909; previously cited) as evidenced by Sigma (Parafilm M. Product insert. Sigma; 2022; previously cited).
The teachings of Murrary, Shahin-Shamsabadi and Kikuchi in combination were recited in the above 35 U.S.C. 103 rejection as applied to claim 1 of which claims 11-14 depend.
Regarding claim 11, Shahin-Shamsabadi teaches forming heterogenous sheets of C2C12 cells and HUVECs, NIH/3T3 fibroblasts, SH-SY5Y neuroblastoma cells, or 3T3-L1 preadipocytes (p. 5348, right col. para. 4; page 5354, left col. para. 3; Figure 5B and S9).
Neither Murray, Shahin-Shamsabadi, nor Kikuchi teach:
that the cells are seeded in different areas of the substrate,
the seeded cells are divided by an insert on the substrate,
that the insert has a thickness of 0.1 to 10 mm.
Regarding i)-iii), Javaherian teaches co-culture cell patterns where two different cell types are seeded on different areas of a substrate divided by Parafilm (i.e., insert as claimed) (Figure 1; p. 3, right col. para 2; Fig. 3). Javaherian continues to teach Parafilm M to generate the inserts.
Regarding claim 14, Javaherian teaches seeding a first cell type then seeding a second cell type and incubation for one hour to allow the second cell type to adhere to the surface and that the Parafilm insert can be removed at any desired time point (p. 4, right col. para 3; p. 7, left col. para 3).
Javaherian continues to teach that co-culture with Parafilm is simple, cheap, fast and ensures reproducible patterning (page 1, right col. paragraph 1).
Javaherian continues to teach that micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale and regulation of these spatial parameters is important for controlling cell fate and cell function (Abstract; p. 1, left col.).
Javaherian continues to teach that the method using Parafilm for micropatterning can be used for studying cell behaviors in co-culture and during dynamic cell re-organization during wound healing (p. 2, right col. para. 2; p. 7, left col. last para.).
The teachings of Sigma are relied upon as evidencing that Parafilm M has a thickness of 0.13 mm (left col. paragraph 3).
Therefore, it would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to form a cell sheet on a PDMS surface as taught by Murray, Shahin-Shamsabadi, and Kikuchi in combination, where a Parafilm insert is used for separate co-culture of two cell types on a substrate as taught by Javaherian, to arrive at the claimed invention. Javaherian teaches the method using Parafilm for micropatterning can be used for studying cell behaviors in co-culture and during dynamic cell re-organization during wound healing. One of ordinary skill in the art would have motivated to modify the method of Murray, Shahin-Shamsabadi, and Kikuchi in combination to include a parafilm divider as taught by Javaherian with a reasonable expectation of success of providing direct control over the spatial organization of cells at the sub-mm scale and controlling cell fate and cell function as taught by the prior art.
Response to Arguments
Applicant has not specifically challenged the teachings of Javaherian as they relate to claims 11-14, thus the rejection is maintained.
Claim(s) 16-19 is/are rejected under 35 U.S.C. 103 as being unpatentable Murray in view of Shahin-Shamsabadi, Kikuchi (see rejections above) and Hasegawa et al. (Journal of Biomedical Materials Research Part A 103.12 (2015): 3825-3833; previously cited).
The teachings of Murray, Shahin-Shamsabadi, and Kikuchi were recited in the above 35 U.S.C. 103 rejection of claims 1-2, 4, 6-10, and 15. Those teachings are incorporated by reference herein regarding the production of cell sheets and stacking them (see above).
However, neither Murray, Shahin-Shamsabadi, nor Kikuchi teach:
that the stacked cell sheets fuse when cultured from 10 minutes to 10 hours and at a temperate of 25-39℃.
Regarding (i), Hasegawa teaches a method of using cell sheet engineering to form three-dimensional tissues where C2C12 cell sheets are stacked on each other at 37 °C for 10, 15, or 20 minutes in media (p. 3826, left col. last para. and right col. para. 1 – 2; p. 3828, left col. last para. and right col. para. 1; Table II; Figure 3D – F and 4C – D). Hasegawa teaches after 20 minutes there were hardly any spaces observed between the layered cell sheets (p. 3828, right col. para. 3). Hasegawa also teaches a method for rapid fabrication of multi-layered cell sheets by centrifugation (Figure 1; p. 3826, right col. para. 2) but does not teach incubation combined with centrifugation as shown in Table II. Hasegawa teaches after centrifugation, spaces between C2C12 sheets were hardly detected and centrifugation demonstrates tight attachment will promote a stronger cell adhesion process (p. 3828, right col. para. 3). Hasegawa teaches decreases in fabrication time of transplantable tissues may be brought about by improvements in the centrifugal method (p. 3831, left col. para. 1). Hasegawa teaches the effectiveness of cell sheet-therapy has been shown in various models of damaged heart and in the first clinical trial, quadruple-layered myoblast sheets were transplanted onto the heart and after treatment the patient was discharged without the need for ventricular assisting system (p. 3831, left col. para. 2). Hasegawa teaches rapid fabrication of 3D tissues will reduce the already heavy burden on patients (p. 3831, right col. para. 1).
Therefore, it would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to form a cell sheet on a PDMS surface as taught by Murray, Shahin-Shamsabadi, and Kikuchi in combination, where the sheets cultured under temperature and time conditions permissive for fusing as taught by Hasegawa to arrive at the claimed invention. One of ordinary skill would have been motivated to combine the teachings of Murray, Shahin-Shamsabadi, Kikuchi, and Hasegawa to rapidly form cell sheets for tissue regeneration since Hasegawa teaches rapid fabrication of 3D tissues will reduce the already heavy burden on patients with a reasonable expectation of success as taught by the prior art.
Response to Arguments
Applicant has not specifically challenged the teachings of Hasegawa as they relate to claims 11-14, thus the rejection is maintained.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 2, 4, and 6-19 remain provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 20 of copending Application No. 18751954.
Instant claim 1 recites a method of forming a cell sheet, comprising: seeding cells with a culture medium onto a substantially flat, non-adherent substrate; and incubating the cells on the substrate under an incubation time and temperature conditions to form a cell sheet; wherein the cells are seeded on the substrate to a monolayer density of at least 65% and wherein the incubation time ranges from 20 minutes to 10 hours.
Reference claim 1 recites a method of forming a cell sheet, comprising: seeding cells with a culture medium on to a substantially flat substrate, the substrate having low surface energy and/or ultralow adhesion; and incubating cells on the substrate under time and temperature conditions to form a cell sheet; wherein the cells are seeded on the substrate to a monolayer density of at least 65%.
Instant claim 16 recites a method of constructing a multi-layer cell sheet, comprising: stacking two or more of the cell sheets produced by the method of claim 1 in a culture medium, wherein the cell sheets mutually fuse under time and temperature conditions
Reference claim 16 recites a method of constructing a multi-layer cell sheet, comprising: stacking two or more of the cell sheets produced by the method of claim 1 in a culture medium, wherein
the cell sheets mutually fuse under time and temperature conditions.
Instant claim 18 recites a method of constructing a two-dimensional (2D) or a three-dimensional (3D) cell construct comprising: overlapping the edges of two or more of the cell sheets produced by
the method of claim 1 in a culture medium, wherein the edges mutually fuse under time and
temperature conditions.
Reference claim 18 recites a method of constructing a two-dimensional (2D) or a three-dimensional (3D) cell construct comprising: overlapping the edges of two or more of the cell sheets produced by the method of claim 1 in a culture medium, wherein the edges mutually fuse under time and temperature conditions.
Reference claim 1 differs from instant claim 1 in that reference claim 1 broadly recites “under time” “conditions” whereas instant claim 1 recites “wherein the incubation time ranges from 20 minutes to 10 hours”. However, reference claim 3 recites “wherein the incubation time ranges from 20 minutes to 10 hours”. Therefore, instant claim 1 is anticipated by reference claim 1 because the incubation time recited in instant claim 1 is encompassed by reference claim 1.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 2, 4, and 6-19 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 24 of copending Application No. 18012743 (reference application). in view of Murray (Journal of biomedical materials research Part A 104.7 (2016): 1638-1645; previously cited), Shahin-Shamsabadi (ACS Biomaterials Science & Engineering 6.9 (2020): 5346-5356; previously cited; in the IDS filed 03/24/2025) and Kikuchi (WO2019177146-A1; Filed 03/15/2019; Published 09/19/2019; previously cited).
Instant claim 1 recites a method of forming a cell sheet, comprising: seeding cells with a culture medium onto a substantially flat, non-adherent substrate; and incubating the cells on the substrate under an incubation time and temperature conditions to form a cell sheet; wherein the cells are seeded on the substrate to a monolayer density of at least 65% and wherein the incubation time ranges from 20 minutes to 10 hours.
Reference claim 1 recites a method of making a cell construct, comprising: a) plating a plurality of cells on a substantially flat surface; b) growing the plurality of cells to at least 80% confluence to form a cell sheet with intercellular linkages; c) applying a culture medium having a pH of about 5 to about 6.8 to the cell sheet; d) replacing the culture medium of step c) with a culture medium having a pH of about 7.5 to about 8.5; and e) replacing the culture medium of step d) with a culture medium having a pH of about 7 to about 7.7, to obtain a substantially planar untethered cell sheet.
Reference claim 1 lacks “non-adherent” of instant claim 1.
Murray teaches a method of forming a cell sheet comprising seeding cells with a culture medium onto a substantially flat PDMS substrate (page 1639, left col. para. 4 and 7; page 1640, right col. last para.; page 1641, left col. para. 4; page 1643, left col. para. 1; Figure 8; page 1645, left col. para. 2). Murray teaches PDMS is hydrophobic and because of the hydrophobicity, the sheet was unable to maintain adhesion (page 1643, right col. para. 2; page 1644, left col. para. 2). Murray teaches that the ability to form sheets on PDMS has interesting implications for PDMS substrates as biomaterials for potential ex vivo tissue development models and regenerative wound healing applications (page 1645, left col. para. 2).
Shahin-Shamsabadi teaches seeding C2C12 myoblasts at 90% confluence (0.235 x 106 cells per well in 24 well plates or at 0.48 x 106 cells per well in 12 well plates) on flat plates in DMEM medium results in the formation of cell sheets (p. 5347, right col. para. 2; p. 5349, left col. and right col. para. 1; Figure 2A).
Shahin-Shamsabadi continues to teach it is also possible to form sheets using 6 well plates and 1.15 x 106 cells and 10 cm dishes and 6.3 x 106 cells (p. 5349, right col. para. 1). Due to the high cell number and metabolic activity of the cells, the medium was refreshed to prevent the medium from becoming acidic (page 5349, left col.).
Shahin-Shamsabadi teaches the ability to form tissue-like constructs that have high cell density with proper cell-cell and cell-ECM interactions is critical for many applications including tissue models for drug discovery and tissue regeneration (Abstract). Shahin-Shamsabadi teaches methods using self-assembly can be used to create tissue-like constructs with high cellular density and well-defined microstructure in the form of cell sheets (Abstract). Shahin-Shamsabadi teaches cell sheets have a particularly interesting architecture in the context of tissue regeneration and repair as they can be applied as patches to integrate with surrounding tissues (Abstract; page 5346, left col.). Shahin-Shamsabadi teaches the cell sheets generated by the method can be stacked to form thick grafts that can be used for regeneration purposes or as in vitro models (Abstract).
Shahin-Shamsabadi teaches that the cell sheets can adhere to each other within a short incubation time (Abstract). Shahin-Shamsabadi continues to teach incubating stacks of cell sheets for one hour followed by imaging (p. 5348, left col. para. 1 – 2; p. 5349, left col. last para.). When the layers were stacked on each other, the cells formed interlayer attachments attributed to cell-cell, cell-ECM or ECM-ECM adherence between the different sheets (p. 5349, right col. para. 1).
Kikuchi teaches a method of forming a cell sheet comprising seeding myoblasts and fibroblasts obtained from skeletal muscle with a culture medium onto a substantially flat substrate and incubating the cells at 37 °C for 1 hour, 2 hours, 4 hours, 5.5 hours, 6 hours, 11.5 hours, and 12 hours (p. 69 – 70, para. 0077; Figure 1). Kikuchi teaches that when the incubation time was 2 hours or more, a sheet was formed (p. 70, para. 0078; Figure 1; p. 8, para. 0010). Kikuchi teaches a sheet-shaped cell culture of sufficient strength can be formed by incubating for about 2 hours after seeding and that the sheet naturally detaches (p. 5, para. 0007; p. 7, para. 0009; p. 34, para. 0038). Kikuchi teaches the intercellular adhesive forces of the cells that make up the sheet increase as the sheet is formed, exceeding the adhesive forces between the cells and the substrate (p. 5, para. 0007). Kikuchi teaches the time required for sheet formation varies depending on the type of cells contained in the seeded population and the state of the cell (p. 34, para. 0037 – 0038). Kikuchi teaches the cell sheet can be used in the treatment of various disease including grafts and therapeutic applications of cell sheets have already reached the stage of clinical application (p. 2, para. 0001; p. 3, para. 0005; p. 4, para. 0006). Kikuchi teaches there is a growing demand for sheet-shaped cell cultures that are of higher quality, easier to handle and can be produced simply (p. 4, para. 0007).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention as claimed to have modified the surface recited in claim 1 of the reference application to include non-adherent as taught by Murray, where the cells are cultured for 1-10 hours as taught by Shahin-Shamsabadi and Kikuchi to provide a method to produce cell sheets for regenerative wound healing applications.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to Arguments
Applicant argues on p. 9-10 of Remarks that there is no TSM in Murray to increase cell density not reduce incubation time.
While this argument has been fully considered, it is not persuasive. As recited above, both Shahin-Shamsabadi and Kikuchi teach incubating cells on a cell sheet for at least 1 hour and seeding cells at 90% confluency. Thus, Applicant’s argument is not persuasive.
Conclusion
No claim is allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Kim et al (US20150125952A1, 3/15/2013; published 5/7/2015), which teaches tissue engineered compositions comprising a nanotextured substrate which is structurally configured for growth of cells in an anatomically correct adult phenotype in vitro. In particular, described herein are nanotextured substrates which are structurally configured for the anisotropic organization, maturation, and growth of in vitro-differentiated muscle cells, such as cardiomyocytes, and methods for the production and use thereof in varying sizes, nanotextures and substrate rigidities (abstract). Unlabeled figure of Kim shows cell sheets stacked and discusses the use of PDMS molds to produce these cell sheets.
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/DAVID A MONTANARI/Examiner, Art Unit 1632
/G.R./Examiner, Art Unit 1632
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