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
1. The amendment filed 01/30/2026 has been entered. Claims 1, 2, and 4 – 19 remain pending and are under consideration. Claims 3 and 20 have been canceled.
Priority
2. This application is a continuation of U.S. patent application no. 18/751954 filed on June 24, 2024, which claims the benefit of priority from copending U.S. provisional patent application no. 63/522,795 filed on June 23, 2023.
Withdrawn Claim Rejections
3. The rejection of claim 20 under 35 U.S.C. 101 is rendered moot in view of Applicant’s cancellation of the claim.
4. The rejection of claim 3 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
5. The rejection of claim 20 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
6. The rejection of claims 1 – 2, 4 – 10, and 15 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
7. The rejection of claim 3 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
8. The rejection of claims 11 – 14 under 35 U.S.C. 103 103 is withdrawn in view of Applicant’s amendment to claim 1.
9. The rejection of claims 16 – 17 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
10. The rejection of claims 18 and 19 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
11. The rejection of claim 3 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 20 of copending Application No. 18751954 is rendered moot in view of Applicant’s cancellation of the claim.
12. The rejection of claim 20 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 20 of copending Application No. 18751954 is rendered moot in view of Applicant’s cancellation of the claim.
13. The rejection of claim 3 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 20 of copending Application No. 18012743 is rendered moot in view of Applicant’s cancellation of the claim.
14. The rejection of claim 20 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 20 of copending Application No. 18012743 is rendered moot in view of Applicant’s cancellation of the claim.
Claim Interpretation
15. 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”.
Claim Rejections Necessitated by Amendment
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.
16. Claim(s) 1 – 2, 4 – 10, and 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Murray (Murray, L. M., et al. Journal of biomedical materials research Part A 104.7 (2016): 1638-1645.), hereinafter Murray in view of Shahin-Shamsabadi (Shahin-Shamsabadi, Alireza, et. al. ACS Biomaterials Science & Engineering 6.9 (2020): 5346-5356; previously cited), hereinafter Shahin-Shamsabadi which is cited on the IDS filed 03/24/2025 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), hereinafter Selvaganapathy and Imashiro (Imashiro C, et. al. Int J Mol Sci. 2021 Jan 3;22(1):425), hereinafter Imashiro in view of Kikuchi (WO-2019177146-A1; Filed 03/15/2019; Published 09/19/2019), hereinafter Kikuchi. A machine translation of Kikuchi is provided. The translation was performed on 03/10/2026 of pages 3 – 35 of the original document.
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 teaches 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) but does not teach “seeded on the substrate to a monolayer density of at least 65%” or “the incubation time ranges from 20 minutes to 10 hours”.
Regarding claim 4, Murray teaches 37 °C (page 1639, left col. para. 7 and right col. para. 1).
Regarding claim 5, Murray teaches in Figure 8 the sheet is a 3D sheet (page 1641, right col. para. 3; Figure 3).
Regarding claim 6, Murray teaches the cells are seeded on the PDMS surface in a 6-well plate (page 1639, left col. para. 4 and 7).
Regarding claim 7, Murray teaches the PDMS substrate is patterned (page 1640, left col. and right col. para. 1; page 1641, right col. para. 4; page 1642, left col. para. 2; Figure 4).
Regarding claims 8 and 9, Murray teaches the non-adherent substrate is a silicone polymer (claim 8) that is PDMS (claim 9) and that the surface is unmodified (page 1639, left col. para. 4).
Regarding claim 10, Murray teaches the cells are C2C12 (“animal”) (page 1638, right col. last para.; page 1639, left col. para. 7).
Regarding claim 15, Murray teaches the cell culture medium is DMEM (page 1639, left col. para. 8).
Murray teaches 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) but does not teach “seeded on the substrate to a monolayer density of at least 65%” or “the incubation time ranges from 20 minutes to 10 hours” of claim 1 or “seeding on the non-adherent substrate to a monolayer density of 90% to 100%” of claim 2. However, Murray teaches in the Figure 8 legend that the cells are monolayer culture that peeled off the PDMS. Murray teaches 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 (page 1645, left col. para. 2; page 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; page 1638, left col. para. 1). Murray teaches the culture environment can be designed or engineered to modify or regulate muscle cell functions (Abstract). 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 (page 1643, right col. last para.; page 1645, right col. para. 1). Murray teaches on patterns of pillars, cells grew between an over the features on PDMS because of its hydrophobicity (page 1644, left col. para. 2; page 1645, left col. last para.; Figure 4 and 6). Murray teaches C2C12 cells cultured on PDMS reached ~90% confluence after 72 hours (page 1641, left col. para. 4; page 1643, left col. last para.).
Regarding “seeded on the substrate to a monolayer density of at least 65%” of claim 1 and “seeding on the non-adherent substrate to a monolayer density of 90% to 100%” of claim 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 (page 5347, right col. para. 2; page 5349, left col. and right col. para. 1; Figure 2A). Shahin-Shamsabadi teaches 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 (page 5349, right col. para. 1). Shahin-Shamsabadi teaches that 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 does not teach “the incubation time ranges from 20 minutes to 10 hours” of claim 1. However, Shahin-Shamsabadi teaches the cell sheets can adhere to each other within a short incubation time (Abstract). Shahin-Shamsabadi teaches incubating stacks of cell sheets for one hour followed by imaging (page 5348, left col. para. 1 – 2; page 5349, left col. last para.). Shahin-Shamsabadi teaches 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 (page 5349, right col. para. 1). Therefore, Shahin-Shamsabadi teaches C2C12 cells can adhere to each other in one hour. Skeletal muscle cells grown to confluence connect with each other, generate ECM by themselves and forma a contiguous sheet that is connected with each other as evidenced by Selvaganapathy (minute 33:03 – 33:32; page 61 – 62 of the transcript). 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).
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 a method of chemical delamination of the cell sheets from the plate using changes in pH (Figure 1B; page 5347, right col. last para.; page 5348, left col. para. 1), which contrasts the method of Murray where the sheets naturally detach from the PDMS surface. 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.
Regarding “the incubation time ranges from 20 minutes to 10 hours” of claim 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 (page 69 – 70, para. 0077; Figure 1). Kikuchi teaches that when the incubation time was 2 hours or more, a sheet was formed (page 70, para. 0078; Figure 1; page 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 (page 5, para. 0007; page 7, para. 0009; page 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 (page 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 (page 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 (page 2, para. 0001; page 3, para. 0005; page 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 (page 4, para. 0007).
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 combine the teachings of Murray regarding a method of forming a cell sheet on a PDMS surface with the teachings of Shahin-Shamsabadi regarding a method of forming a cell sheet by plating cells at near confluence and forming multi-layer cell sheets where C2C12 cells can adhere to each other in one hour with the teachings of Kikuchi regarding a method of forming a cell sheet by incubating seeded myoblasts for 2 hours to arrive at the claimed method 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. One 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 and 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 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 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 and 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.
17. Claim(s) 11 – 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Murray (Murray, L. M., et al. Journal of biomedical materials research Part A 104.7 (2016): 1638-1645.), hereinafter Murray in view of Shahin-Shamsabadi (Shahin-Shamsabadi, Alireza, et. al. ACS Biomaterials Science & Engineering 6.9 (2020): 5346-5356; previously cited), hereinafter Shahin-Shamsabadi which is cited on the IDS filed 03/24/2025 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), hereinafter Selvaganapathy and Imashiro (Imashiro C, et. al. Int J Mol Sci. 2021 Jan 3;22(1):425), hereinafter Imashiro in view of Kikuchi (WO-2019177146-A1; Filed 03/15/2019; Published 09/19/2019), hereinafter Kikuchi as applied to claims 1 – 2, 4 – 10, and 15 above, and further in view of Javaherian (Javaherian S, et. al. PLoS One. 2011;6(6):e20909; previously cited), hereinafter Javaherian as evidenced by Sigma (Parafilm M. Product insert. Sigma; 2022; previously cited), hereinafter Sigma.
Murry in view of Shahin-Shamsabadi and Kikuchi make obvious the limitations of claim 1 as set forth above.
Regarding claim 11, Shahin-Shamsabadi teaches a method of forming heterogenous sheets of C2C12 cells and HUVECs, NIH/3T3 fibroblasts, SH-SY5Y neuroblastoma cells, or 3T3-L1 preadipocytes (page 5348, right col. para. 4; page 5354, left col. para. 3; Figure 5B and S9) but does not teach “cells are seeded and incubated at different areas of the non-adherent substrate”.
Murray and Shahin-Shamsabadi do not teach “cells are seeded and incubated at different areas divided by an insert on the non-adherent substrate” of claim 12, “wherein the insert has a thickness that ranges from 0.1mm to 10 mm” of claim 13, or “wherein the insert is used to segment the non-adherent substrate area for selective seeding of cells and is removed within 10 minutes to 1 hour of cell seeding” of claim 14. However, Murray teaches the culture environment can be designed or engineered to modify or regulate muscle cell functions (Abstract). Murray teaches the PDMS substrate patterned with 5 µM periodic gratings, cells align specifically along the grated features (Figure 4B; page 1641, right col. 4). Murray teaches C2C12 morphology can be controlled by culture topography through patterning with dimensions comparable to cell features (page 1644, right col. para. 2). Shahin-Shamsabadi teaches skeletal muscle cells in vivo have symbiotic interactions with other cells and tissues including vascular tissue, connective tissue, and nervous tissue that may have significance in disease models and in modulating their function (page 5354, left col. para. 3). Shahin-Shamsabadi teaches fibroblasts are important in providing the stability for the skeletal muscle tissue through secretion of ECM components; endothelial cells and neuronal cells are important for vasculogenesis and formation of neuromuscular junctions (page 5354, left col. para. 3). Shahin-Shamsabadi teaches cell sheet engineering allows noninvasive and nonenzymatic harvest of cells and if transplanted in vivo, then the cells within the sheets will be regulated according to host environment to secrete appropriate growth factors and cytokines (page 5346, left col.).
Javaherian teaches co-culture cell patterns where two different cell types are seeded on different areas of a substrate divided by Parafilm (“insert” of claims 12 – 14) (Figure 1; page 3, right col. paragraph 2; Figure 3). Javaherian teaches Parafilm M to generate the inserts which has a thickness of 0.13 mm (claim 13) as evidenced by Sigma (left col. paragraph 3). 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 (claim 14) (page 4, right col. paragraph 3; page 7, left col. paragraph 3). Javaherian teaches co-culture with Parafilm is simple, cheap, fast and ensures reproducible patterning (page 1, right col. paragraph 1). Javaherian teaches 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; page 1, left col.). 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 (page 2, right col. para. 2; page 7, left col. last para.).
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 combine the teachings of Murray regarding a method of forming a cell sheet on a PDMS surface with the teachings of Shahin-Shamsabadi regarding a method of forming a cell sheet with multiple cell types with the teachings of Javaherian regarding a Parafilm insert for separate co-culture of two cell types to arrive at the claimed invention wherein two or more types of cells are seeded and incubated at different areas of the non-adherent substrate. One would have been motivated to combine the teachings of Murray, Shahin-Shamsabadi, and Javaherian to form cell sheets for tissue regeneration as Murray teaches the culture environment can be designed or engineered to modify or regulate muscle cell functions, Shahin-Shamsabadi teaches skeletal muscle cells in vivo have symbiotic interactions with other cells and tissues including vascular tissue, connective tissue, and nervous tissue that may have significance in disease models and in modulating their function, and 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 would have a reasonable expectation of success in combining the teachings as Shahin-Shamsabadi teaches forming heterogenous sheets of C2C12 cells and HUVECs, NIH/3T3 fibroblasts, SH-SY5Y neuroblastoma cells, or 3T3-L1 preadipocytes and Javaherian teaches co-culture with Parafilm is simple, cheap, fast and ensures reproducible patterning.
18. Claim(s) 16 – 17 is/are rejected under 35 U.S.C. 103 as being unpatentable Murray (Murray, L. M., et al. Journal of biomedical materials research Part A 104.7 (2016): 1638-1645.), hereinafter Murray in view of Shahin-Shamsabadi (Shahin-Shamsabadi, Alireza, et. al. ACS Biomaterials Science & Engineering 6.9 (2020): 5346-5356; previously cited), hereinafter Shahin-Shamsabadi which is cited on the IDS filed 03/24/2025 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), hereinafter Selvaganapathy and Imashiro (Imashiro C, et. al. Int J Mol Sci. 2021 Jan 3;22(1):425), hereinafter Imashiro in view of Kikuchi (WO-2019177146-A1; Filed 03/15/2019; Published 09/19/2019), hereinafter Kikuchi as applied to claims 1 – 2, 4 – 10, and 15 above, and further in view of Hasegawa (Hasegawa, Akiyuki, et al. Journal of Biomedical Materials Research Part A 103.12 (2015): 3825-3833; previously cited), hereinafter Hasegawa.
Murry in view of Shahin-Shamsabadi and Kikuchi make obvious the limitations of claim 1 as set forth above.
Regarding “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” of claim 16 and wherein the time ranges from 10 minutes to 10 hours and/or wherein the temperature ranges from 25°C to 39°C of claim 17, Shahin-Shamsabadi teaches a method of forming stacks of multiple sheets in culture media at 37 °C for 5 minutes (page 5348, left col. para. 1; Figure 1B; page 5349, left col. last para. and right col. para. 1). Shahin-Shamsabadi teaches when multiple sheets were stacked on each other, the cells formed interlayer attachments (page 5349, right col. para. 1). Shahin-Shamsabadi does not teach “from 10 minutes to 10 hours” of claim 17.
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 (Abstract; page 3826, left col. last para. and right col. para. 1 – 2; page 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 (page 3828, right col. para. 3). Hasegawa also teaches a method for rapid fabrication of multi-layered cell sheets by centrifugation (Figure 1; page 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 (page 3828, right col. para. 3). Hasegawa teaches decreases in fabrication time of transplantable tissues may be brought about by improvements in the centrifugal method (page 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 (page 3831, left col. para. 2). Hasegawa teaches rapid fabrication of 3D tissues will reduce the already heavy burden on patients (page 3831, right col. para. 1).
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 combine the teachings of Murray regarding a method of forming a cell sheet on a PDMS surface with the teachings of Shahin-Shamsabadi regarding a method of forming a cell sheet with multiple cell types with the teachings of Hasegawa regarding a methods of forming multi-layer cell sheets by incubation or centrifugation to arrive at the claimed invention 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. One would have been motivated to combine the teachings of Murray, Shahin-Shamsabadi, and Hasegawa to rapidly form cell sheets for tissue regeneration 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 tissue regeneration and Hasegawa teaches rapid fabrication of 3D tissues will reduce the already heavy burden on patients. One would have a reasonable expectation of success in combining the teachings as Shahin-Shamsabadi teaches when multiple sheets were stacked on each other, the cells formed interlayer attachments and Hasegawa teaches 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.
19. Claim(s) 18 and 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Murray (Murray, L. M., et al. Journal of biomedical materials research Part A 104.7 (2016): 1638-1645.), hereinafter Murray in view of Shahin-Shamsabadi (Shahin-Shamsabadi, Alireza, et. al. ACS Biomaterials Science & Engineering 6.9 (2020): 5346-5356; previously cited), hereinafter Shahin-Shamsabadi which is cited on the IDS filed 03/24/2025 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), hereinafter Selvaganapathy and Imashiro (Imashiro C, et. al. Int J Mol Sci. 2021 Jan 3;22(1):425), hereinafter Imashiro in view of Kikuchi (WO-2019177146-A1; Filed 03/15/2019; Published 09/19/2019), hereinafter Kikuchi as applied to claims 1 – 2, 4 – 10, and 15 above, and further in view of Shimizu (Shimizu, Tatsuya, et al. Circulation research 90.3 (2002): e40-e48; previously cited), hereinafter.
Murry in view of Shahin-Shamsabadi and Kikuchi make obvious the limitations of claim 1 as set forth above.
Regarding “overlapping the edges of two or more of the cell sheets produced by the method of claim 1 in a culture medium” of claim 18 and “wherein the time ranges from 10 minutes to 10 hours and/or wherein the temperature ranges from 25°C to 39°C of claim 19, Shahin-Shamsabadi teaches a method of forming stacks of multiple sheets in culture media at 37 °C for 5 minutes (page 5348, left col. para. 1; Figure 1B; page 5349, left col. last para. and right col. para. 1). Shahin-Shamsabadi teaches when multiple sheets were stacked on each other, the cells formed interlayer attachments (page 5349, right col. para. 1). Shahin-Shamsabadi does not teach “overlapping the edges of two or more of the cell sheets” of claim 18 or “from 10 minutes to 10 hours” of claim 19.
Regarding “overlapping the edges of two or more of the cell sheets” of claim 18 and “from 10 minutes to 10 hours” of claim 19, Shimizu teaches a method of forming cardiac tissue grafts by stacking sheets allowing adhesion by incubating for 30 minutes (claim 19) of cardiomyocytes where the edges overlap as shown in Figure 2 (claim 18) (page 2, right col. para. 3). Shimizu teaches when 4 sheets were layered, macroscopic pulsation was observed (page 4, left col. para. 3). Shimizu teaches transplanting the layered cardiomyocyte sheets into rats and pulsation was observed (page 4, left col. last para. and right col.; page 5, left col. para. 1). Shimizu teaches in order to more effectively restore heart dysfunction due to myocardial infarction, transplantation of layered cardiomyocyte sheets onto the myocardial scar may be beneficial (page 7, left col. last para.). Shimizu teaches placing sheets onto wide areas of impaired heart tissue may prove a safe and supportive therapeutic intervention (page 7, right col. para. 1). Shimizu teaches the formation of cardiomyocyte sheets should have enormous potential for constructing in vitro 3D heart tissue models and improving viable functional graft material for clinical tissue repair (page 8, right col. para. 2).
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 combine the teachings of Murray regarding a method of forming a cell sheet on a PDMS surface with the teachings of Shahin-Shamsabadi regarding a method of forming a stacked cell sheets with the teachings of Shimizu regarding a method of forming cardiomyocyte sheet grafts by overlapping cell sheets to arrive at the claimed invention 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 cell sheets mutually fuse under time and temperature conditions. One would have been motivated to combine the teachings of Murray, Shahin-Shamsabadi, and Shimizu to rapidly form cell sheets for tissue regeneration 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 tissue regeneration and Shimizu teaches the formation of cardiomyocyte sheets should have enormous potential for constructing in vitro 3D heart tissue models and improving viable functional graft material for clinical tissue repair. One would have a reasonable expectation of success in combining the teachings as Shahin-Shamsabadi teaches when multiple sheets were stacked on each other, the cells formed interlayer attachments and Shimizu teaches transplanting the layered cardiomyocyte sheets into rats and pulsation was observed.
Maintained Claim Rejections
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.
20. Claims 1, 2, and 4 – 19 remain provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 20 of copending Application No. 18751954. Although maintained, the rejection is revised in light of the amendments to the claims.
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.
21. Claims 1, 2, and 4 – 19 remain 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 (Murray, L. M., et al. Journal of biomedical materials research Part A 104.7 (2016): 1638-1645; previously cited), hereinafter Murray. Although maintained, the rejection is revised in light of the amendments to the claims.
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). 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 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.
Applicant’s Arguments/ Response to Arguments
22. Applicant’s Arguments: On page 7, para. 6 – 7, Applicant asserts that the method of Murray does not result in a non-adherent cell sheet and that the cells in Murray are incubated on PDMS for at least 24 hours. On page 8, Applicant asserts that the long incubation time in Murray leads to non-specific protein adsorption on PDMS from media components and in Murray the seeding density is low, which allows the cells to grow and spread over time which require adhesion to the substrate.
Response to Arguments: This is not found persuasive because the claims recite “a cell sheet” and do not recite that the cell sheet is non-adherent and 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 (page 1643, right col. last para.; page 1645, right col. para. 1). Murray teaches in the Figure 8 legend that the cells are monolayer culture that peeled off the PDMS. Therefore, Murray teaches forming a cell sheet on a non-adherent substrate that does not adhere to the substrate (PDMS). Murray teaches plating 5 x 103 C2C12 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). 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 Murray teaches the C2C12 cell sheet peels off the PDMS substrate because the intercell forces were stronger than the forces of attachment between the cells and the PDMS substrate.
Applicant’s Arguments: On page 9, paragraph 1 – 2, Applicant asserts that there is no teaching or suggestion in Murray to increase cell density and/or decrease incubation time to avoid adherence on the PDMS substrate.
Response to Arguments: In response, 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 (page 1643, right col. last para.; page 1645, right col. para. 1) and Murray teaches in the Figure 8 legend that the cells are monolayer culture that peeled off the PDMS. Therefore, Murray teaches a monolayer of C2C12 cells (cell density) peel off from the PDMS. Further, 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). The monolayer of Murray did not require trypsin treatment for detachment from the PDMS surface. Additionally, in the new rejection set forth above, Kikuchi teaches a sheet-shaped cell culture of sufficient strength can be formed by incubating for about 2 hours after seeding myoblasts and fibroblasts from skeletal muscle and that the sheet naturally detaches (page 5, para. 0007; page 7, para. 0009; page 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 (page 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 (page 34, para. 0037 – 0038).
Applicant’s Arguments: On page 9, paragraph 5, Applicant requests that the provisional rejection over claims 1 – 20 of Application 18/751,954 to be held in abeyance until the present claims are in condition for allowance.
Response to Arguments: With respect to holding the rejection in abeyance, MPEP 804 states a complete response to a nonstatutory double patenting (NSDP) rejection is either a reply by applicant showing that the claims subject to the rejection are patentably distinct from the reference claims, or the filing of a disclaimer in accordance with 37 CFR 1.321 in the pending application(s) with a reply to the Office action (see MPEP § 1490 for a discussion of terminal disclaimers). Such a response is required even when the nonstatutory double patenting rejection is provisional. Only objections or requirements as to form not necessary for further consideration of the claims may be held in abeyance until allowable subject matter is indicated. Accordingly, the obvious double patenting rejection is maintained.
Applicant’s Arguments: On page 10, paragraph 1 – 5, Applicant disagrees with the provisional rejection of the claims over application 18/012,743 because the method of Murray does not result in a non-adherent cell sheet and there is no teaching or suggestion in Murray to increase cell density and/or decrease incubation time as recited in claim 1.
Response to Arguments: Applicant is directed to the preceding Response to Arguments addressing these arguments.
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 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.
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/Z.M.B./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632