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 .
Examiner’s Note
Upon further consideration, the limitations of claim 23 are not allowable because the claimed cell dispersion made obvious by combining the teachings of Chen and Mollamohammadi would be capable of a viability of at least 95% when thawed absent any arguments to the contrary because the combined teachings of Chen and Mollamohammadi make obvious the limitations of claim 5.
This action is non-Final.
Claim Status
1. The Amendment filed 10/01/2025 has been entered. Claims 1, 4 – 5, 9 – 11, 13, 15 -18, 20 and new claims 21 – 23 are pending.
Election/Restrictions
2. Applicant's election with traverse of Group II (claims 5 – 16) in the reply filed on 12/19/2023 is acknowledged. The traversal is on the ground(s) that claim 1 has been amended to depend from elected claim 5 and thus Applicant respectfully requests rejoinder of claim 1 and its dependent claims. This is not found persuasive because claim 1 step (3) requires replacing the medium of step (2) containing the ROCK inhibitor with a medium for cryopreservation, however claim 5 requires a cell dispersion consisting of a dispersion medium, and pluripotent stem cells dispersed in the medium and a ROCK inhibitor.
The requirement is still deemed proper and is therefore made FINAL.
3. Claims 1, 4, and 17 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 12/19/2023.
4. Claims 5, 9 – 11, 13, 15 – 18, and 20 – 22 are under consideration.
Priority
5. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in parent Application No. JP2018-130985, filed on 07/10/2018.
6. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Withdrawn Claim Rejections
7. The rejection of claims 15 and 23 under 35 U.S.C. 103 are rendered moot by Applicant’s cancellation of these claims.
Maintained Claim Rejections
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.
8. Claim(s) 5, 13, 16, 18, and 20 remain rejected under 35 U.S.C. 103 as being unpatentable over Chen (CN105087472A; previously cited), hereinafter Chen in view of Mollamohammadi (Mollamohammadi S, et. al. Hum Reprod. 2009 Oct;24(10):2468-76; previously cited), hereinafter Mollamohammadi. A machine translation of CN105087472A is provided. The translation was performed on 03/11/2025 on pages 1 – 7 of the original document.
Regarding claim 5 and 16, Chen teaches a frozen cell dispersion consisting of a dispersion medium comprising IMDM/F12 and induced pluripotent stem cells (iPSCs) dispersed in the dispersion medium and a ROCK inhibitor wherein the concentration of ROCK inhibitor is 1 ng – 1000 ng per 100 mL (0.01 ng/mL to 10 ng/mL) with a preferred embodiment of 1.1 ng to 100 ng per 100 mL (0.011 ng/mL to 1 ng/mL) (page 3; page 4, paragraph 3 – 4). Chen teaches the concentration of the iPSCs is 1 to 10 x 106/mL (1000 – 10,000 cells/µL) (page 4, paragraph 4). Chen teaches a ROCK inhibitor (page 3, paragraph 9) but does not teach “Y-27632” of claim 5. Chen teaches the cells are induced pluripotent stem cells but does not teach “human” of claim 5.
Regarding “airtight container” claim 5 and 16, Chen teaches the frozen cells are in a cryotube that is placed in a cryobox and by properly storing a certain amount of cells, it is possible to prevent the cells from being contaminated due to contamination or other unexpected events of the cells being cultured (page 2, paragraph 7; page 4, paragraph 4; page 5, 14 – 16).
Regarding claim 13, Chen teaches the density of the frozen cells is 1 to 10 x 106/mL in a volume of 1.5 mL per tube, which is 1.5 x 106 cells to 15 x 106 cells (page 4, paragraph 4). Chen does not teach “airtight container”. Chen teaches 1500 µL (1.5 mL) but does not teach 250 µL to 1000 µL.
Regarding claim 18, Chen teaches the cell dispersion comprises IMDM/F12 and does not teach KOSR (page 3).
Regarding claim 20, Chen teaches a preferred embodiment of 3 to 10 v/v% DMSO (page 3).
Chen does not teach “Y-27632” or “human” iPS cells of claim 5 or 250 µL to 1000 µL of claim 13. However, Chen teaches the use of iPSCs to treat disease is the ultimate goal and iPSCs can be used for differentiation and transplantation and provide disease models to study the mechanisms of disease formation, screen new drugs, and develop new treatments and are therefore of great value in research and clinical applications (page 2, paragraph 6). Chen teaches cell cryopreservation can preserve cell characteristics so that the cells are resuscitated for experimentation when needed (page 2, paragraph 7). Chen teaches to minimize cell damage during cryopreservation and resuscitation, chemical and temperature manipulations must be further optimized (page 2, paragraph 8). Chen teaches the cryopreservation solution used to disperse iPSCs ensures the safety of the cells (page 3, last paragraph).
Regarding “Y-27632” and “human” of claim 5, Mollamohammadi teaches cryopreserved human iPSCs (hiPSCs) with ROCK inhibitor Y-27632 retained typical morphology, stable karyotype, expression of pluripotency markers, and the potential to differentiate into derivatives of all three germ layers (Abstract).
Regarding cell density of claim 13, Mollamohammadi teaches frozen dissociated hiPSCs with Y-27632 at 1 – 2 x 106 cells per 250 µl freezing medium in a vial stored in liquid nitrogen (page 2469, right col. paragraph 2). Mollamohammadi teaches effective freezing/thawing of single dissociated hiPSCs in a feeder-free culture in the presence of Y-27632 (page 2473, left col. paragraph 1; page 2475, left col. paragraph 2). Mollamohammadi teaches Y-27632 is safe for use with pluripotent stem cells (page 2473, left col. paragraph 1). Mollamohammadi teaches Y-27632 no only increased the survival rate but also the adhesion of frozen-thawed dissociated single pluripotent stem cells (page 2473, right col. paragraph 2). Mollamohammadi teaches applications involving hiPSCs-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation (page 2469, left col. paragraph 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 Chen regarding a frozen cell dispersion of iPSCs comprising a ROCK inhibitor with the teachings of Mollamohammadi regarding cryopreserved hiPSCs with Y-27632 to arrive at the claimed composition of frozen IMDM/F12, 5000 hiPSCs per µL, and 1 ng/mL Y-27632 in an airtight container. One would have been motivated to combine the teachings of Chen and Mollamohammadi to arrive at the claimed composition as Chen teaches iPSCs provide disease models to study the mechanisms of disease formation, screen new drugs, and develop new treatments and are therefore of great value in research and clinical applications and Chen teaches to minimize cell damage during cryopreservation and resuscitation, chemical and temperature manipulations must be further optimized and Mollamohammadi teaches applications involving hiPSCs-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation. One would have a reasonable expectation of success in combining the teachings as Chen teaches the cryopreservation solution used to disperse iPSCs ensures the safety of the cells which showed 91% viability when thawed and Mollamohammadi teaches effective freezing/thawing of single dissociated hiPSCs in the presence of Y-27632 and Mollamohammadi teaches Y-27632 is safe for use with pluripotent stem cells.
9. Claim(s) 9 – 11 remain rejected under 35 U.S.C. 103 as being unpatentable over Chen (CN105087472A; previously cited), hereinafter Chen in view of Mollamohammadi (Mollamohammadi S, et. al. Hum Reprod. 2009 Oct;24(10):2468-76; previously cited), hereinafter Mollamohammadi as applied to claims 5, 13, 16, 18, and 20 above, and further in view of FDA (Content and Review of Chemistry, Manufacturing, and Control Information for Human Somatic Cell Therapy Investigational New Drug Applications, 2008; previously cited), hereinafter FDA, in view of USP-71 (USP 71 Sterility Tests, 2012; previously cited), hereinafter USP-71, in view of USP-671 (USP 671 Containers Performance Testing, 2007; previously cited), hereinafter USP-671, in view of SKS (Torque Guide, June 28, 2017; previously cited), hereinafter SKS.
Chen in view of Mollamohammadi make obvious the limitations of claim 5 as set forth above but do not teach claims 9 – 11. However, Chen teaches the frozen cells are in a cryotube that is placed in a cryobox and by properly storing a certain amount of cells, it is possible to prevent the cells from being contaminated due to contamination (page 2, paragraph 7; page 4, paragraph 4; page 5, 14 – 16).
Regarding claims 9 and 10, FDA teaches product testing for cellular therapies include, but not limited to, microbiological testing (including sterility, mycoplasma, and adventitious viral agent testing) to ensure safety (page 14). FDA teaches product testing is an integral part of ensuring control of the manufacturing process and lot to lot consistency and therefore it is important to identify critical parameters in the manufacturing process and critical product attributes to ensure the desired clinical effect of the final product (page 14). FDA teaches performing microbiological testing on cell banks, in-process intermediates, and the final product (page 14). FDA teaches suitable sterility tests include the test described in United States Pharmacopoeia (USP) <71> Sterility Tests. USP-71 teaches the test is applied to substances, preparations, or articles which, according to the Pharmacopeia, are required to be sterile (page 69). USP-71 teaches if no evidence of microbial growth is found, the product to be examined complies with the test for sterility (page 74).
Regarding claim 11, USP-671 teaches standards for the functional properties of plastic containers and their components used to package regulated articles (pharmaceuticals, biologics, dietary supplements, and devices) (page 1). USP-671 teaches standards and tests measure the functional and performance characteristics of plastic containers used to package aqueous products by measuring the liquid water weight loss as a percent of contents (page 3). USP-671 teaches applying a torque that is within the range specified in Table 1 if using screw closures (page 3). USP-671 teaches unit-dose containers for liquids meet the requirements of a tight container if the average water weight loss is less than or equal to 2.5% loss per year (page 3). SKS teaches over-torquing may cause some points on the cap to have more pressure than others, which could result in an improper seal and if the cap is applied with too little torque, the container may leak or the closure could come off too easily (page 1). SKS teaches as a general rule, the amount of torque is about half of the diameter of the cap (page 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 Chen regarding a frozen cell dispersion of iPSCs comprising a ROCK inhibitor with the teachings of Mollamohammadi regarding cryopreserved hiPSCs with Y-27632 with the teachings of FDA regarding product testing for cellular therapies include, but not limited to, microbiological testing to ensure safety with the teachings USP-671 regarding teaches applying a torque if using screw closures with the teachings of SKS regarding the amount of torque is about half of the diameter of the cap of to arrive at the claimed composition of frozen IMDM/F12, 5000 hiPSCs per µL, and 1 ng/mL Y-27632 in an airtight container that is sterile and satisfies airtightness criterion of applied torque of 6.0 to 16.9 in[Symbol font/0xB7]oz. One would have been motivated to combine the teachings of Chen, Mollamohammadi, FDA, USP-671, and SKS to arrive at the claimed composition as Chen teaches iPSCs can be used for differentiation and transplantation and are of great value in clinical applications and by properly storing a certain amount of cells, it is possible to prevent the cells from being contaminated due to contamination and Mollamohammadi teaches applications involving hiPSCs-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation and FDA teaches it is important to identify critical parameters in the manufacturing process and critical product attributes to ensure the desired clinical effect of the final product. One would have a reasonable expectation of success in combining the teachings as Chen teaches the cryopreservation solution used to disperse iPSCs ensures the safety of the cells which showed 91% viability when thawed.
10. Claim(s) 5, 13, 16, 18, 20, and 21 remain rejected under 35 U.S.C. 103 as being unpatentable over Chen (CN105087472A; previously cited), hereinafter Chen in view of Mollamohammadi (Mollamohammadi S, et. al. Hum Reprod. 2009 Oct;24(10):2468-76; previously cited), hereinafter Mollamohammadi.
Chen in view of Mollamohammadi make obvious the limitations of claim 5 as set forth above. Chen teaches a cryopreservation medium comprising DMSO (“cryoprotectant”) (page 5, Example 3 – 4) but does not teach “does not comprise a culture medium” of claim 21.
Mollamohammadi teaches freezing cells in 10% DMSO plus 90% FCS (condition A) or 10% DMSO plus 90% KOSR (condition B) or 10% DMSO plus culture medium (condition C) (page 2469, right col. para. 2). Mollamohammadi teaches the survival rate of pluripotent stem cells were highest in condition A in Figure 1 (page 2470, right col. para. 2). Mollamohammadi teaches one problem in the development of pluripotent stem cell culture is that these cells are vulnerable to apoptosis upon cellular detachment and dissociation (page 2469, left col. para. 2). Mollamohammadi teaches future applications involving pluripotent stem cell-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation (page 2469, left col. para. 2). Mollamohammadi teaches current freezing approaches of pluripotent stem cells have an extremely poor survival rate, are labor intensive and unsuited for handling bulk quantities of cells (page 2469, left col. para. 2). Mollamohammadi teaches ROCK inhibition with Y-27632 significantly improves the recovery of cryopreserved pluripotent stem cells and their growth upon culture (page 2469, left 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 substitute the culture media of Chen with the FCS of Mollamohammadi to arrive at the claimed dispersion medium comprising DMSO and FCS. One would have been motivated to make such a substitution because Mollamohammadi teaches future applications involving pluripotent stem cell-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation current freezing approaches of pluripotent stem cells have an extremely poor survival rate, are labor intensive and unsuited for handling bulk quantities of cells. One would have a reasonable expectation of success in carrying out the substitution because Mollamohammadi teaches condition A results in a higher cell survival rate than condition C comprising a cell culture media.
11. Claim(s) 5, 13, 16, 18, 20, and 22 remain rejected under 35 U.S.C. 103 as being unpatentable over Chen (CN105087472A; previously cited), hereinafter Chen in view of Mollamohammadi (Mollamohammadi S, et. al. Hum Reprod. 2009 Oct;24(10):2468-76; previously cited), hereinafter Mollamohammadi.
Chen in view of Mollamohammadi make obvious the limitations of claim 5 as set forth above. Chen teaches thiazovivin is a ROCK inhibitor and teaches pluripotent stem cells dispersed in medium comprising thiazovivin, culture media, and DMSO for cryopreservation (page 5, Example 3 – 4). Therefore, Chen does not teach the medium excludes thiazovivin of claim 22. Chen teaches in Table 1 that the medium comprising thiazovivin results in a survival rate of iPS cells after resuscitation ranging from 92.4% to 84.9% (page 6, para. 7 – 8; Table 1).
Mollamohammadi teaches ROCK inhibition with Y-27632 significantly improves the recovery of cryopreserved pluripotent stem cells and their growth upon culture (page 2469, left col. para. 2). Mollamohammadi teaches freezing cells in a medium with DMSO and Y-27632 (page 2469, right col. para. 2). Mollamohammadi teaches the survival rate of greater than 90% of pluripotent stem cells in FCS + DMSO + Y-27632 in Figure 1 (page 2470, right col. para. 2). Mollamohammadi teaches one problem in the development of pluripotent stem cell culture is that these cells are vulnerable to apoptosis upon cellular detachment and dissociation (page 2469, left col. para. 2). Mollamohammadi teaches future applications involving pluripotent stem cell-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation (page 2469, left col. para. 2). Mollamohammadi teaches current freezing approaches of pluripotent stem cells have an extremely poor survival rate, are labor intensive and unsuited for handling bulk quantities of cells (page 2469, left col. para. 2). Mollamohammadi teaches ROCK inhibition with Y-27632 significantly improves the recovery of cryopreserved pluripotent stem cells and their growth upon culture (page 2469, left 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 substitute the culture media comprising thiazovivin of Chen with the culture media comprising Y-27632 of Mollamohammadi to arrive at the claimed dispersion medium comprising Y-27632 and excluding thiazovivn. One would have been motivated to make such a substitution because Mollamohammadi teaches future applications involving pluripotent stem cell-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation current freezing approaches of pluripotent stem cells have an extremely poor survival rate, are labor intensive and unsuited for handling bulk quantities of cells. One would have a reasonable expectation of success in carrying out the substitution because Mollamohammadi teaches Y-27632 + FCS + DMSO is able to consistently preserve cells with greater than 90% survival rate without thiazovivn.
New Claim Rejections
Claim Rejections - 35 USC § 103
12. Claim(s) 5, 13, 16, 18,and 20 – 22 are rejected under 35 U.S.C. 103 as being unpatentable over Martin (Martin-Ibanez, R., et al. Human reproduction 23.12 (2008): 2744-2754.), hereinafter Martin in view of Mollamohammadi (Mollamohammadi S, et. al. Hum Reprod. 2009 Oct;24(10):2468-76; previously cited), hereinafter Mollamohammadi in view of Chen (CN105087472A; previously cited), hereinafter Chen in view of Nishishita (Nishishita N, et. al. Am J Stem Cells. 2015 Mar 15;4(1):38-49), hereinafter Nishishita. A machine translation of CN105087472A is provided. The translation was performed on 03/11/2025 on pages 1 – 7 of the original document.
Regarding claim 5 and 16, Martin teaches frozen human embryonic stem cells in culture medium supplemented with DMSO in a cryovial (“a dispersion medium”, “pluripotent stem cells dispersed in the dispersion medium”, “airtight container”, “in a frozen state”, “human embryonic stem (ES) cells”) (page 2745, left col. last para. and right col. para.1). Martin teaches the method for obtaining the cells for cryopreservation included harvesting hESCs obtained by splitting hESCs into single cells in the presence of Y-27632 ROCK inhibitor (“ROCK inhibitor comprises Y-27632”; Figure 2). Martin teaches freezing between 30,000 – 100,000 hESCs (page 2745, left col. last para.). Martin does not teach “a concentration of Y-27632 in the cell dispersion is 0.5 ng/mL to 1 ng/mL, and a concentration of the pluripotent stem cells per unit volume of the cell dispersion is 5 x 102 cells/µL to 5 x 103 cells/µL” of claim 5.
Regarding claim 20, Martin teaches 10% DMSO (page 2745, left col. last para.). Because the taught percentage of DMSO is so close to the claimed range, one of ordinary skill in the art would have expected the taught percentage and claimed percentage to have the same properties with respect to acting as a cryoprotectant (see MPEP 2144.05 (I)).
Regarding claim 22, Martin teaches the ROCK inhibitor is Y-27632 and not thiazovivin (page 2745, left col. last para.).
Martin does not teach “a concentration of Y-27632 in the cell dispersion is 0.5 ng/mL to 1 ng/mL, and a concentration of the pluripotent stem cells per unit volume of the cell dispersion is 5 x 102 cells/µL to 5 x 103 cells/µL” of claim 5 or “1 x 105 cells to 2 x 106 cells, and a volume of the cell dispersion per airtight container is 250 µL to 1000 µL” of claim 13 or “the dispersion medium excludes knock out serum replacement (KOSR)” of claim 18 or “the medium for cryopreservation does not comprise a culture medium” of claim 21. However, Martin teaches the hESCs frozen with residual Y-27632 showed approximately 30% survival when thawed relative to the number of frozen cells (Figure 3A; page 2747, left col. para. 3). Martin teaches Y-27632 plays a role in cell adhesion and the cells frozen with residual Y-27632 showed attached cells and the unattached cells were viable (page 2747, left col. para. 4; Figure 3B). Martin teaches hESCs frozen with residual Y-27632 formed colonies when thawed and cultured with Y-27632 (condition 3-/+ in Figure 3C-D; page 2747, left col. last para. and right col.). Martin teaches Y-27632 treatment after thawing produced a greater effect on colony formation that its presence only in the freezing medium since the number of colonies obtained in condition 3-/+ was significantly higher that in condition 2+/- (Y-27632 added to freezing medium at 10 µm) (page 2748, left col. para. 1; Figure 2 and 3C-D; page 2745, left col. para. 2). Therefore, Martin teaches a cell dispersion of frozen hESCs with residual Y-27632 that are viable, adhesive, and form colonies when thawed and addition of Y-27632 to thawed cells improves viability with increased colony formation. Martin teaches cells were frozen at a rate of -1 °C/min until -80 °C and then stored in liquid nitrogen (page 2745, right col. para. 1).
Regarding “a concentration of Y-27632 in the cell dispersion is 0.5 ng/mL to 1 ng/mL, and a concentration of the pluripotent stem cells per unit volume of the cell dispersion is 5 x 102 cells/µL to 5 x 103 cells/µL” of claim 5, Mollamohammadi teaches hESCs and hiPSCs at 1 – 2 x 106 cells per 250 µL freezing medium were cryopreserved (page 2469, right col. para. 2) which is 4000 – 8000 cells/µL meeting the limitation of the claimed concentration of the pluripotent stem cells per unit volume of the cell dispersion. Mollamohammadi teaches the cells were collected for freezing by the method of Martin (page 2469, right col. para. 2) but does not teach the concentration of Y-27632. Mollamohammadi does not teach “a concentration of Y-27632 in the cell dispersion is 0.5 ng/mL to 1 ng/mL”.
Regarding or “1 x 105 cells to 2 x 106 cells, and a volume of the cell dispersion per airtight container is 250 µL to 1000 µL” of claim 13, Mollamohammadi teaches each vial contained hESCs and hiPSCs at 1 – 2 x 106 cells per 250 µL (page 2469, right col. para. 2).
Regarding “the dispersion medium excludes knock out serum replacement (KOSR)” of claim 18, Mollamohammadi teaches freezing pluripotent stem cells in 10% DMSO with 90% FCS (page 2469, right col. para. 2; page 2470, right col. para. 2).
Regarding “the medium for cryopreservation does not comprise a culture medium” of claim 21, Mollamohammadi teaches freezing pluripotent stem cells in 10% DMSO with 90% FCS and in 10% DMSO with 90% KOSR (page 2469, right col. para. 2; page 2470, right col. para. 2).
Mollamohammadi does not teach “a concentration of Y-27632 in the cell dispersion is 0.5 ng/mL to 1 ng/mL” of claim 5. However, Mollamohammadi teaches in Figure 1 post-thawing viability of pluripotent stem cells cryopreserved in 10% DMSO plus 90% FCS was greater than 90% where cell viability was measured after recovery of cryopreserved cells by culturing in media containing 10 µM Y-27632 (page 2469, right col. para. 3 – 4). Mollamohammadi teaches exposure to Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs but when ROCK inhibitor is added to both pre- and post-thaw culture media there was an enhancement in the survival rate (Abstract; page 2469, right col. para. 2). Therefore, Mollamohammadi teaches in Figure 1 that addition of 10 µM Y-27632 in a freezing medium containing hESC culture medium with KOSR reduces post-thaw viability (page 2469, right col. para. 2; page 2470, right col. para. 2). Mollamohammadi also teaches in Figure 1 that post-thaw viability in the same media varies between the different pluripotent stem cells. Mollamohammadi teaches an essential prerequisite for the future widespread application of hiPSCs and hESCs is the development of efficient cryopreservation methods to facilitate their storage and transportation (Abstract; page 2469, left col. para. 2). Mollamohammadi teaches one problem in the development of hiPSC and hESC culture is that these cells are vulnerable to apoptosis upon cellular detachment and dissociation (page 2469, left col. para. 2). Mollamohammadi teaches that Y-27632 significantly improves the recovery of cryopreserved hiPSCs and their growth upon subculture (page 2469, left col. para. 2). One would have been motivated to combine the teachings of Martin and Mollamohammadi because both teach the same method for harvesting pluripotent stem cells for cryopreservation and because Mollmohammadi teaches freezing in 90% FCS results in greater than 90% viability when the cells are thawed.
Regarding “a concentration of Y-27632 in the cell dispersion is 0.5 ng/mL to 1 ng/mL” of claim 5, Chen teaches a frozen cell dispersion consisting of a dispersion medium comprising and iPSCs dispersed in the dispersion medium and a ROCK inhibitor thiazovivin wherein the concentration of ROCK inhibitor is 1 ng – 1000 ng per 100 mL (0.01 ng/mL to 10 ng/mL) with a preferred embodiment of 1.1 ng to 100 ng per 100 mL (0.011 ng/mL to 1 ng/mL) (page 3; page 4, paragraph 3 – 4). Chen teaches the concentration of the iPSCs is 1 to 10 x 106/mL (1000 – 10,000 cells/µL) (page 4, paragraph 4). Chen teaches 2 – 20% DMSO in the dispersion medium (page 3, para. 3). Chen teaches after 3 months of cryopreservation the viability of iPS cells frozen in the cryopreservation solution was 91% or more (page 4, paragraph 5). One would have been motivated to combine the teachings of Martin, Mollamohammadi, and Chen because Martin and Mollamohammadi teach cryopreservation of pluripotent stem cells with residual Y-27632 but do not teach the concentration of the residual Y-27632 and Mollamohammadi teaches 10 µM Y-27632 (which is ~2473 ng/mL based on the molecular weight of 247.3 g/mol) in freezing medium containing culture medium reduces post-thaw survival and Chen teaches a dispersion medium with 0.011 ng/mL to 1 ng/mL ROCK inhibitor maintains 91% viability of cells post-thaw that were cryopreserved for 3 months. It is noted that Martin and Mollamohammadi teach the ROCK inhibitor Y-27632 but do not teach the concentration and Chen teaches the ROCK inhibitor thiazovivin and a concentration of thiazovivin that is 0.011 ng/mL to 1 ng/mL (~0.032 – 3.2 nM based on the molecular weight of thiazovivin of 311.36 g/mol). However, Martin and Mollamohammadi teach that 10 µM Y-27632 added to the freezing medium reduces post-thaw viability (Figure 3C condition 2+/- of Martin and Figure 1 of Mollamohammadi). Further, Mollamohammadi teaches residual Y-27632 in the freezing medium of FCS/DMSO yields much higher post-thaw viability (Figure 1). Therefore, one of ordinary skill in the art would recognize that the residual concentration of Y-27632 in the freezing medium of FCS/DMSO following the method of cryopreservation as taught by Martin and Mollamohammadi is significantly less than 10 µM and would be in the nanomolar range as taught by Chen.
Nishishita teaches dispersions of frozen pluripotent stem cells in Cryovials and methods for finding optimal conditions for cryopreservation to recover viable cells post-thaw (page 39, left col. last para. and right col.; Figure 1; page 43, left col. para. 2). Nishishita teaches cells were treated with 5 µM Y-27632 for 2 hours before dissociation and resuspending the cells in freezing medium (page 39, right col. para. 1). Nishishita teaches selection of optimum dissociation reagent that can generate single cell suspension with minimal cell damage; selection of cryopreservation medium that produces a high recovery rate after thawing of cells; selection of thawing methods that can prevent differentiation and minimize apoptosis of thawed hPSCS (page 43, left col. para. 2). Nishishita teaches all combinations were tested and the best combination of dissociation buffer and freezing medium was used for selecting thawing methods (page 43, left col. para. 2 and right col. para. 1). Nishishita teaches dissociation of KhES-1 cells with x0.75 TrypLE Select followed by cryopreservation with CryoStem showed the best recovery rate for hPSCs after thawing (page 43, right col. para. 3; page 45, left col.). Nishishita teaches the thawing procedure includes warming the cryovials, diluting the cells with medium containing Y-27632, pelleting the cells, and resuspending the cells in fresh media with Y-27632 (page 39, right col. last para.). Nishishita teaches single cell cryopreservation will facilitate quality control of cells (page 47, left col. para. 1). Nishishita teaches loss of cell-cell contact of hPSCs during dissociation into single cells may confer stress to the cytoskeleton structure in addition to the frost damage during freezing and thawing manipulation (page 47, left col. para. 2). Nishishita teaches the dissociation reagent selected would minimize the time needed for dissociation and consequently minimize cell damage in hPSCs during dissociation (page 47, right col. last para.). Nishishita teaches two types of cryopreservation methods are currently used for hPSCs: vitrification and slow-freezing (page 38, right col. para. 2). Nishishita teaches vitrification requires skill in rapid freezing and is not suitable for the cryopreservation of large numbers of hPSCs (page 38, right col. para. 2). Nishishita teaches slow-freezing methods do not require special skills and allow for the cryopreservation of large numbers of hPSCs in a single batch (page 38, 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 Martin regarding a cell dispersion consisting of a frozen dispersion medium and hESCs dispersed in the dispersion medium and Y-27632 in a cryovial with the teachings of Mollamohammadi regarding a cell dispersion of frozen hESCs or hiPSCs in FCS/DMSO with Y-27632 with the teachings of Chen regarding a frozen cell dispersion of pluripotent stem cells where the dispersion contains a ROCK inhibitor at 0.011 ng/mL to 1 ng/mL with the teachings of Nishishita regarding dispersions of frozen pluripotent stem cells with Y-27632 to arrive at the claimed a cell dispersion consisting of a dispersion medium, and pluripotent stem cells dispersed in the dispersion medium and a ROCK inhibitor, wherein the ROCK inhibitor comprises Y-27632, wherein a concentration of Y-27632 in the cell dispersion is 0.5 ng/ml to 1 ng/ml, and a concentration of the pluripotent stem cells per unit volume of the cell dispersion is 5 x 102 cells/μL to 5 x 103 cells/μL, wherein the cell dispersion is contained in an airtight container, wherein the cell dispersion is in a frozen state, wherein the pluripotent stem cells are human induced pluripotent stem (iPS) cells or human embryonic stem (ES) cells, and wherein when the pluripotent stem cells in the cell dispersion in a frozen state are thawed, viability of the pluripotent stem cells is at least 95%. One would have been motivated to combine the teachings of Martin, Mollamohammadi, Chen, and Nishishita in a frozen cell dispersion of pluripotent stem cells as Mollamohammadi teaches an essential prerequisite for the future widespread application of hiPSCs and hESCs is the development of efficient cryopreservation methods to facilitate their storage and transportation and one problem in the development of hiPSC and hESC culture is that these cells are vulnerable to apoptosis upon cellular detachment and dissociation and Nishishita teaches single cell cryopreservation will facilitate quality control of cells. A rationale for arriving at Y-27632 at a concentration of 0.5 ng/mL to 1 ng/mL through routine optimization comes from the teachings of Mollamohammadi, Chen, and Nishishita where Mollamohammadi teaches 10 µM Y-27632 does not enhance post-thaw viability but residual Y-27632 along with a freezing medium of FCS/DMSO results in greater than 90% post-thaw viability and Chen teaches 0.11 ng/mL to 1 ng/mL of ROCK inhibitor thiazovivin results in a cell dispersion that when thawed after 3 months has 91% viability. Nishishita teaches optimizing the cell dispersion medium and method of dissociating cells by selection of optimum dissociation reagent that can generate single cell suspension with minimal cell damage; selection of cryopreservation medium that produces a high recovery rate after thawing of cells; selection of thawing methods that can prevent differentiation and minimize apoptosis of thawed hPSCS. Taken together, Mollamohammadi, Chen, and Nishishita provide a rationale for one of ordinary skill in the art to arrive at the claimed concentration of Y-27632 with a reasonable expectation of success as Mollamohammadi teaches residual Y-27632 in pluripotent stem cells frozen in FCS/DMSO results in cells that when thawed have greater than 90% viability and Chen teaches the range of ROCK inhibitor for cryopreservation of pluripotent stem cells also result in cells that when thawed have greater than 90% viability. Further, a rationale for arriving at a frozen cell dispersion that when thawed has a viability of at least 95% through routine optimization comes from the teachings of Martin, Mollamohammadi, and Nishishita where each teach that post-thaw cell viability was measured after culturing cells with Y-27632: Martin teaches a cell dispersion of frozen hESCs with residual Y-27632 that are viable, adhesive, and form colonies when thawed and addition of Y-27632 to thawed cells improves viability with increased colony formation; Mollamohammadi teaches post-thawing cell viability was measured after recovery of cryopreserved cells by culturing in media containing 10 µM Y-27632; Nishishita teaches the thawing procedure includes warming the cryovials, diluting the cells with medium containing Y-27632, pelleting the cells, and resuspending the cells in fresh media with Y-27632. Taken together, Martin, Mollamohammadi, and Nishishita provide a rationale for one of ordinary skill in the art to arrive at the claimed frozen cell dispersion that when thawed has a viability of at least 95% with a reasonable expectation of success as each teach that cell viability is measured after addition of Y-27632 to media containing the thawed cells and culturing the cells. One would have a reasonable expectation of success in combining the teachings as Mollamohammadi teaches in Figure 1 post-thawing viability of pluripotent stem cells cryopreserved in 10% DMSO plus 90% FCS with Y-27632 was greater than 90%.
13. Claim(s) 9 – 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Martin (Martin-Ibanez, R., et al. Human reproduction 23.12 (2008): 2744-2754.), hereinafter Martin in view of Mollamohammadi (Mollamohammadi S, et. al. Hum Reprod. 2009 Oct;24(10):2468-76; previously cited), hereinafter Mollamohammadi in view of Chen (CN105087472A; previously cited), hereinafter Chen in view of Nishishita (Nishishita N, et. al. Am J Stem Cells. 2015 Mar 15;4(1):38-49), hereinafter Nishishita as applied to claims 5, 13, 16, 18,and 20 – 22 above, and further in view of FDA (Content and Review of Chemistry, Manufacturing, and Control Information for Human Somatic Cell Therapy Investigational New Drug Applications, 2008; previously cited), hereinafter FDA, in view of USP-71 (USP 71 Sterility Tests, 2012; previously cited), hereinafter USP-71, in view of USP-671 (USP 671 Containers Performance Testing, 2007; previously cited), hereinafter USP-671, in view of SKS (Torque Guide, June 28, 2017; previously cited), hereinafter SKS.
Martin in view of Mollamohammadi, Chen, and Nishishita make obvious the limitations of claim 5 as set forth above. Martin, Mollamohammadi, Chen, and Nishishita do not teach “airtightness satisfying a criterion requiring that bacterial proliferation not be observed in a sterility test method” of claim 9 or “the sterility test method is a sterility test method carried out by process simulation” of claim 10 or “a measured value with an application torque measuring device be 6.0 to 16.9 in[Symbol font/0xB7]oz” of claim 11. However, Chen teaches the frozen cells are in a cryotube that is placed in a cryobox and by properly storing a certain amount of cells, it is possible to prevent the cells from being contaminated (page 2, paragraph 7; page 4, paragraph 4; page 5, 14 – 16).
Regarding claims 9 and 10, FDA teaches product testing for cellular therapies include, but not limited to, microbiological testing (including sterility, mycoplasma, and adventitious viral agent testing) to ensure safety (page 14). FDA teaches product testing is an integral part of ensuring control of the manufacturing process and lot to lot consistency and therefore it is important to identify critical parameters in the manufacturing process and critical product attributes to ensure the desired clinical effect of the final product (page 14). FDA teaches performing microbiological testing on cell banks, in-process intermediates, and the final product (page 14). FDA teaches suitable sterility tests include the test described in United States Pharmacopoeia (USP) <71> Sterility Tests. USP-71 teaches the test is applied to substances, preparations, or articles which, according to the Pharmacopeia, are required to be sterile (page 69). USP-71 teaches if no evidence of microbial growth is found, the product to be examined complies with the test for sterility (page 74).
Regarding claim 11, USP-671 teaches standards for the functional properties of plastic containers and their components used to package regulated articles (pharmaceuticals, biologics, dietary supplements, and devices) (page 1). USP-671 teaches standards and tests measure the functional and performance characteristics of plastic containers used to package aqueous products by measuring the liquid water weight loss as a percent of contents (page 3). USP-671 teaches applying a torque that is within the range specified in Table 1 if using screw closures (page 3). USP-671 teaches unit-dose containers for liquids meet the requirements of a tight container if the average water weight loss is less than or equal to 2.5% loss per year (page 3). SKS teaches over-torquing may cause some points on the cap to have more pressure than others, which could result in an improper seal and if the cap is applied with too little torque, the container may leak or the closure could come off too easily (page 1). SKS teaches as a general rule, the amount of torque is about half of the diameter of the cap (page 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 Martin regarding a cell dispersion consisting of a frozen dispersion medium and hESCs dispersed in the dispersion medium and Y-27632 in a cryovial with the teachings of Mollamohammadi regarding a cell dispersion of frozen hESCs or hiPSCs in FCS/DMSO with Y-27632 with the teachings of Chen regarding a frozen cell dispersion of pluripotent stem cells where the dispersion contains a ROCK inhibitor at 0.011 ng/mL to 1 ng/mL with the teachings of Nishishita regarding dispersions of frozen pluripotent stem cells with Y-27632 with the teachings of FDA regarding product testing for cellular therapies include, but not limited to, microbiological testing to ensure safety with the teachings USP-671 regarding teaches applying a torque if using screw closures with the teachings of SKS regarding the amount of torque is about half of the diameter of the cap of to arrive at the claimed composition wherein airtightness of the airtight container satisfies a criterion requiring that bacterial proliferation not be observed in a sterility test method and satisfies an airtightness criterion of applied torque of 6.0 to 16.9 in[Symbol font/0xB7]oz. One would have been motivated to combine the teachings of Martin, Mollamohammadi, Chen, Nishsishita, FDA, USP-671, and SKS to arrive at the claimed composition as Chen teaches it is possible to prevent the cells frozen in a cryotube from being contaminated with proper handling and Mollamohammadi teaches applications involving hiPSCs-derived cells or tissues depend upon establishment of an efficient cryopreservation protocol to facilitate their storage and transportation and Nishishita teaches single cell cryopreservation will facilitate quality control of cells and FDA teaches it is important to identify critical parameters in the manufacturing process and critical product attributes to ensure the desired clinical effect of the final product. One would have a reasonable expectation of success in combining the teachings as Mollamohammadi and Chen teach cryopreserved pluripotent stem cells with ROCK inhibitor showed viability greater than 90% when thawed.
Applicant’s Arguments/ Response to Arguments
14. Applicant Argues: On page 5, Applicant asserts that the pending claims are not obvious as amended.
Response to Arguments: Upon further consideration, the limitations of claim 23 are not allowable because the claimed cell dispersion made obvious by combining the teachings of Chen and Mollamohammadi would be capable of a viability of at least 95% when thawed absent any arguments to the contrary because the combined teachings of Chen and Mollamohammadi make obvious the limitations of claim 5. Further, in the new claim rejections set forth above, Martin, Mollamohammadi, and Nishishita teaches that viability of thawed pluripotent cells are measured after culturing with Y-27632 and that it is the addition of Y-27632 post-thaw and the nature of the cell freezing medium and method of cell freezing that is primarily responsible for the increased cell viability because Martin teaches cells frozen with residual or 10 µM Y-27632 in culture media have at maximum 50% cell viability relative to the number of cells frozen; Mollamohammadi teaches cells frozen in FCS/DMSO with residual Y-27632 and cultured with 10 µM Y-27632 post-thaw have cell viability greater than 90%; and Nishishita teaches optimization of dissociation agent, cryopreservation medium, and thawing methods are responsible for post-thaw cell viability. Therefore, given the teachings of Martin and Mollamohammadi regarding freezing cells in residual Y-27632 in FCS/DMSO and Chen regarding ng/mL concentration of ROCK inhibitor for cryopreservation, one would have reached the claimed concentration of Y-27632 through routine optimization. Further, given the teachings of Martin, Mollamohammadi, and Nishishita regarding post-thaw culturing in Y-27632, one would have reached the claimed concentration of Y-27632 in the cell dispersion medium resulting cell viability of at least 95% when cells are thawed and recovered through routine optimization.
Conclusion
No claims allowed.
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/Z.M.B./Examiner, Art Unit 1632
/ANOOP K SINGH/Primary Examiner, Art Unit 1632