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 .
1. Claims 1 – 11, 16, 18, 19, and 21 – 31 are pending and under consideration.
Priority
2. This application claims priority to JP2020-120695 filed on 07/14/2020.
3. 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.
Information Disclosure Statement
4. The information disclosure statement (IDS) submitted on 12/23/2025 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Withdrawn Specification Objection
5. The objection to the specification is withdrawn in view of Applicant’s amendment to the specification.
Withdrawn Claim Objection
6. The objection to claim 10 is withdrawn in view of Applicant’s amendment to the claim.
7. The objection to claim 18 is withdrawn in view of Applicant’s amendment to the claim.
Withdrawn Claim Rejections
8. The rejection of claims 1, 3, 5 – 11, and 16 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to claim 1.
9. The rejection of claim 5 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to the claim.
10. The rejection of claim 6 under 35 U.S.C. 112(b) is rendered moot in view of Applicant’s cancellation of the claim.
11. The rejection of claim 7 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to the claim.
12. The rejections of claim 9 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to the claim.
13. The rejection of claim 16 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to the claim.
14. The rejection of claims 2 and 17 – 19 under 35 U.S.C. 102(a)(1) is withdrawn in view of Applicant’s amendment to claim 2 reciting “human pluripotent stem cells” because Bartholoma teaches chicken cardiomyocytes.
15. The rejection of claims 6 and 17 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of these claims.
16. The rejection of claims 1 – 5, 7 – 11, 16, 18, and 19 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to independent claims 1 and 2 and dependent claim 16.
Maintained Claim Objections
17. Claim 1 remains objected to because the claim is awkwardly constructed, making it difficult to discern and because in line 5, “on and orbital” should read “on an orbital”. Appropriate correction is required.
Claim Interpretation
18. For the purpose of applying prior art, claim 1 is interpreted as comprising a single active method step of aggregation of dissociated cardiomyocytes under suspension conditions in a substantially flat-bottomed container on an orbital shaker with a rotation speed of less than or equal to 200 rpm. Recitation of “after a period of stabilization culture which occurs after thawing” is interpreted as conditional limitations and not active steps.
19. For the purpose of applying prior art, “flow under suspension conditions” of claims 1 and 2 is interpreted as dynamic culturing conditions and not static culturing.
20. For the purpose of applying prior art, “cardiomyocytes are derived from human pluripotent stem cells” is interpreted as the cardiomyocytes are human and “derived from…” is interpreted as a product-by-process limitation.
21. For the purpose of applying prior art, “a non-annular container” of claim 2 is interpreted to include a petri dish, a 6-well plate, a 12-well plate, and a flask as shown in Applicant’s Figure 1 and at page 6, 0013 of Applicant’s specification.
22. For the purpose of applying prior art, “substantially flat-bottomed” of claim 1 and 2 is interpreted to exclude round-bottom and V-bottom plates and include a petri dish based on Applicant’s specification at page 6, 0013 and Figure 1.
23. For the purpose of applying prior art, the “wherein” clause of claims 3 and 4 are not given patentable weight as claim 1 does not recite an active step of stabilization culture.
24. For the purpose of applying prior art, claim 5 is interpreted as the time for formation of beating cardiomyocyte spheroids is within 216 hours.
25. For the purpose of applying prior art, claim 10 and 18 are interpreted as suspension culturing for 26 hours or more in a medium that does not contain one or more components selected from the group consisting of insulin, transferrin, selenium, bFGF, EGF, PDGF-BB, and ET-1 based on Applicant’s specification at page 3, para. 4 and page 13, 0026.
26. For the purpose of applying prior art, the “wherein” limitation of claims 11 and 19 are not given patentable weight as the limitation “derived from a pluripotent stem cell(s)” is a product-by-process limitation (see MPEP 2113).
27. For the purpose of applying prior art, claim 16 is interpreted as the method of claim 2 further comprising a stabilization culture step before the aggregation step wherein the stabilization culture is adherent culture.
28. For the purpose of applying prior art, “derived from a pluripotent stem cells” of claim 25 is interpreted as a product-by-process limitation (see MPEP 2113).
29. For the purpose of applying prior art, “the cells” in line 3 of claim 27 is interpreted as cardiomyocytes and the cardiomyocytes are human where “derived from human pluripotent stem cells” is a product-by-process limitation.
30. For the purpose of applying prior art, “adherent stabilization” is interpreted as adherent culturing and the time for formation of beating cardiomyocyte spheroids is within 216 hours.
Rejections Necessitated by Amendment
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
31. Claim 19 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 19 fails to further limit claim 2 because claim 2 has been amended to recite “derived from human pluripotent stem cells” and claim 19 recites “derived from a pluripotent stem cell”. Therefore, claim 19 broadens claim 2 because claim 2 recites “human pluripotent stem cell” while claim 19 broadly recites “a pluripotent stem cell”. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
32. Claim 25 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 25 fails to further limit claim 22 because claim 2 has been amended to recite “derived from human pluripotent stem cells” and claim 25 recites “derived from a pluripotent stem cell”. Therefore, claim 25 broadens claim 22 because claim 2 recites “human pluripotent stem cell” while claim 25 broadly recites “a pluripotent stem cell”. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
33. Claim(s) 1, 3, 4, 5, 8, 9, 10, 11 and 29 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Bartholoma (Bartholomä, P., et al. Journal of biomolecular screening 10.8 (2005): 814-822; previously cited), hereinafter Bartholoma.
Claim 1 is drawn to method for producing cardiomyocyte spheroids, comprising an aggregation step of allowing dissociated cardiomyocytes, after a period of stabilization culture which occurs after thawing said dissociated cardiomyocytes and before production of said cardiomyocyte spheroids, to flow under suspension conditions in a substantially flat-bottomed container on and orbital shaker with a rotation speed of not more than 200 rpm to aggregate the cells.
Regarding claims 1, 3, 4, 8, and 11, Bartholoma teaches a method of forming cardiomyocyte spheroids by reaggregating (“an aggregation step” of claim 1, 3, 4) a suspension of cardiomyocytes that were dissociated from hearts of chicken embryos (“dissociated cardiomyocytes” of claim 1, 3, 4, and 11) in 35-mm petri dishes (“substantially flat-bottomed” of claim 1, 3, 4) on a gyratory shaker (“flow under suspension conditions” of claim 1, 3, 4) at a rotation speed of 75 rpm (“not more than 200 rpm” of claim 1, 3, 4 and claim 8) at a 12.3 mm radius (claim 9) (page 814, right col. last para.; page 815, left col. para. 1; page 816, right col. para. 2; Figure 2B – D; page 818, right col. para. 2 and last para.).
Regarding claim 5, Bartholoma teaches beating spheroids 24 hours to 48 hours after reaggregation (page 816, right col. para. 2; page 819, right col.).
Regarding claim 10, Bartholoma teaches the composition of the media during suspension culturing that was changed daily does not contain bFGF, EGF, PDGF-BB or ET-1 (page 815, left col. para. 1).
Regarding claim 29, Bartholoma teaches 2.4 x 106 cells were reaggregated (page 815, left col. para. 1; Figure 1).
Therefore, Bartholoma anticipates claims 1, 3, 4, 5, 8, 9, 10, 11 and 29.
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.
34. Claim(s) 2, 7, 18, 19, 22, 25, and 30 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bartholoma (Bartholomä, P., et al. Journal of biomolecular screening 10.8 (2005): 814-822; previously cited), hereinafter Bartholoma in view of Kido (Kido, Koji, et al. Journal of anesthesia 32.1 (2018): 120-131.), hereinafter Kido.
Regarding claims 2, 7, 19, 22, and 25, Bartholoma teaches a method of forming cardiomyocyte spheroids by reaggregating (“an aggregation step” of claim 2, 19, 25) a suspension of cardiomyocytes that were dissociated from hearts of chicken embryos (“dissociated cardiomyocytes” of claim 2, 19, 25) in 35-mm petri dishes (“substantially flat-bottomed” of claim 2, 19, 25 and claim 7) on a gyratory shaker (“flow under suspension conditions” of claim 2, 19, 25) at a rotation speed of 75 rpm (“not more than 200 rpm” of claim 2, 19, 25, and claim 22) at a 12.3 mm radius (page 814, right col. last para.; page 815, left col. para. 1; page 816, right col. para. 2; Figure 2B – D; page 818, right col. para. 2 and last para.). Bartholoma does not teach human cardiomyocytes of claim 2.
Regarding claim 18, Bartholoma teaches the composition of the media during suspension culturing that was changed daily does not contain bFGF, EGF, PDGF-BB or ET-1 (page 815, left col. para. 1).
Regarding claim 30, Bartholoma teaches 2.4 x 106 cells were reaggregated (page 815, left col. para. 1; Figure 1).
Bartholoma does not teach human cardiomyocytes of claim 2 or “stabilization culture” of claim 16. However, Bartholoma teaches to understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are required (Abstract; page 814, left col. para. 1). Bartholoma teaches the creation of 3D cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo (Abstract). Bartholoma teaches therapeutically directed studies profit from the use of 3D cell aggregates, allowing the investigation of the effects of various novel drug-mediated therapies (page 814, left col. para. 1). Bartholoma teaches beating spheroids 24 hours to 48 hours after reaggregation (page 816, right col. para. 2; page 819, right col.). Bartholoma teaches the spheroid model produced by the method was used for investigating the effect of isoproterenol on the contractibility of the spheroids and isoproterenol influences the contractility of the heart muscle in humans (Abstract; page 818, right col. para. 2).
Regarding human cardiomyocytes of claim 2, Kido teaches human iPSC-derived cardiomyocytes (ReproCardio2) were used to understand the effect of propofol on human cardiomyocytes (Abstract; page 121, left col. last para. and right col. para. 2 – 3).
Regarding “stabilization culture” of claim 16, Kido teaches culturing thawed ReproCardio2 cells and adherent culture prior to treating the cells with propofol (page 121, left col. last para. and right col. para. 2 – 3).
Kido teaches compared with experimental models using human tissues or embryonic stem cell lines, models to test drug toxicity using iPSC cell lines can be easily established with fewer ethical concerns (page 121, left col. para. 2). Kido teaches propofol infusion syndrome (PRIS) is a rare but lethal condition caused by prolonged propofol overdose (page 120, left col.). Kido teaches the limited availability of human samples and ethical issues have meant that the mechanism of PRIS has not been completely elucidated (Abstract; page 120, right col. para. 1). Kido teaches a need for high-throughput assays for human drug toxicity (page 127, left col.). Kido teaches a study reported that genes associated with mitochondrial energy metabolism in iPSC-derived cardiomyocytes showed lower expression levels than native cardiac tissue but these cells had potential as a screening model for drug-induced mitochondrial toxicity (page 127, right col.). Kido teaches propofol-induced cytotoxicity in human iPSC-derived cardiomyocytes may be associated with mitochondrial dysfunction via downregulated PGC1alpha-regulated genes (Abstract).
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 chicken cardiomyocytes of Bartholoma with the ReproCardio2 human iPSC-derived cardiomyocytes of Kido to arrive at the claimed method for producing cardiomyocyte spheroids, comprising an aggregation step of allowing dissociated cardiomyocytes to flow under suspension conditions in a non-annular substantially flat-bottomed container to aggregate the cells, wherein the cardiomyocytes are derived from human pluripotent stem cells, and wherein aggregation occurs in said container while said container is rotated on an orbital shaker with a rotation speed of not more than 200 rpm. One would have been motivated to make the substitution to study drug toxicity on human cardiomyocyte spheroids because Bartholoma teaches to understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are and Bartholoma teaches the creation of 3D cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo and Bartholoma teaches therapeutically directed studies profit from the use of 3D cell aggregates, allowing the investigation of the effects of various novel drug-mediated therapies and Kido teaches a need for high-throughput assays for human drug toxicity. One would have a reasonable expectation of success in making the substitution as Bartholoma teaches the method produced beating cardiomyocyte spheroids that were used to investigate the effect of isoproterenol on the contractibility of the spheroids and isoproterenol influences the contractility of the heart muscle in humans.
35. Claim(s) 1, 3, 4, 5, 8, 9, 10, 11, 21, 24, 28, and 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bartholoma (Bartholomä, P., et al. Journal of biomolecular screening 10.8 (2005): 814-822; previously cited), hereinafter Bartholoma in view of Muller (Muller, Natalie, et al. Biotechnology and bioengineering 89.4 (2005): 400-406.), hereinafter Muller in view of Kido (Kido, Koji, et al. Journal of anesthesia 32.1 (2018): 120-131.), hereinafter Kido.
Bartholoma anticipates claims 1, 3, 4, 5, 8, 9, 10, 11 and 29 as set forth above. Batholomoa does not teach “a rotation speed of 90 rpm or more and less than 120 rpm” of claim 21 or “orbit is 20 mm or more” of claim 24, or “adherent stabilization culture” of claim 28. However, Bartholoma teaches to understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are required (Abstract; page 814, left col. para. 1). Bartholoma teaches the creation of 3D cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo (Abstract). Bartholoma teaches therapeutically directed studies profit from the use of 3D cell aggregates, allowing the investigation of the effects of various novel drug-mediated therapies (page 814, left col. para. 1). Bartholoma teaches beating spheroids 24 hours to 48 hours after reaggregation (page 816, right col. para. 2; page 819, right col.). Bartholoma teaches the spheroid model produced by the method was used for investigating the effect of isoproterenol on the contractibility of the spheroids and isoproterenol influences the contractility of the heart muscle in humans (Abstract; page 818, right col. para. 2).
Regarding “a rotation speed of 90 rpm or more and less than 120 rpm” of claim 21 or “orbit is 20 mm or more” of claim 24, Muller teaches mammalian cell culture using an orbital shaker at 120 rpm and 105 rpm, and speeds below 100 rpm (claim 21) at a rotational diameter of 2.5 cm (claim 24) (page 401, right col. last para.; page 402, left col. para. 2 and right col. para. 2 – 3). Muller teaches observations have shown that 120 rpm prevented both the settling of cells and the foaming of the medium (page 401, right col. last para.). Muller teaches culture volume is critical because the headspace serves as a reservoir for oxygen and carbon dioxide and it affects foam formation (page 402, left col. para. 2). Muller teaches culture volumes less than 300 mL yielded good growth when the speed was decreased from 120 rpm to 105 rpm (page 402, left col. para. 2). Muller teaches agitation speed can improve the rate of oxygen transfer and at agitation speeds below 100 rpm, cells settled on the bottom of the vessel (page 402, right col. para. 2). Muller teaches a rotation diameter of 5 cm, volume of 300 – 400 mL and agitation speed of 110 rpm, which showed the same cell growth as 130 rpm with a rotational diameter of 2.5 cm (page 403, left col.). Muller teaches cell lines other than HEK293 can be used with the culture system (page 404, right col. para. 1). One would have been motivated to combine the teachings of Bartholoma and Muller for improved cell growth as both teach culturing on orbital shakers and Muller teaches agitation speed can improve the rate of oxygen transfer and at agitation speeds below 100 rpm, cells settled on the bottom of the vessel. Muller does not teach “adherent stabilization culture” of claim 28.
Regarding “adherent stabilization culture” of claim 28, Kido teaches culturing thawed ReproCardio2 (human iPSC-derived cardiomyocytes) cells and adherent culture prior to treating the cells with propofol (page 121, left col. last para. and right col. para. 2 – 3).
Kido teaches compared with experimental models using human tissues or embryonic stem cell lines, models to test drug toxicity using iPSC cell lines can be easily established with fewer ethical concerns (page 121, left col. para. 2). Kido teaches propofol infusion syndrome (PRIS) is a rare but lethal condition caused by prolonged propofol overdose (page 120, left col.). Kido teaches the limited availability of human samples and ethical issues have meant that the mechanism of PRIS has not been completely elucidated (Abstract; page 120, right col. para. 1). Kido teaches a need for high-throughput assays for human drug toxicity (page 127, left col.). Kido teaches a study reported that genes associated with mitochondrial energy metabolism in iPSC-derived cardiomyocytes showed lower expression levels than native cardiac tissue but these cells had potential as a screening model for drug-induced mitochondrial toxicity (page 127, right col.). Kido teaches propofol-induced cytotoxicity in human iPSC-derived cardiomyocytes may be associated with mitochondrial dysfunction via downregulated PGC1alpha-regulated genes (Abstract).
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 Bartholoma regarding a method of producing cardiomyocyte spheroids by culturing cardiomyocytes in an orbital shaker at 75 rpm with the teachings of Muller regarding optimizing cell growth on an orbital shaker at different culture volumes where the agitation speed is 100 – 120 rpm and rotational orbit is 2.5 cm to prevent cells from settling with the teachings of Kido regarding thawing and adherent culture of human iPSC-derived cardiomyocytes for drug testing to arrive at the claimed method wherein the flow is performed at a rotation speed of 90 rpm or more and less than 120 rpm and wherein aggregation occurs in said container while said container is rotated on said orbital shaker where the orbit is 20 mm or more. One would have been motivated to combine the teachings of Bartholoma, Muller, and Kido in a method of producing human cardiomyocyte spheroids to study drug toxicity as Bartholoma teaches to understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are and Bartholoma teaches the creation of 3D cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo and Bartholoma teaches therapeutically directed studies profit from the use of 3D cell aggregates, allowing the investigation of the effects of various novel drug-mediated therapies and Kido teaches a need for high-throughput assays for human drug toxicity. One would have a reasonable expectation of success in making the substitution as Bartholoma teaches the method produced beating cardiomyocyte spheroids that were used to investigate the effect of isoproterenol on the contractibility of the spheroids and isoproterenol influences the contractility of the heart muscle in humans and Muller teaches cell lines other than HEK293 can be used with the culture system.
36. Claim(s) 23 and 26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bartholoma (Bartholomä, P., et al. Journal of biomolecular screening 10.8 (2005): 814-822; previously cited), hereinafter Bartholoma in view of Kido (Kido, Koji, et al. Journal of anesthesia 32.1 (2018): 120-131.), hereinafter Kido as applied to claims 2, 7, 18, 19, 22, 25, and 30 above, and further in view of Muller (Muller, Natalie, et al. Biotechnology and bioengineering 89.4 (2005): 400-406.), hereinafter Muller.
Bartholoma in view of Kido make obvious the limitations of claims 2 and 22 as set forth above. Bartholoma and Kido do not teach “a rotation speed of 90 rpm or more and less than 120 rpm” of claim 23 or “orbit is 20 mm or more” of claim 26.
Regarding “a rotation speed of 90 rpm or more and less than 120 rpm” of claim 23 or “orbit is 20 mm or more” of claim 26, Muller teaches mammalian cell culture using an orbital shaker at 120 rpm and 105 rpm, and speeds below 100 rpm (claim 23) at a rotational diameter of 2.5 cm (claim 26) (page 401, right col. last para.; page 402, left col. para. 2 and right col. para. 2 – 3). Muller teaches observations have shown that 120 rpm prevented both the settling of cells and the foaming of the medium (page 401, right col. last para.). Muller teaches culture volume is critical because the headspace serves as a reservoir for oxygen and carbon dioxide and it affects foam formation (page 402, left col. para. 2). Muller teaches culture volumes less than 300 mL yielded good growth when the speed was decreased from 120 rpm to 105 rpm (page 402, left col. para. 2). Muller teaches agitation speed can improve the rate of oxygen transfer and at agitation speeds below 100 rpm, cells settled on the bottom of the vessel (page 402, right col. para. 2). Muller teaches a rotation diameter of 5 cm, volume of 300 – 400 mL and agitation speed of 110 rpm, which showed the same cell growth as 130 rpm with a rotational diameter of 2.5 cm (page 403, left col.). Muller teaches cell lines other than HEK293 can be used with the culture system (page 404, right col. para. 1). One would have been motivated to combine the teachings of Bartholoma and Muller for improved cell growth as both teach culturing on orbital shakers and Muller teaches agitation speed can improve the rate of oxygen transfer and at agitation speeds below 100 rpm, cells settled on the bottom of the vessel.
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 Bartholoma regarding a method of producing cardiomyocyte spheroids by culturing cardiomyocytes in an orbital shaker at 75 rpm with the teachings of Kido regarding thawing and adherent culture of human iPSC-derived cardiomyocytes for drug testing with the teachings of Muller regarding optimizing cell growth on an orbital shaker at different culture volumes where the agitation speed is 100 – 120 rpm and rotational orbit is 2.5 cm to prevent cells from settling to arrive at the claimed method wherein the flow is performed at a rotation speed of 90 rpm or more and less than 120 rpm and wherein aggregation occurs in said container while said container is rotated on said orbital shaker where the orbit is 20 mm or more. One would have been motivated to combine the teachings of Bartholoma, Kido, and Muller in a method of producing human cardiomyocyte spheroids to study drug toxicity as Bartholoma teaches to understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are and Bartholoma teaches the creation of 3D cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo and Bartholoma teaches therapeutically directed studies profit from the use of 3D cell aggregates, allowing the investigation of the effects of various novel drug-mediated therapies and Kido teaches a need for high-throughput assays for human drug toxicity. One would have a reasonable expectation of success in making the substitution as Bartholoma teaches the method produced beating cardiomyocyte spheroids that were used to investigate the effect of isoproterenol on the contractibility of the spheroids and isoproterenol influences the contractility of the heart muscle in humans and Muller teaches cell lines other than HEK293 can be used with the culture system.
37. Claim(s) 27 and 31 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bartholoma (Bartholomä, P., et al. Journal of biomolecular screening 10.8 (2005): 814-822; previously cited), hereinafter Bartholoma in view of Kido (Kido, Koji, et al. Journal of anesthesia 32.1 (2018): 120-131.), hereinafter Kido in view of Muller (Muller, Natalie, et al. Biotechnology and bioengineering 89.4 (2005): 400-406.), hereinafter Muller.
Regarding claim 27, Bartholoma teaches a method of forming cardiomyocyte spheroids by reaggregating (“an aggregation step”) a suspension of cardiomyocytes that were dissociated from hearts of chicken embryos (“dissociated cardiomyocytes”) in 35-mm petri dishes (“substantially flat-bottomed”) on a gyratory shaker (“flow under suspension conditions”) at a rotation speed of 75 rpm at a 12.3 mm radius (page 814, right col. last para.; page 815, left col. para. 1; page 816, right col. para. 2; Figure 2B – D; page 818, right col. para. 2 and last para.). Bartholoma does not teach human cardiomyocytes, “the orbit is 20 mm to 100 mm” or “the rotation speed of the orbital shaker is 90 to 120 rpm”.
Regarding claim 31, Bartholoma teaches 2.4 x 106 cells were reaggregated (page 815, left col. para. 1; Figure 1).
Bartholoma does not teach human cardiomyocytes, “the orbit is 20 mm to 100 mm” or “the rotation speed of the orbital shaker is 90 to 120 rpm” of claim 27. However, Bartholoma teaches to understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are required (Abstract; page 814, left col. para. 1). Bartholoma teaches the creation of 3D cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo (Abstract). Bartholoma teaches therapeutically directed studies profit from the use of 3D cell aggregates, allowing the investigation of the effects of various novel drug-mediated therapies (page 814, left col. para. 1). Bartholoma teaches beating spheroids 24 hours to 48 hours after reaggregation (page 816, right col. para. 2; page 819, right col.). Bartholoma teaches the spheroid model produced by the method was used for investigating the effect of isoproterenol on the contractibility of the spheroids and isoproterenol influences the contractility of the heart muscle in humans (Abstract; page 818, right col. para. 2).
Regarding human cardiomyocytes of claim 27, Kido teaches human iPSC-derived cardiomyocytes (ReproCardio2) were used to understand the effect of propofol on human cardiomyocytes (Abstract; page 121, left col. last para. and right col. para. 2 – 3).
Kido teaches compared with experimental models using human tissues or embryonic stem cell lines, models to test drug toxicity using iPSC cell lines can be easily established with fewer ethical concerns (page 121, left col. para. 2). Kido teaches propofol infusion syndrome (PRIS) is a rare but lethal condition caused by prolonged propofol overdose (page 120, left col.). Kido teaches the limited availability of human samples and ethical issues have meant that the mechanism of PRIS has not been completely elucidated (Abstract; page 120, right col. para. 1). Kido teaches a need for high-throughput assays for human drug toxicity (page 127, left col.). Kido teaches a study reported that genes associated with mitochondrial energy metabolism in iPSC-derived cardiomyocytes showed lower expression levels than native cardiac tissue but these cells had potential as a screening model for drug-induced mitochondrial toxicity (page 127, right col.). Kido teaches propofol-induced cytotoxicity in human iPSC-derived cardiomyocytes may be associated with mitochondrial dysfunction via downregulated PGC1alpha-regulated genes (Abstract). Kido does not teach “the orbit is 20 mm to 100 mm” or “the rotation speed of the orbital shaker is 90 to 120 rpm” of claim 27. One would have been motivated to substitute the chicken cardiomyocytes of Bartholoma with the human iPSC-derived cardiomyocytes of Kido because both teach the use of cardiomyocytes for testing drug toxicity.
Regarding “a rotation speed of 90 rpm or more and less than 120 rpm” “orbit is 20 mm or more” of claim 27, Muller teaches mammalian cell culture using an orbital shaker at 120 rpm and 105 rpm, and speeds below 100 rpm at a rotational diameter of 2.5 cm (page 401, right col. last para.; page 402, left col. para. 2 and right col. para. 2 – 3). Muller teaches observations have shown that 120 rpm prevented both the settling of cells and the foaming of the medium (page 401, right col. last para.). Muller teaches culture volume is critical because the headspace serves as a reservoir for oxygen and carbon dioxide and it affects foam formation (page 402, left col. para. 2). Muller teaches culture volumes less than 300 mL yielded good growth when the speed was decreased from 120 rpm to 105 rpm (page 402, left col. para. 2). Muller teaches agitation speed can improve the rate of oxygen transfer and at agitation speeds below 100 rpm, cells settled on the bottom of the vessel (page 402, right col. para. 2). Muller teaches a rotation diameter of 5 cm, volume of 300 – 400 mL and agitation speed of 110 rpm, which showed the same cell growth as 130 rpm with a rotational diameter of 2.5 cm (page 403, left col.). Muller teaches cell lines other than HEK293 can be used with the culture system (page 404, right col. para. 1). One would have been motivated to combine the teachings of Bartholoma and Muller for improved cell growth as both teach culturing on orbital shakers and Muller teaches agitation speed can improve the rate of oxygen transfer and at agitation speeds below 100 rpm, cells settled on the bottom of the vessel.
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 Bartholoma regarding a method of producing cardiomyocyte spheroids by culturing cardiomyocytes in an orbital shaker at 75 rpm with the teachings of Kido regarding thawing and adherent culture of human iPSC-derived cardiomyocytes for drug testing with the teachings of Muller regarding optimizing cell growth on an orbital shaker at different culture volumes where the agitation speed is 100 – 120 rpm and rotational orbit is 2.5 cm to prevent cells from settling to arrive at the claimed method wherein the flow is performed at a rotation speed of 90 rpm or more and less than 120 rpm and wherein aggregation occurs in said container while said container is rotated on said orbital shaker where the orbit is 20 mm or more. One would have been motivated to combine the teachings of Bartholoma, Kido, and Muller in a method of producing human cardiomyocyte spheroids to study drug toxicity as Bartholoma teaches to understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are and Bartholoma teaches the creation of 3D cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo and Bartholoma teaches therapeutically directed studies profit from the use of 3D cell aggregates, allowing the investigation of the effects of various novel drug-mediated therapies and Kido teaches a need for high-throughput assays for human drug toxicity. One would have a reasonable expectation of success in making the substitution as Bartholoma teaches the method produced beating cardiomyocyte spheroids that were used to investigate the effect of isoproterenol on the contractibility of the spheroids and isoproterenol influences the contractility of the heart muscle in humans and Muller teaches cell lines other than HEK293 can be used with the culture system.
Applicant’s Arguments/Response to Arguments
38. Applicant Argues: Applicant asserts that Bartholoma does not teach a rotation range of 20 mm or more.
Response to Argument: The amendments to the claims necessitated new rejections in which Muller teaches a rotation range greater than 20 mm for culturing cells on an orbital shaker.
Applicant Argues: Applicant asserts that the present invention demonstrates unexpected significant effects where suspension culture of human cardiomyocytes with a rotation range of 20 mm or more with a specific-shaped container produce high-quality spheroid formation and drastically reduce cell death.
Response to Argument: As Muller teaches the culture volume, rotation speed, and rotational diameter affect the oxygen transfer rate in suspension culturing in a bottle, the results of high-quality spheroid formation and drastically reduced cell death would have been expected by one of ordinary skill in the art as Muller teaches changing the volume, speed, and diameter results in changes to cell viability.
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
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