DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 22-23, 27, 29, 31, 33, 43, 46, 48-49, 51-52, 55-56, 58, 60, 64, 162-163, 373, 375 and 406 are pending.
Claims 22-23, 27, 29, 31, 33, 43, 46, 48-49, 51-52, 55-56, 58, 60, 64, 162-163, 373, 375 and 406 are under examination.
Examiner’s Remark
Claim 26 is labeled as “cancelled” and also includes the entire text of claim 26 marked as removed. Because it is clear that claim 26 is intended to be cancelled, it is treated as cancelled for the sake of compact prosecution (see also Applicant’s remarks filed 2nd July 2026 which lists claim 26 as cancelled and does not include claim 26 on the list of claims that are pending or under Examination).
All claims being currently amended must be presented with markings to indicate the changes that have been made relative to the immediate prior version. See MPEP § 714(II)(c) and (f)
Withdrawn Claim Objections
The objections to claims 51 and 406 due to informalities as set forth in the previous office action are withdrawn in view of Applicant’s amendments.
Moot Claim Rejections - 35 USC § 112(b)
The rejection of claims 26, 34-36, 38, and 40-42 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite as set forth in the previous office action is moot in view of the cancellation of this claim.
Withdrawn Claim Rejections - 35 USC § 112(b)
The rejection of claims 22-23, 26-27, 29, 43, 46, 48-49, 51-52, 55-56, 60, 64, 162-163, 373, 375 and 406 as written as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 23, 31, 33 and 58 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 23 recites “the pluripotent cell” is “a fibroblast, a gastric epithelial cell, a ductal cell, or a hepatocyte” which makes the scope of the claim indefinite because it is unclear how a cell can be both a pluripotent cell, which can differentiate into a specific type, while also being a specific differentiated cell type.
Claim 23 recites “the pluripotent stem cell is a stem cell.” Which include the broader limitation of a “stem cell” together with the narrow limitation of a “pluripotent cell” which is a type of stem cell. A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) is considered indefinite, since the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). Note the explanation given by the Board of Patent Appeals and Interferences in Ex parte Wu, 10 USPQ2d 2031, 2033 (Bd. Pat. App. & Inter. 1989), as to where broad language is followed by "such as" and then narrow language. The Board stated that this can render a claim indefinite by raising a question or doubt as to whether the feature introduced by such language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Note also, for example, the decisions of Ex parte Steigewald, 131 USPQ 74 (Bd. App. 1961); Ex parte Hall, 83 USPQ 38 (Bd. App. 1948); and Ex parte Hasche, 86 USPQ 481 (Bd. App. 1949).
Claim 31 recites “the differentiating step b) takes place after the pluripotent cell differentiates into a progenitor hepatocyte-like cell.” However, step b of claim 22, upon which claim 31 depends, recites “differentiating the pluripotent cell in vitro to an immature hepatocyte.” Because step b) requires a pluripotent cell, it is unclear what the metes and bounds of this step encompass if the pluripotent cell has already been differentiated into an immature hepatocyte.
Similar, claim 33 recites “the differentiating step b) takes place during the differentiation of the immature hepatocyte-like cell to the mature hepatocyte-like cell.” However, step b of claim 22, upon which claim 33 depends, recites “differentiating the pluripotent cell in vitro to an immature hepatocyte.” Because step b) requires a pluripotent cell, it is unclear what the metes and bounds of this step encompass if the pluripotent cell has already been differentiated into an immature hepatocyte.
Claim 58 recites “the culture medium further comprises hepatocyte growth factor (HGF) and oncostatin-M (OSM), is free of hepatocyte growth factor (HGF), or both.” The scope of the claim is indefinite because it is unclear how the claim can include “both” being “free of hepatocyte growth factor (HGF)” and comprising hepatocyte growth factor (HGF).
Generally, when the claims are indefinite, vague or unclear, they cannot be construed without speculation or conjecture; therefore, the indefinite claims are not treated on the merits with respect to prior art. See In re Steele, 305 F.2d 859, 862 (CCPA 1962) (A prior art rejection cannot be sustained if the hypothetical person of ordinary skill in the art would have to make speculative assumptions concerning the meaning of claim language.); see also In re Wilson, 424 F.2d 1382, 1385 (CCPA 1970) ("If no reasonably definite meaning can be ascribed to certain terms in the claim, the subject matter does not become obvious-the claim becomes indefinite."). Notwithstanding Steele, the Office has made every attempt to construe the claims in what the Office believes is the intent of the Applicants in the interest of compact prosecution.
Moot Claim Rejections - 35 USC § 112
Improper Markush
The rejection of claim 26 on the basis that it contains an improper Markush grouping of alternatives as set forth in the previous office action is moot in view of the cancellation of this claim.
Maintained Claim Rejections - 35 USC § 112
Improper Markush
Claim 29 stands rejected on the basis that it contain an improper Markush grouping of alternatives.
See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
Regarding claim 29, the Markush grouping of carbamoylphosphate synthetase I (CPS 1), omithine transcarbamylase (OTC), argininosuccinic acid synthetase (ASS1), argininosuccinic acid lyase (ASL), arginase (ARG1), N-acetyl glutamate synthetase (NAGS), ornithine translocase (ORNT1), citrin is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons: The listed alternatives do not share a single structural similarity.
Furthermore, regarding claim 29, the recitation of “comprise” includes open language and is improper Markush language. Applicant is directed to MPEP 2117 which states claim language defined by a Markush grouping requires selection from a closed group "consisting of" the alternative members. Id. at 1280, 67 USPQ2d at 1196.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
Response to Arguments
Applicant’s arguments, filed 2nd, July, 2026, have been fully considered but are not found persuasive.
Applicant argues “Applicant respectfully disagrees and submits that the groupings recited in both claims 26 and 29 are members of an art-recognized class of compounds” (pg. 9-10). Applicant specifically further argues “The specification also teaches that carbamoylphosphate synthetase I (CPS1 ), ornithine transcarbamylase (OTC), argininosuccinic acid synthetase (ASS1 ), argininosuccinic acid lyase (ASL), arginase (ARGI), N-acetyl glutamate synthetase (NAGS), ornithine translocase (ORNTI), and citrin,
as recited in claim 29 are all urea cycle pathway enzymes. See, e.g., as-filed specification
at ,i [00224]. Each of these groupings are members of an art-recognized class of compounds and/or are compounds that will achieve the same intended result if substituted with each other. MPEP § 2117 ("A recognized physical class, a recognized chemical class, or an art-recognized class is a class wherein there is an expectation from the knowledge in the art that members of the class will behave in the same way in the context of the claimed invention. In other words, each member could be substituted one for the other, with the expectation that the same intended result would be achieved." the skilled artisan would understand that the urea cycle pathway enzymes recited in claim 29 belong to the same art-recognized class of urea cycle pathway enzymes.” (pg. 9-10).
In response, regarding Art-recognized classes, Applicant is directed to MPEP 2117 (II)(A) which states that Members of a Markush group share a "single structural similarity" when they belong to the same recognized physical or chemical class or to the same art-recognized class. A recognized physical class, a recognized chemical class, or an art-recognized class is a class wherein there is an expectation from the knowledge in the art that members of the class will behave in the same way in the context of the claimed invention. In other words, each member could be substituted one for the other, with the expectation that the same intended result would be achieved. For example, in the context of a claim covering a disposable diaper, a limitation "the fastener selected from the group consisting of a pressure sensitive adhesive and complementary release material, a complementary hook and loop structure, a snap, and a buckle" would likely be considered an art recognized class because a review of the prior art would establish that it was well known that each member could be substituted for each other with the expectation that the intended result (repositionable and refastenable) would occur. (see MPEP 2117 (II)(A)). In the instant case, the listed members of claim 29 do not belong to the same art recognized class because the intended result would not be the same. The result of expressing structurally and functionally distinct members of urea cycle pathway enzymes would be physiologically distinct in a hepatocyte and is not the same intended result.
Withdrawn Claim Rejections - 35 USC § 102
The rejection of claims 22-23, 27, 29, 31, 33, 48-49, 56, 58, 60, 64 and 406 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) as evidenced by Nitzahn et al. (Mol Genet Metab. 2020 Oct 10;131(3):289–298.; henceforth “Nitzahn”) as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
Moot Claim Rejections - 35 USC § 102
The rejection of claims 26, 34-36, 38 and 42 under 35 U.S.C. 102(a)(1) as being anticipated by Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) as evidenced by Nitzahn et al. (Mol Genet Metab. 2020 Oct 10;131(3):289–298.; henceforth “Nitzahn”) as set forth in the previous office action is moot in view of the cancellation of these claims.
Moot Claim Rejections – 35 USC § 102/103
The rejection of claim 40 under 35 U.S.C. 102(a)(1) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) as evidenced by Soofi et al. (J Struct Biol. 2009 Sep;167(3):216-9. Epub 2009 May 27.; henceforth “Soofi”) as set forth in the previous office action is moot in view of the cancellation of the claim.
Moot Claim Rejections - 35 USC § 103
The rejection of claim 41 under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Schmitt et al. (Adv Healthc Mater. 2015 May 20;4(10):1555–1564.; henceforth “Schmitt”) as set forth in the previous office action is moot in view of the cancellation of the claim.
Withdrawn Claim Rejections - 35 USC § 103
The rejection of claim 43 under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Shirahama et al. (J Vis Exp. 2016 Aug 27:(114):54331.; see IDS filed 14th, July, 2023; henceforth “Shirahama”) as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
The rejection of claim 46 under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Wang et al. (Toxicol Res (Camb). 2017 Sep 28;7(1):13–21.; henceforth “Wang”) as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
The rejection of claim 51 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Yishai et al. (Hepatology. 2015 Jul;62(1):265-78. Epub 2015 Apr 22.; henceforth “Yishai”) as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
The rejection of claims 52 and 55 under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Blackford et al. (Stem Cells Transl Med. 2019 Feb;8(2):124-137. Epub 2018 Nov 19.; see IDS filed 14th, July, 2023; henceforth “Blackford”) as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
The rejection of claims 162-163 under 35 U.S.C. 103 as being unpatentable over Blackford et al. (Stem Cells Transl Med. 2019 Feb;8(2):124-137. Epub 2018 Nov 19.; henceforth “Blackford”) in view of Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”), Liu et al. (J Biosci Bioeng. 2019 Apr;127(4):506-514. Epub 2018 Oct 12.; henceforth “Liu”) and Cellseco (accessed at : https://web.archive.org/web/20190918095637/https://www.cellseco.com/product-category/cell-culture/fetalbovineserum/ on 3rd, February, 2026; Date 18th, September, 2019) as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
The rejection of claims 373 and 375 under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Shirahama et al. (J Vis Exp. 2016 Aug 27:(114):54331.; see IDS filed 14th, July, 2023; henceforth “Shirahama”) and Lee et al. (Acta Biomater. 2017 Dec:64:67-79. Epub 2017 Sep 28.; henceforth “Lee”) as set forth in the previous office action is withdrawn in view of Applicant’s amendments.
New Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 22-23, 27, 31, 33, 48-49, 56, 58, 60, 64 and 406 are rejected under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”).
Regarding claims 22, 27, and 406, Keller discloses a method of generating a mature hepatocyte-like cell from a pluripotent cell, comprising:
providing a pluripotent cell (“pluripotent stem cells”; hESCs or hiPSCs; abstract; para. [0001-0002, 0006, 0021, 0023, 0027, 0050, 0068, 0081-0082, 0084-0085, 0096, 00151-00154, 00177, 00180, 00183, 00223-00228, 00230, 00319-00321]; Figures 7, 9; Example 9; Claims 4-5, 10, 15, 25)
differentiating the pluripotent cell in vitro to an immature hepatocyte (see “aggregates are generated from a monolayer of the cell population comprising hepatocyte” para. [0012]; see also Figure 11).
culturing the immature hepatocyte in at least one culture medium comprising an agent that increases intracellular cyclic AMP (“cAMP and/or a cAMP analog” para. [0014] see also “Inducing maturation of cell aggregates with cAMP treatment” Example 5 and Example 1; abstract; para. [0015, 0017, 0024, 0027, 0031-0032, 0034, 0048-0050, 0052, 0056, 0068, 00102-00105, 00107-00108, 00110, 00119, 00127, 00152-00153, 00157-00158, 00161, 00164, 00168, 00170, 00174, 00176, 00177, 00178, 00183, 00210-00213, 00216-00219, 00262-00263, 00311-00318, 00320, 00322-00323, 00326-003312, 00335, 00351-00352, 00357, 00367, 00369-00370, 00375]; Figures 1, 5-7, 9, 11 ; claims 9-12 and 15), wherein the pluripotent cell differentiates into a mature hepatocyte-like cell (“mature hepatocytes” para. [0005]; see also abstract; para. [0013-0015, 0021, 0023-0024, 0027-0028, 0032, 0034, 0044, 0047-0048, 0052, 0054-0056, 0068-0071, 0093, 0096, 00100-00110, 00113, 00115-00116, 00118, 00120, 00128, 00151, 00153, 00157, 00161, 00164, 00166, 00168-00169, 00172, 00174, 00177-00179, 00183, 00189, 00210, 00214-00217, 00220, 00222, 00243-00245, 00259, 00261, 00297, 00309, 00311, 00316-00317, 00323-00332, 00353, 00392,
Figures 1, 4, 5, 9, 11-13; Examples 1, 5, 7, 8; claims 1,3 ,5, 9-12, 14-15, 19, 23, 26 and 24) and
recovering (“enriching or isolating” para. [0039]; see also para. [0039-0040, 00237-00239, 00243, 00247, 00266-00267, 00288, 00291, 00294]) the mature hepatocyte-like cell.
(see also Figure 1A, 4; para. [0044, 0047, 00107, 0309-0310]),
Wherein the agent is forskolin (para. [00104, 00212]) (instant claim 27).
However, regarding claims 22 and 406, although Keller teaches three-dimensional (3D) culture steps (para. [0044, 0047, 0056, 0059, 0061-0062, 0074, 00177, 00189, 00308, 00316, 00326, 00328]; Figures 4, 9, 13, 16, 18-19; claim 29), Keller does not teach three-dimensional (3D) culture for the formation of hepatocytes from pluripotent stem cells.
Nevertheless, regarding claims 22 and 406, Freyer teaches method steps of differentiation of pluripotent stem cells into hepatocytes from pluripotent stem cells (“dynamic three-dimensional
(3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures” abstract; see also Material and Methods pf. 236-237 “Hepatic differentiation of hiPSC in 3D bioreactors or 2D cultures”). Freyer teaches the hepatic maturation of hiPSC-derived HLC was improved in 3D bioreactors compared with 2D cultures in terms of albumin secretion, CYP2B6 activity, and formation of tissue-like structures with cell–cell contacts and the 3D bioreactor could be used to investigate approaches to improve the maturation of hiPSC-derived hepatocytes and generate fully differentiated hepatocytes from hiPSC in a physiological-like 3D environment in the future (pg. 247 col. 1 “Conclusion).
Therefore, regarding claims 22 and 406, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method of Keller, and combine the known prior art element of the method steps of differentiation of pluripotent stem cells into hepatocytes from pluripotent stem cells of Freyer to obtain the predictable result of hepatocyte differentiation of pluripotent stem cells in a 3D culture system. One of ordinary skill would have been motivated to do so as taught by Freyer to improve the maturation of hiPSC-derived HLC and to use dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures (abstract and conclusion). Regarding the reasonable expectation of success, Freyer evidence method steps of differentiating pluripotent stem cells into hepatocyte like cells in a bioreactor (Material and Methods pg. 236-237 “Hepatic differentiation of hiPSC in 3D bioreactors or 2D cultures”).
Regarding claims 22, 56 and 406, concerning the wherein clauses which state “having enhanced in vitro ureagenesis capability relative to a mature hepatocyte-like cell differentiated in the absence of one or more agent,” (instant claims 22 and 406) and has increased RNA expression of the one or more urea cycle pathway enzymes and increases protein expression of the one or more urea cycle pathway enzymes” (instant claim 56) while Keller is silent to an increase in vitro ureagenesis capability or RNA or protein expression, Nissim teaches that it is well established that ureagenesis is up-regulated by cAMP (pg. 15063 col. 1).
Therefore, regarding claims 22, 56 and 406, because the suggested method includes increasing intracellular cyclic AMP, and Nissim evidences ureagenesis is up-regulated by cAMP (pg. 15063 col. 1), the recited result of “having enhanced in vitro ureagenesis capability relative to a mature hepatocyte-like cell differentiated in the absence of one or more agent,” as well as the recited result of “increased RNA expression of the one or more urea cycle pathway enzymes and increases protein expression of the one or more urea cycle pathway enzymes” would be an obvious and expected result of the suggested method. Furthermore, instant claims only required “enhanced in vitro ureagenesis capability”(instant claims 22 and 406) and “increased RNA expression” and “increases protein expression” (instant claim 56) and do not require a specific amount of enhancement or increase and therefore even a small increase is encompassed by instant claims.
Regarding claim 23, further to the discussion of claim 22 above, Keller teaches the pluripotent stem cells can be obtained from fibroblasts (para. [00280]) and the pluripotent stem cells taught by Keller above are a type of stem cell.
Regarding claim 31, further to the discussion of claim 22 above, Keller teaches the differentiating step b) takes place after the pluripotent cell differentiates into a progenitor hepatocyte-like cell (see Figure 1A, the step with the cAMP activator “cAMP” of ““Hepatic Maturation B” occurs after “Hepatic Specification” and “Hepatic Maturation A” in which the source cell differentiates into a progenitor hepatocyte-like cell).
Regarding claim 33, further to the discussion of claim 22 above, Keller teaches the pluripotent cell is differentiated into an immature hepatocyte cell and the differentiating step b) takes place during the differentiation of an immature hepatocyte-like cell to the mature hepatocyte-like cell (see “Hepatic Maturation B”; Figure 1A).
Regarding claims 48-49, further to the discussion of claim 22 above, Keller teaches the differentiating step b) is carried out at “a 5% CO2 ambient air environment” (“Aggregation cultures were maintained in a 5% CO2 ambient air environment” para. [00332]), which is an oxygen condition (instant claim 48), and is a normoxic condition (instant claim 49).
Regarding claim 58, further to the discussion of claim 22 above, Keller teaches the culture medium does not comprise and is therefore free of HGF (see figure 1a “Hepatic Maturation B” which does not include HGF).
Regarding claim 60, further to the discussion of claim 22 above, Keller teaches activating the cAMP pathway increases ALB (albumin) gene expression (increases the proportion of Albumin positive cells at para.[0048]) and therefore (i) increased expression of ALB is an obvious and expected result of the suggested method which includes activating the cAMP pathway with a cAMP activator as taught by Keller above. Keller teaches the presence of albumin positive cells (para.[0048]) and therefore the produced cells would be expected to (ii) secrete albumin as an obvious and expected result of the suggested method which includes activating the cAMP pathway with a cAMP activator as taught by Keller above. Keller teaches the cells have (Iiii) cytochrome p450 activity, wherein the cytochrome p450 activity comprises increased activity of one or more cytochrome p450 family members comprising CYP2C19, CYP2B6, CYP2D6, CYP3A4, CYP3A7 relative to cells produced without activating the cAMP pathway (para. [0017]) and therefore this is an obvious an expected result of the suggested method which includes activating the cAMP pathway with a cAMP activator as taught by Keller above.
Regarding claim 64, further to the discussion of claim 22 above, as stated above Keller teaches obtaining functional mature hepatocytes (“functional hepatocytes” para. [0001]; see also claim 22 rejection above), that are hepatocytes with known hepatocyte functions and markers, and lipid storage capability is a known regular function of hepatocytes, having some lipid storage capability would be an obvious and expected result of the hepatocyte obtained by the suggested method.
Regarding claim 406, Keller teaches 3D culture system steps that include (i) a fetal bovine serum (FBS) free substrate (Collagen type 1 gel which does not include FBS because Keller is silent to including FBS).
Hence, the claimed invention as a whole was prima facie obvious.
Claim 29 is rejected under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”) as applied to claim 22 above, in further view of Nitzahn et al. (Mol Genet Metab. 2020 Oct 10;131(3):289–298.; henceforth “Nitzahn”).
The teachings of Keller, Freyer, and Nissim above are incorporated herein in their entirety.
Regarding claim 29, further to the discussion of claim 22 above, as stated above, (see claims 22 rejection above), Keller teaches the agent increases intracellular cyclic AMP (“cAMP and/or a cAMP analog” para. [0014] see also “Inducing maturation of cell aggregates with cAMP treatment” Example 5 and Example 1; abstract; para. [0015, 0017, 0024, 0027, 0031-0032, 0034, 0048-0050, 0052, 0056, 0068, 00102-00105, 00107-00108, 00110, 00119, 00127, 00152-00153, 00157-00158, 00161, 00164, 00168, 00170, 00174, 00176, 00177, 00178, 00183, 00210-00213, 00216-00219, 00262-00263, 00311-00318, 00320, 00322-00323, 00326-003312, 00335, 00351-00352, 00357, 00367, 00369-00370, 00375]; Figures 1, 5-7, 9, 11 ; claims 9-12 and 15), and Keller discloses specific agents (see also para. [00104]). Nitzahn teaches increased cAMP increases the expression of the urea cycle pathway enzyme CPS 1 (“Glucagon signaling via cAMP also increases the expression of PGC1α, which rapidly induces increased Sirt5 expression to deacetylate, and thus activate, CPS1” pg. 4) and therefore an increase in the urea cycle pathway enzyme CPS 1 is an obvious and expected result of the suggested method.
Hence, the claimed invention as a whole was prima facie obvious.
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”) as applied to claim 22 above, and in view of Shirahama et al. (J Vis Exp. 2016 Aug 27:(114):54331.; see IDS filed 14th, July, 2023; henceforth “Shirahama”).
The teachings of Keller, Freyer and Nissim above are hereby incorporated in their entirety.
Regarding claim 43, further to the discussion of claim 22 above, although Keller teaches a 3D culture system that comprises ECM components (collagen type 1 gel), Keller, Freyer, and Nissim are silent to a 3D culture system that comprises an inverse colloidal crystal scaffold, wherein the inverse colloidal crystal scaffold is coated with an extracellular matrix (ECM) component.
Nevertheless, regarding claim 43, Shirahama teaches a 3D culture system that comprises an inverse colloidal crystal scaffold, wherein the inverse colloidal crystal scaffold (ICC cell culture platform) is coated with an extracellular matrix (ECM) component for liver tissue culture (abstract). Shirahama teaches the ICC cell culture platform addresses the void in the art for a scaffold with controlled porosity and high spatial organization (pg. 9 last para.). Shirahama also teaches that the further collagen coating enhances cell behavior (pg. 9 last para.).
Therefore, regarding claim 43, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Keller, Freyer and Nissim, and simply substitute the known prior art element of the ICC cell culture platform of Shirahama, which comprises an inverse colloidal crystal scaffold that is coated with an ECM component to obtain the predictable result of a method for culturing hepatocytes in 3D culture. One of ordinary skill would have been motivated to do so as taught by Shirahama to control porosity, have high spatial organization, and enhance cell behavior (pg. 9 last para.). Regarding the reasonable expectation of success, Shirahama evidences culturing hepatocytes in a 3D culture system with the ICC cell culture platform and a collagen coating (pg. 3 “3. Huh-7.5 Cell Culture and Seeding”).
Hence, the claimed invention as a whole was prima facie obvious.
Claim 46 is rejected under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”) as applied to claim 22 above, and in view of Wang et al. (Toxicol Res (Camb). 2017 Sep 28;7(1):13–21.; henceforth “Wang”).
The teachings of Keller, Freyer and Nissim above are hereby incorporated in their entirety.
Regarding claim 46, further to the discussion of claim 22 above, although Keller discloses the
differentiating in the presence of an endothelial cells (Example 8), and Keller teaches co-culturing with endothelial cells results in an increased level of CYP3A4 (Example 89; p pg. 77 1st para.), Keller, Freyer and Nissam are silent to using HUVEC with the method.
Nevertheless, regarding claim 46, Wang teaches a method for culturing hepatocytes comprising co-culturing hepatocytes with HUVECs (pg. 12 col. 2 3rd para. “The co-culture of hiHeps and HUVECs on paper”). Wang teaches the hiHeps co-cultured with HUVECs exhibited a 3D like morphology and maintained the liver specific functions of producing albumin and urea, maintained a higher expression of cytochrome P450 gene, revealing a marked enhancement of hepatic functions in the 3D liver co-culture model and exhibited near physiological hepatotoxic responses (abstract). Wang teaches that by integrating valid human hepatocyte lineages with other non-parenchymal cells, the established low-cost and simple paper-based array could serve as in vitro tissue/organ models and provide great opportunities for developing further flexible platforms for evaluating drug induced cytotoxicity more accurately (Conclusion; pg. 19 col. 1 2nd para.).
Therefore, regarding claim 46, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Keller in view of Freyer and Nissim, and combine the known prior art element of co-culturing with HUVECs of Keller to obtain the predictable result of a method of culturing hepatocytes. One of ordinary skill would have been motivated to do so as taught by Wang to enhance hepatocyte functions, such as producing albumin and urea, to maintain a higher expression of cytochrome P450 gene, and to develop further flexible platforms for evaluating drug induced cytotoxicity more accurately (Abstract; Conclusion; pg. 19 col. 1 2nd para.). One of ordinary skill would have been specifically motivated to perform the co-culture during the differentiation step because Keller teaches co-culture with endothelial cells during differentiation resulted in hepatocytes which expressed substantially higher levels of CYP3A4 (pg. 77 1st para.). Regarding the reasonable expectation of success, Wang evidences a method for culturing hepatocytes comprising co-culturing hepatocytes with HUVECs (pg. 12 col. 2 3rd para. “The co-culture of hiHeps and HUVECs on paper”).
Hence, the claimed invention as a whole was prima facie obvious.
Claim 51 is rejected under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”) as applied to claim 22 above, and in view of Yishai et al. (Hepatology. 2015 Jul;62(1):265-78. Epub 2015 Apr 22.; henceforth “Yishai”).
The teachings of Keller, Freyer and Nissim above are hereby incorporated in their entirety.
Regarding claim 51, further to the discussion of claim 22 above, although Keller teaches the cell culture mediums may contain vitamins (para. [00138]), Keller, Freyer and Nissam are silent to whether the mature hepatocyte-like cell is contacted with vitamin K wherein the vitamin K is Vitamin K2.
Nevertheless, regarding claim 51, Yishai teaches contacting a mature hepatocyte-like cell with Vitamin K2 (menaquinone-4 (MK4)) (pg. 267 col. 1 2nd para.; pg. 267 col. 2 1st para.; pg. 270; Figures 2-3) to drive expression of liver-specific genes, including urea cycle enzymes, such as ASS and ARG1, as
well as A1AT and CYP1A2 (Figure 3). Yishai teaches hepatocytes treated with Vitamin K2 MK4 exhibit higher CYP450 activity (Figure 5). Yishai teaches hepatocytes treated with Vitamin K2 MK4 produce
CYP450-inducible cells that show an exceptional ability to predict TC50 values of a diverse group of hepatotoxic compounds (Figs. 5 and 6; Discussion pg. 276 col. 1 3rd. para.).
Therefore, regarding claim 51, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Keller in view of Freyer and Nissam, and combine the known prior art element of contacting the hepatocytes with Vitamin K2 of Yishai to obtain the predictable result of a method for culturing hepatocytes. One of ordinary skill would have been motivated to do so as taught by Yishai to drive expression of liver-specific genes, including urea cycle enzymes, such as ASS and ARG1, as well as A1AT and CYP1A2 (Figure 3) and to produce CYP450-inducible cells that show an exceptional ability to predict TC50 values of a diverse group of hepatotoxic compounds (Figs. 5 and 6; Discussion pg. 276 col. 1 3rd. para.). Regarding the reasonable expectation of success, Yishai evidences methods of culturing hepatocytes comprising contacting the hepatocytes with Vitamin K2 (menaquinone-4 (MK4)) (pg. 267 col. 1 2nd para.; pg. 267 col. 2 1st para.; pg. 270; Figures 2-3).
Hence, the claimed invention as a whole was prima facie obvious.
Claims 52 and 55 are rejected under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”) as applied to claim 22 above, and in view of Blackford et al. (Stem Cells Transl Med. 2019 Feb;8(2):124-137. Epub 2018 Nov 19.; see IDS filed 14th, July, 2023; henceforth “Blackford”).
The teachings of Keller, Freyer and Nissim above are hereby incorporated in their entirety.
Regarding claims 52 and 55, further to the discussion of claim 22 above, Keller, Freyer and Nissam are silent to a step d) encapsulating the mature hepatocyte-like cell in a polymer matrix, wherein the polymer matrix comprises a matrix component, wherein the matrix component comprises alginate (instant claim 52), and Keller is silent to culturing the mature hepatocyte-like cell into a spheroid (instant claim 53).
Nevertheless, regarding claims 52 and 55, Blackford teaches a method comprising the step of d) encapsulating a mature hepatocyte-like cell in a polymer matrix comprising an alginate matrix component (“Alginate Encapsulation of hPSC-Derived Hepatocyte Spheroids” pg. 126 col 2 last para and pg. 127 col. 1 1st para.) where the mature hepatocyte-like cell is cultured into a spheroid (“hPSC-Derived Hepatocyte Spheroids” pg. 126 col 2 last para and pg. 127 col. 1 1st para.; see also Figure 1 and “Alginate microencapsulated hepatocyte spheroids” pg. 127 col. 1 3rd para.). Blackford teaches encapsulating into the alginate matrix component for transplantation (pg. 127 col. 1 3rd para.). Blackford teaches hepatocytes remained viable after transplantation into the immune-privileged material alginate and remained viable as well as functional upon transplantation into immune competent mice (abstract), and that the alginate encapsulated hepatocyte spheroids were suitable for acute liver failure bridging therapy (Figure 6).
Therefore, regarding claims 52 and 55, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Keller in view of Freyer and Nissim and combine the known prior art elements of encapsulating the mature hepatocyte-like cell in a polymer matrix, wherein the polymer matrix comprises a matrix component, wherein the matrix component comprises alginate (instant claim 52), and culturing the mature hepatocyte-like cell into a spheroid (instant claim 55) of Blackford to obtain the predictable result of a method for culturing alginate encapsulated hepatocyte spheroids. One of ordinary skill would have been motivated to do so as taught by Blackford to create alginate encapsulated hepatocyte spheroids suitable for acute liver failure bridging therapy that included viable as well as functional hepatocytes (pg. 127 col. 1 3rd para.; Figure 6). Regarding the reasonable expectation of success, Blackford evidences a method comprising the step of d) encapsulating a mature hepatocyte-like cell in a polymer matrix comprising an alginate matrix component (“Alginate Encapsulation of hPSC-Derived Hepatocyte Spheroids” pg. 126 col 2 last para and pg. 127 col. 1 1st para.) where the mature hepatocyte-like cell is cultured into a spheroid (“hPSC-Derived Hepatocyte Spheroids” pg. 126 col 2 last para and pg. 127 col. 1 1st para.; see also Figure 1 and “Alginate microencapsulated hepatocyte spheroids” pg. 127 col. 1 3rd para.).
Hence, the claimed invention as a whole was prima facie obvious.
Claims 162-163 are rejected under 35 U.S.C. 103 as being unpatentable over Blackford et al. (Stem Cells Transl Med. 2019 Feb;8(2):124-137. Epub 2018 Nov 19.; henceforth “Blackford”) in view of Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”), Liu et al. (J Biosci Bioeng. 2019 Apr;127(4):506-514. Epub 2018 Oct 12.; henceforth “Liu”), Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”) and Cellseco (accessed at : https://web.archive.org/web/20190918095637/https://www.cellseco.com/product-category/cell-culture/fetalbovineserum/ on 3rd, February, 2026; Date 18th, September, 2019)
Regarding claim 162, Blackford discloses a method for producing a mature hepatocyte-like cell providing a source cell (human pluripotent stem cells);
differentiating the source cell in vitro in at least one culture medium (defined four-step hepatic differentiation protocol);and
recovering the mature hepatocyte-like cell,
wherein the differentiating is initially carried out on a first substrate (pg. 127 col. 2 “Differentiation of cGMP-Compliant Stem Cells Toward hPSC-Derived Hepatocytes” to obtain hPSC-Heps) comprising gelatin (“gelatin coated tissue culture dishes”) and transferred to a second substrate comprising collagen (day 21 hPSC-heps “We first seeded the day 21 hPSC-Heps onto collagen- 1 coated tissue culture plastic” pg. 130 col. 1 1st para.; day 21 is encompassed by the instantly claimed “about day 14”)) (see also Figure 1; Materials and Methods pg. 125-127)
However, regarding claim 162, Blackford does not disclose differentiating includes at least one culture medium comprising an agent that increases intracellular cyclic AMP.
Nevertheless, regarding claim 162, Keller teaches a method for producing a mature hepatocyte-like cell comprising culturing in a differentiation medium comprising an agent that increases intracellular cyclic AMP (“cAMP and/or a cAMP analog” para. [0014] see also “Inducing maturation of cell aggregates with cAMP treatment” Example 5 and Example 1; abstract; para. [0015, 0017, 0024, 0027, 0031-0032, 0034, 0048-0050, 0052, 0056, 0068, 00102-00105, 00107-00108, 00110, 00119, 00127, 00152-00153, 00157-00158, 00161, 00164, 00168, 00170, 00174, 00176, 00177, 00178, 00183, 00210-00213, 00216-00219, 00262-00263, 00311-00318, 00320, 00322-00323, 00326-003312, 00335, 00351-00352, 00357, 00367, 00369-00370, 00375]; Figures 1, 5-7, 9, 11 ; claims 9-12 and 15), wherein the source cell differentiates into a mature hepatocyte-like cell (“mature hepatocytes” para. [0005]; see also abstract; para. [0013-0015, 0021, 0023-0024, 0027-0028, 0032, 0034, 0044, 0047-0048, 0052, 0054-0056, 0068-0071, 0093, 0096, 00100-00110, 00113, 00115-00116, 00118, 00120, 00128, 00151, 00153, 00157, 00161, 00164, 00166, 00168-00169, 00172, 00174, 00177-00179, 00183, 00189, 00210, 00214-00217, 00220, 00222, 00243-00245, 00259, 00261, 00297, 00309, 00311, 00316-00317, 00323-00332, 00353, 00392) Figures 1, 4, 5, 9, 11-13; Examples 1, 5, 7, 8; claims 1,3 ,5, 9-12, 14-15, 19, 23, 26 and 24). Keller teaches activating the cAMP pathway induces maturation of the hepatocytes (para. [0024, 0048]; Figure 5), increase the proportion of Albumin positive cells (para.[0048]) and increases metabolic enzyme activity (para. [0049]; Figure 6). Keller teaches the cAMP activator is forskolin (para. [00104, 00212])
Therefore, regarding claim 162, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method of Blackford, and combine the known prior art element of culturing in a maturation medium comprising a cAMP activator of Forskolin of Keller to obtain the predicable result of a method for culturing hepatocytes. One of ordinary skill would have been motivated to do so as taught by Keller to induces maturation of the hepatocytes (para. [0024, 0048]; Figure 5), increase the proportion of Albumin positive cells (para.[0048]) and increases metabolic enzyme activity (para. [0049]; Figure 6). Regarding the reasonable expectation of success, Keller evidences a method for culturing hepatocytes comprising culturing in a maturation medium with a cAMP activator (see Figure 1a; Examples).
However, regarding claim 162, although Blackford teaches three-dimensional (3D) culture steps (“3D Maturation of hPSC-Heps Within a PEG-DA-Based Scaffold Suitable for Biomedical Applications” pg. 130 col. 2), Blackford and Keller are silent to three-dimensional (3D) culture for the formation of hepatocytes from pluripotent stem cells.
Nevertheless, regarding claim 162, Freyer teaches method steps of differentiation of pluripotent stem cells into hepatocytes from pluripotent stem cells (“dynamic three-dimensional
(3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures” abstract; see also Material and Methods pf. 236-237 “Hepatic differentiation of hiPSC in 3D bioreactors or 2D cultures”). Freyer teaches the hepatic maturation of hiPSC-derived HLC was improved in 3D bioreactors compared with 2D cultures in terms of albumin secretion, CYP2B6 activity, and formation of tissue-like structures with cell–cell contacts and the 3D bioreactor could be used to investigate approaches to improve the maturation of hiPSC-derived hepatocytes and generate fully differentiated hepatocytes from hiPSC in a physiological-like 3D environment in the future (pg. 247 col. 1 “Conclusion).
Therefore, regarding claim 162, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Blackford in view of Keller, and combine the known prior art element of the method steps of differentiation of pluripotent stem cells into hepatocytes from pluripotent stem cells of Freyer to obtain the predictable result of hepatocyte differentiation of pluripotent stem cells in a 3D culture system. One of ordinary skill would have been motivated to do so as taught by Freyer to improve the maturation of hiPSC-derived HLC and to use dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures (abstract and conclusion). Regarding the reasonable expectation of success, Freyer evidence method steps of differentiating pluripotent stem cells into hepatocyte like cells in a bioreactor (Material and Methods pg. 236-237 “Hepatic differentiation of hiPSC in 3D bioreactors or 2D cultures”).
Regarding claims 162, concerning the wherein clauses which state “having enhanced in vitro ureagenesis capability relative to a mature hepatocyte-like cell differentiated in the absence of one or more agent,” while Keller is silent to an increase in vitro ureagenesis capability Nissim teaches that it is well established that ureagenesis is up-regulated by cAMP (pg. 15063 col. 1).
Therefore, claim 162 because the suggested method includes increasing intracellular cyclic AMP, and Nissim evidences ureagenesis is up-regulated by cAMP (pg. 15063 col. 1), the recited result of “having enhanced in vitro ureagenesis capability relative to a mature hepatocyte-like cell differentiated in the absence of one or more agent,” as well as the recited result of “increased RNA expression of the one or more urea cycle pathway enzymes and increases protein expression of the one or more urea cycle pathway enzymes” would be an obvious and expected result of the suggested method. Furthermore, instant claims only required “enhanced in vitro ureagenesis capability” and do not require a specific amount of enhancement or increase and therefore even a small increase in vitro ureagenesis capability is encompassed by instant claims.
However, regarding claims 162-163, Blackford, Keller, Freyer and Nissim are silent to including FBS in the medias.
Nevertheless, regarding claims 162-163, Liu teaches a method of culturing hepatocytes comprising culturing in mediums with FBS (abstract; pg. 507 col. 2; pg. 501 col. 1 1-2nd para.; pg. 509; pg. 510 col. 1; pg. 513 col. 1).
Additionally, regarding claims 162-163, Cellseco teaches the most common type of serum used for cell growth is foetal bovine serum (FBS)(pg. 1 1st para.). Cellseco teaches using FBS helps to ensure high quality and consistency essential for successful cell culture (Title). Cellseco teaches in cell culture, serum provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water - insoluble components, and other compounds necessary for in vitro growth of cells, such as hormones and attachment factor and also adds buffering capacity to the medium and binds or neutralises components that may be toxic (pg. 1 2nd para.). Cellseco teaches FBS promotes steady, healthy cell growth in culture (pg. 3).
Therefore, regarding claims 162-163, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Blackford in view of Keller, Freyer and Nissim, and combine the known prior art element of the FBS of Liu and Cellseco to both substrates taught by Blackford obtain the predictable result of a method of culturing hepatocytes. One of ordinary skill would have been motivated to do so as taught by Cellseco to ensure high quality and consistency essential for successful cell culture (Title), to provide a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water - insoluble components, and other compounds necessary for in vitro growth of cells, such as hormones and attachment factor, to add buffering capacity to the medium and binds or neutralize components that may be toxic (pg. 1 2nd para.), and to promote steady, healthy cell growth in culture (pg. 3). Regarding the reasonable expectation of success, Liu evidences methods of culturing hepatocytes with FBS and Liu evidences optimization of FBS amounts for hepatocyte culture (abstract; pg. 507 col. 2; pg. 501 col. 1 1-2nd para.; pg. 509; pg. 510 col. 1; pg. 513 col. 1).
Hence, the claimed invention as a whole was prima facie obvious.
Claims 373 and 375 are rejected under 35 U.S.C. 103 as being unpatentable over Keller et al. (WO-2014/124527-A1; see IDS filed 14th, July, 2023; henceforth “Keller”) in view of Freyer et al. (Biores Open Access. 2016 Aug 1;5(1):235-48.; henceforth “Freyer”) and Nissim et al. (J Biol Chem. 2008 May 30;283(22):15063-71. Epub 2008 Mar 28.; henceforth “Nissim”) as applied to claim 22 above, and in view of Shirahama et al. (J Vis Exp. 2016 Aug 27:(114):54331.; see IDS filed 14th, July, 2023; henceforth “Shirahama”) as applied to claim 43 above and in view of Lee et al. (Acta Biomater. 2017 Dec:64:67-79. Epub 2017 Sep 28.; henceforth “Lee”).
The teachings of Keller, Freyer, Nissim and Shirahama above are hereby incorporated in their entirety.
Regarding claims 373 and 375, further to the discussion of claim 42 above, as stated above (see claim 43 rejection above), Shirahama teaches and makes obvious an ICC cell culture platform which comprises an inverse colloidal crystal scaffold that is coated with an ECM component. The ICC cell culture platform of Shirahama comprises a soft hydrogel substrate (instant claim 373), which is poly(ethylene glycol) (PEG) (instant claim 375) (abstract) (pg. 1 introduction 2nd para.; pg. 2 1st-2nd para.; Protocol “2. Preparing bare and ECM-coated PEGDA scaffolds”).
However, regarding claims 373 and 375, although Shirahama teaches preparation of the ICC cell culture platform which comprises the soft hydrogel substrate comprising PEG, Shirahama is silent to the elastic modulus of the substrate.
Nevertheless, regarding claims 373 and 375, Lee teaches elastic modulus values for PEG hydrogel substrates that are suitable for culturing hepatocyte. Specifically, Lee teaches a microenvironment affording low mechanical compliance (from 0.4 to 2 kPa range) is considered suitable to enhance hepatocyte function and differentiation (pg. 68 col. 1 2nd para.).
Therefore, regarding claims 373 and 375, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Keller in view of Freyer, Nissim and Shirahama, and combine the known prior art element of the elastic modulus range of from 0.4 to 2 kPa to obtain the predicable result of a method for culturing hepatocytes. One of ordinary kill would have been motivated to do so as taught by Lee because the microenvironment affording low mechanical compliance (from 0.4 to 2 kPa range) is considered suitable to enhance hepatocyte function and differentiation (pg. 68 col. 1 2nd para.). Regarding the reasonable expectation of success, Lee evidences preparation of hydrogels with elastic moduli and methods of culturing cells using the hydrogels (see Figures 3-4 and of 74 “3.5. Optimal 3D microenvironment enhances drug-metabolizing enzyme induction, Alb secretion, and drug metabolism” ; see also Materials and Methods pg. 68-69).
Additionally, regarding claims 373 and 375, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use, since it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. See M.P.E.P. §2144.05. In the instant case, the elastic modulus range of from 0.4 to 2 kPa range suggested by Lee above overlaps with the instantly claimed range of from about 1 kPa to about 8 kPa and thereby makes it obvious.
Hence, the claimed invention as a whole was prima facie obvious.
Conclusion
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.
No claim is allowable.
Correspondence
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/BRIANA N EBBINGHAUS/Examiner, Art Unit 1632
/EMILY A CORDAS/Primary Examiner, Art Unit 1632
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