DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Applicant’s Request for Continued Examination, Amendment and Arguments/Remarks received on 03 December 2025 have been entered.
Claims 1-13 were previously pending in the application. Claims 1-12 have been cancelled, and new claims 14-25 have been added by Applicant. Claims 13-25 are currently pending in the application. Claims 13, 23, and 24 are independent claims.
The election of Group I, drawn to a method of producing a chondrocyte culture with high tissue-regenerating ability and a chondrocyte culture produced by the method, remains in effect in the instant application. The following election of species remains in effect in the instant application:
1) genes which the chondrocyte culture has the ability to expresses: SOX9.
Claim 24 is newly withdrawn from consideration as being directed to a nonelected invention, there being no allowable generic or linking claim. Claim 16 is newly withdrawn from consideration as being directed to a nonelected species, there being no allowable generic or linking claim.
Claims 13-15, 17-23, and 25 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/JP2020/015896, filed 08 April 2020, which claims priority to JAPAN 2019-073783, filed 08 April 2019. Filing of a certified copy of the JAPAN 2019-073783, filed 08 April 2019, is acknowledged.
Thus, the earliest possible priority for the instant application is 08 April 2019.
Information Disclosure Statement
The information disclosure statement filed 16 December 2025 has been considered by the Examiner. Examiner notes the filing of IDS Size Fee assertions for the IDS filed 16 December 2025, as required under 37 CFR 1.98, along with payment of the appropriate Size Fee, as required under 37 CFR 1.17(v)(1).
Claim Objections
The objection to cancelled and amended claims 1 and 13 for reciting, “compared to an expressing of”, is withdrawn in view of Applicant’s cancellation of claim 1 and the amendment to claim 13 now reciting, “compared to an expression of one or more genes in a second chondrocyte culture that is created without the thermoreversible gelation polymer by merely adding the basal media to the chondrocyte cell population for a second culturing of the second chondrocyte culture to obtain a second chondrocyte tissue regeneration.
Amended and new claims 13 and 17 are newly objected to because of the following informalities:
Claim 13 recite, “wherein,” in line 19 such that the “wherein” is modifying a single clause. The comma following “wherein” should be removed.
Claims 13 and 17 each recite, “second chondrocyte culture that is the created without” in line 22 and 9, respectively, wherein recitation of “the” prior to “created” appears to be a typographical error.
New claim 17 also recites “wherein,” in line 1, such that the “wherein” is modifying each of three subsequent clauses. As such, “wherein” should be followed by a colon (“:”) rather than a common (“,”).
Additionally, new claim 17 recites “a somatic stem cells” in lines 2-3, which has a grammatical mismatch of a singular article “a” with a plural noun “cells”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The rejection of cancelled and amended claims 1-2, 4-8, and 11-13 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for multiple issues of indefiniteness is withdrawn in view of Applicant’s cancellation of claims 1-2, 4-8, and 11-12 and amendments to the claim 13.
Amended claim 13-15, 17-23, and 25 are newly 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.
Amended independent claim 13 has multiple issues of indefiniteness.
Amended independent claim 13 recites “the polymer” in line 4. There is insufficient antecedent basis for this limitation in the claim. Claim 13 has a prior recitation of “a thermoreversible gelation polymer” in line 4, but not of “a polymer”.
Amended independent claim 13 newly recites, “obtaining a thermoreversible gelation polymer, the polymer being a product of a polymerization reaction of N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate as a hydrophilic block in an aqueous solution” in lines 4-6, which is indefinite because it is unclear how the components are being grouped and which phrases modify which elements. For example, it is unclear whether “the thermoreversible gelation polymer” of line 4 is the product of polymerization of just the N-isopropyl acrylamide or the product of polymerization of all three of the N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol. Additionally, it is unclear whether the thermoreversible gelation polymer is “as a hydrophilic block”, whether the polyethylene glycol is “as a hydrophilic block”. Further, it is unclear whether the thermoreversible gelation is meant to be in an aqueous solution, whether the polymerization reaction is meant to be in an aqueous solution, or whether just the hydrophilic block is meant to be in an aqueous solution.
Claim 13 recites the limitation "the chondrocyte culture" in line 10. There is insufficient antecedent basis for this limitation in the claim. Independent claim 13 has a prior recitation of “a first chondrocyte culture” in line 9, but does not have a prior recitation of “a chondrocyte culture”.
Claim 13 recites the limitation "the first chondrocyte tissue" in line 13. There is insufficient antecedent basis for this limitation in the claim. Independent claim 13 has a prior recitation of “a first chondrocyte tissue regeneration” but does not have a prior recitation of “a chondrocyte tissue”.
Claim 13 recites the limitation "the gelled first chondrocyte culture" in lines 13-15. There is insufficient antecedent basis for this limitation in the claim. Independent claim 13 has a prior recitation of “gelling the chondrocyte culture” but has not prior recitation of “a gelled first chondrocyte culture”.
Claim 13 recites, “the collecting including cooling the first chondrocyte culture below the sol-gel temperature to convert the gelled first chondrocyte culture back to a sol state for the collecting”, which is indefinite because it is unclear how the collecting can include a step “for the collecting” in that the step recited as “for the collecting” is itself a step of the collecting.
Claim 13 recites, “wherein, the first culturing includes stimulating the first chondrocyte tissue regeneration”, which is indefinite because the first culturing is a step of obtaining a first chondrocyte tissue regeneration. As such, it is unclear how a first chondrocyte tissue regeneration can be stimulated within a step which is still producing the first chondrocyte tissue regeneration.
Additionally, “wherein, the first culturing includes stimulating the first chondrocyte tissue regeneration” is indefinite because it is unclear whether “stimulating” is an active method step or merely the consequence of culturing the first chondrocyte culture comprising the basal media and the thermoreversible gelation without additional active steps.
Claim 13 recites, “one or more genes selected from SOX9, COL2A1, miR140, and miR212”, which is in an improper Markush form, and as such, it is unclear whether the group of genes are a closed or open group of genes.
As such, the metes and bounds of the claim cannot be determined.
New claim 14 recites, “wherein the thermoreversible gelation polymer is a product of a polymerization reaction of 42.0 g of N-isopropyl acrylamide, 4.0 g of n-butyl methacrylate, and 11.5 g of polyethylene glycol dimethacrylate in an aqueous solution”, which is indefinite because it is unclear whether the thermoreversible gelation polymer is in an aqueous solution, whether the polymerization reaction is in an aqueous solution, or whether just the polyethylene glycol dimethacrylate is in an aqueous solution. As such, the metes and bounds of the claim cannot be determined.
New claim 15 has multiple issues of indefiniteness.
New claim 15 recites, “the chondrocyte cell population including stem cells” in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 15 depends on independent claim 13. Neither claim 15 nor claim 13 have any prior recitation of a chondrocyte cell population including stem cells. Therefore, it is unclear whether Applicant intends to recite an additional chondrocyte cell population which comprises stem cells or whether Applicant intends to recite that the chondrocyte cell population recited in claim 13 line 3 comprises stem cells.
Claim 15 recites that the method further comprises “retaining miRNA”, “maintaining the stem cells”, “expanding the stem cells”, or “promoting or retaining hyaluronic acid secretions”, which are indefinite because it is unclear whether the steps of “retaining”, “maintaining”, “expanding”, and “promoting or retaining” are active method step or merely the consequence of culturing/incubating the first chondrocyte culture comprising the basal media and the thermoreversible gelation without additional active steps.
New claim 15 recites, “promoting or retaining hyaluronic acid secretions in the first chondrocyte culture” in line 7. The term “promoting” is indefinite in that it is unclear whether “promoting” requires some kind of increase in hyaluronic acid secretions or a maintenance of hyaluronic acid secretions. To the extent that “promoting” is meant to indicate some kind of relative change or increase in the hyaluronic acid secretions, then “promoting” is a relative term which renders the claim indefinite. The term “promoting” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear what would constitute “promoting”, for example, it is unclear whether the hyaluronic acid secretions would need to be increased compared to a prior state of the culture, merely maintained compared to a prior state of the culture, or increased compared to an otherwise similarly treated control culture over the same duration of culture. Claim 15 recites in lines 9-13 a comparison for promoting to be increased relative to the second chondrocyte culture. However, it is unclear in what way the “promoting” in one culture is compared to, and increased relative to, the “promoting” in another culture. For example, it is unclear whether the first culture must have an increase in hyaluronic acid levels over starting conditions which is higher than an increase in hyaluronic acid conditions of the control culture over the starting conditions of the control culture, or whether the first culture merely needs to have a higher absolute level of hyaluronic acid secretions relative to the absolute levels of hyaluronic acid secretions in the control culture. Additionally, it is unclear whether “increased promoting” requires an increase in the active step of promoting, such as an increased duration of promoting or an increase in an inducer facilitating the promoting.
Additionally, it is unclear whether Applicant intents for “promoting or retaining hyaluronic acid secretions in the first chondrocyte culture” to mean 1) that the secreted hyaluronic acid itself is promoted or retained in the culture such that the culture media is not removed, which would thereby remove the secreted hyaluronic acid, or 2) that the cells themselves have promoted or retained secretion, such that the secreted hyaluronic acid itself may be removed from the culture, such as through media changes, but that the cells will continue to exhibit the promoted secretion/ retained secretion within/during the culture.
Recitation of “indicating a healthy condition” in lines 7-8 is indefinite because it is unclear whether the “promoting or retaining hyaluronic acid secretions” is meant to be an indication of a healthy condition or whether the promoting or retaining is occurring within a first chondrocyte culture which itself is indicating a healthy condition.
Claim 15 recites, “the retaining” in line 9. There is insufficient antecedent basis for this limitation in the claim. Claim 15 has two prior recitations of retaining- in lines 3 and 7.
Claim 15 recites in lines 9-13 a comparison for the “retaining”, “maintaining”, and “expanding” to be increased relative to the second chondrocyte culture, which is indefinite because it is unclear whether Applicant intends for the active steps of “retaining”, “maintaining”, and “expanding” to be increased in the first culture compared to the second culture, such as being carried out for a longer duration or having an increased level of inducer for the retaining, maintaining, or expanding.
To the extent that Applicant may intend for the increased “retaining”, “maintaining”, and “expanding” to merely indicate an increase in the result of the “retaining”, “maintaining”, and “expanding”, such that the miRNA or hyaluronic acid secretions are more retained in the first culture compared to the second culture and the stem cells are more maintained and/or more expanded in the first culture compared to the second culture, then it is unclear which quantitative effects are being increased for retaining hyaluronic acid secretions, maintaining stem cells, or expanding stem cells.
The specification teaches “[t]he ability of retaining miRNA being high refers to that the miRNA is present in the chondrocyte culture at the time of being culture, at a concentration that is, without being limited, 101 % or higher, 105 % or higher, 110 % or higher, 130 % or higher, 140 % or higher, 150 % or higher, 160 % or higher, 170 % or higher, 180 % or higher, 190 % or higher, or 200 % or higher as compared to a control culture at the time of being cultured” [00133]. Therefore, the specification teaches a relative meaning for “retaining miRNA” having a quantitative attribute which can be increased relative to another culture. It is unclear whether Applicant intends for “retaining hyaluronic acid secretions” to likewise indicate a higher absolute level of hyaluronic acid secretions in the first culture compared to the second culture, or whether Applicant intends to indicate a relative level of hyaluronic acid secretions following the retaining vs the level of hyaluronic acid secretions prior to the retaining for the first culture be higher than a relative level of hyaluronic acid secretions following the retaining vs the level of hyaluronic acid secretions prior to the retaining for the second culture.
It is understandable how the result of “expanding” can be quantitated to be increased relative to another culture with different culture conditions, in that expanding refers to a change in a quantitative trait of the culture (i.e., number or fraction of a cell within the culture). However, it is unclear whether the “increased expanding” would require that the number or fraction of stem cells in the first culture over the starting state of the first culture be higher than the number or fraction of stem cells in the second culture over the starting state of the second culture, or whether the number or fraction of stem cells in the first culture merely needs to be higher following the expanding step than the number or fraction of stem cells in the second culture following the expanding step.
Additionally, “maintaining the stem cells” indicates the continued presence of the stem cells within the first chondrocyte culture without indicating any relative or specific quantitative attribute, such as cell numbers or a quantitative stemness characteristic within the cells. The specification does not provide a limiting definition for “maintaining” which would provide a quantitative value which could be compared to, and therefore be increased relative to, another culture. Therefore, it is unclear in what way the maintaining stem cells can be increased relative to another culture condition. Also, it is unclear whether the increased maintaining is meant to be a comparison of a relative maintenance resulting from the maintaining steps for each culture or whether the increased maintaining is meant to be a comparison of an absolute maintenance end point value resulting from the maintaining steps for each culture.
As such, the metes and bounds of the claim cannot be determined.
New claim 17 has multiple issues of indefiniteness.
Claim 17 recites, “the differentiating of the somatic stem cells into chondrocyte cells in the first chondrocyte culture is increased when compared to a second differentiating of the somatic stem cells into chondrocyte cells in the second chondrocyte culture”, which is indefinite because it is unclear in what way “differentiating” can be “increased”. For example, it is unclear whether Applicant intends that a higher number or proportion of stem cells are converted into chondrocytes in the first culture, such that the resultant differentiation is increased relative to the same differentiating step applied to the second culture, or whether the active step of differentiating itself is increased, such as occurring for a longer duration or having an increased level of a differentiation inducer.
Claim 17 as written requires that the stem cells be selected from the group consisting of pluripotent stem cells, somatic stem cells, or a combination of pluripotent and somatic stem cells in lines 2-3. However, claim 17 also requires that the somatic stem cells are differentiated into chondrocyte cells in the first chondrocyte culture in lines 4-5, which limitation conflicts with the prior limitation in lines 2-3 indicating that somatic stem cells are one option for the identity of the stem cells within the first culture.
As such, the metes and bounds of the claim cannot be determined.
New claim 21 has multiple issues of indefiniteness.
Firstly, claim 21 recites, “removing molecular weights of the thermoreversible gelation polymer”, which is indefinite because molecular weight is an attribute of a molecule (e.g., a polymer) and is not a physical entity itself.
Additionally, recitation of “molecular weights… less than 100,000” is indefinite because no unit is indicated.
As such, the metes and bounds of the claim cannot be determined.
In the interest of compact prosecution, Examiner has interpreted the claim to encompass removing thermoreversible gelation polymers having a molecular weight less than 100,000 Daltons.
New claim 23 recites “the method of producing the first chondrocyte culture of Claim 13”. However, claim 13 is a method of producing a chondrocyte culture having an increased tissue-regenerating ability and not a metho of producing the first chondrocyte culture. Note that the method of claim 13 has a step of adding a basal media to the chondrocyte cell mixture to create a first chondrocyte culture as well as additional steps which modify the first chondrocyte cell culture, including a step of culturing the first chondrocyte cell culture to obtain a first chondrocyte tissue regeneration, stimulating the first chondrocyte tissue regeneration, and collecting the first chondrocyte tissue from the first chondrocyte culture. Therefore, it is unclear which method steps of claim 13 are required to produce the chondrocyte culture of claim 23.
As such, the metes and bounds of the claim cannot be determined.
New claim 25 recite, “the disease” in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 25 is dependent on claim 22, which is dependent on independent claim 13. None of 13, 22, nor 25 have any prior recitation of any disease.
As such, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 103
The rejection of cancelled and amended claims 1-2, 4-8, and 11-13 under 35 U.S.C. 103 as being unpatentable over Mizuno [US20040191900A1, published 30 September 2004, cited in a prior action]; in view of da Silva & Loh [1998, Journal of Colloid & Interface Science, 202, 385-390, CS985456]; Rey-Rico et al. [2018, Osteoarthritis and Cartilage, Abstracts, 26, S60-S474]; Nazempour & Van Wie [2016, Annals of Biomedical Engineering, 44(5), 1325-1354]; Tateno et al. [2016, Glycobiology, 26(12), 1328-1337]; and Chen & Hoffman [US6486213B1, published 26 November 2002], is withdrawn.
Amended and new claims 13-15, 17-23, and 25 are newly rejected under 35 U.S.C. 103 as being unpatentable over Mizuno [US20040191900A1, published 30 September 2004, cited in a prior action]; in view of Mori et al [US20080213869A1, published 04 September 2008]; Caron et al. [2012, Osteoarthritis and Cartilage, 20, 1170-1178]; Kumar et al. [2016, Osteoarthritis and Cartilage, abstract #253, 24, S154]; Nazempour & Van Wie [2016, Annals of Biomedical Engineering, 44(5), 1325-1354]; and Tateno et al. [2016, Glycobiology, 26(12), 1328-1337].
Regarding independent claim 13, Mizuno teaches a method of producing a chondrocyte culture having an increased tissue-regenerating ability (i.e., a method for repair and restoration of damaged, injured, diseased, or aged cartilage to a functional cartilage) comprising the steps of
separating a chondrocyte cell population from a cartilage tissue [0045, 0129-136];
obtaining a thermoreversible gelation polymer, the polymer including i) poly-N-isopropyl acrylamide [0072, 0152, 0170-0172], and ii) polyethylene oxide or polyvinyl alcohol as a hydrophilic block [0151, 0710-0172]; wherein the poly-N-isopropyl acrylamide is copolymers with (e.g., bound by) the hydrophilic block [0170-0172];
suspending/dispersing the chondrocyte cell population into a solution of the thermoreversible gelation polymer to create a chondrocyte cell mixture above the sol-gel temperature [0033, 0137, 0143, 0153];
adding a basal culture media (i.e., DMEM/F-12) to the chondrocyte cell mixture to create a first chondrocyte culture [0033, 0049, 0051, 0106, 0137, 0359, 0382];
gelling the chondrocyte culture above the sol-gel temperature [0106, 0214, 0364];
a first culturing of the chondrocyte culture to obtain a neo-cartilage construct/first chondrocyte tissue regeneration [0106, 0188-0190, 0214, 0364-0365]; and
collecting the first chondrocyte tissue regeneration from a sol state of the first chondrocyte culture, wherein the collecting includes cooling the first chondrocyte culture below the sol-gel temperature to convert the gelled first chondrocyte culture back to a sol state of the chondrocyte culture for collecting [0173, 0311];
Mizuno teaches that the chondrocytes culture produced by their method have tissue-regenerating ability [0013, 0064, 0316, 0321, 0324, 0037].
Mizuno does not teach wherein the polymer is a product of a polymerization reaction of N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate in an aqueous solution.
However, Mori teaches producing a thermoreversible gelation polymer (i.e., TGP-5, TGP-6, and TGP-7) for use in 3D human cell culture, wherein the thermoreversible gelation polymer is a product of a polymerization reaction of N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate in an aqueous solution [0193-0201], wherein the thermoreversible gelation polymer has a sol-gel transition temperatures of 18-20 oC, 7 oC, and 37 oC for TGP-5, TGP-6, and TGP-7, respectively [0195, 0198, 0201, 0202, 0205]. Mori also teaches that the hydrogel is gelled at room temperature and at the cell incubation temperature of 37 oC, but allows for removal of the cells from the hydrogel by decreasing the temperature to 4 oC, wherein the hydrogel transitions to a liquid state so that the cells can be diluted with physiological saline, centrifuged, and collected [0202]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use the hydrogel of Mori for 3D culturing human cells to allow for a gelled state at both room temperature and at cell incubation temperature (i.e., 37 oC) while allowing for a liquid state at a refrigerated temperature (i.e., 4 oC), which is commonly used and readily achievable for common laboratory procedures such as centrifugation, thereby facilitating the collection of cells from the hydrogel for downstream applications.
Mizuno also does not teach wherein the first culturing includes stimulating the first chondrocyte tissue regeneration by increasing the expression of a SOX9 gene compared to the expression of a SOX9 gene in a second chondrocyte culture incubated by merely adding the basal media to the chondrocyte cell population for a second culturing of the second chondrocyte culture to obtain a second chondrocyte tissue regeneration.
However, Caron teaches that 3D culturing of primary chondrocytes results in increased expression of the SOX9 gene (i.e., increased SOX9 mRNA levels) compared to SOX9 expression of culture of cells from the same source under 2D conditions (i.e., without a gelation polymer) [abstract, column 3 ¶ 3- column 4 ¶ 1, column 6 ¶ 2, Figure 1]. Caron also teaches that for redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential. Further, Kumar teaches that SOX9 protein levels are increased in 3D chondrocyte culture compared to 2D cultures [column 1 ¶ 7]. Therefore, given the teachings of Caron and Kumar that 3D culture promotes increased SOX9 mRNA and/or increased SOX9 protein levels, an ordinarily skilled artisan at the time of filing the instant application would expect that culturing a chondrocyte cell population in the presence of a thermoreversible gelation polymer, wherein the thermoreversible gelation polymer is in a gelled state to effect a 3D culture, would stimulate a higher SOX9 expression level compared to culturing a chondrocyte cell population merely in basal media without a gelation polymer, thereby effecting a 2D culture.
Regarding claim 14, Mori teaches producing a thermoreversible gelation polymer, TGP-6, wherein the thermoreversible gelation polymer is a product of a polymerization reaction of 42.0 g of N-isopropyl acrylamide, 4.0 g of n-butyl methacrylate, and 11.5 g of polyethylene glycol dimethacrylate in an aqueous solution and has a sol-gel transition temperature of 7 oC [0196-0198].
Regarding claim 15, Mizuno also teaches that the algorithm described for chondrocyte culture is similarly applicable to other types of cells and tissue, such as stem cells capable of differentiation [0257]. Caron teaches that the method of culturing chondrocytes comprises applying a differentiation medium to redifferentiate dedifferentiated chondrocytes [abstract, column 4 ¶ 1].
Mizuno, Mori, Caron, and Kumar do not teach that the method of producing a chondrocyte culture having an increased tissue-regeneration ability further comprises retaining miRNA, maintaining stem cells, expanding stem cells, or promoting or retaining hyaluronic acid secretions compared to a chondrocyte culture in the absence of the thermoreversible gelation polymer.
However, Nazempour teaches that autologous chondrocyte implantation has limitations because of de-differentiation of articular chondrocytes (AChs) upon in vitro expansion, but that de-differentiation can be abated if initial populations of AChs are co-cultured with mesenchymal stem cells (MSCs), which not only undergo chondrogenesis themselves but also support chondrocyte vitality [abstract]. Nazempour teaches that problems in obtaining sufficient articular chondrocytes (AChs) to fill defects and donor site morbidity are causing researchers to consider stem cells (SCs), particularly MSCs, as a promising alternative [column 3 ¶ 2], and that natural articular cartilage comprises both AChs and MSCs, thereby teaching that MSCs are somatic stem cells [column 4 ¶ 2]. Nazempour also teaches that MSCs display significant therapeutic benefits for OA treatment regimens as they not only undergo chondrogenesis but also can prevent cartilage degeneration through secretion of bioactive factors that exert anti-apoptotic, anti-fibrotic, and anti-inflammatory effects [column 19 ¶ 5]. Nazempour further teaches that AChs also have positive effects on inducing MSC chondrogenesis [column 19 ¶ 5]. Nazempour teaches that MSCs have multiple differentiation potential [column 9 ¶ 2].
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to maintain and/or expand stem cells, such as mesenchymal stem cells, in a chondrocyte culture for tissue-regeneration to promote chondrogenesis and prevent cartilage degeneration.
Additionally, Nazempour also teaches that:
“In comparison to monolayer culture in which both AChs and MSCs lose their original phenotype and receptor profiles, 3D environments such as those simulated in scaffolds better mimic the in vivo niche of cells and are shown to maintain cell survival, self-renewal, differentiation phenotype and construct shape and size. Scaffolds have also been shown to permit Chs to expression their differentiated phenotype and to contribute to MSC chondrogenesis. As a result, ” [column 22 ¶ 3].
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have expected that culturing a first chondrocyte cell population comprising stem cells (i.e., MSCs) in the presence of a thermoreversible gelation polymer, wherein the thermoreversible gelation polymer is in a gelled state to effect a 3D culture, would maintain an increased level of stem cells compared to culturing a chondrocyte cell population comprising stem cells merely in basal media without a gelation polymer (i.e., in a 2D culture).
Regarding claim 17, as discussed above, Nazempour teaches that MSCs are somatic stem cells [column 4 ¶ 2]. As also discussed above, Nazempour further teaches that 3D culturing contributes to MSC chondrogenesis. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have expected that culturing a first chondrocyte cell population comprising stem cells (i.e., MSCs) in the presence of a thermoreversible gelation polymer, wherein the thermoreversible gelation polymer is in a gelled state to effect a 3D culture, would increase the differentiation of the stem cells compared to culturing a chondrocyte cell population comprising stem cells merely in basal media without a gelation polymer (i.e., in a 2D culture).
Regarding claim 18, Mizuno, Mori, Caron, Kumar, and Nazempour do not teach to measure the amount of 1-2 fucose or α2-6 sialic acid in the chondrocyte culture to measure the growth of stem cells in the chondrocyte culture.
However, Tateno teaches that α2-6 sialylation is a marker of the differentiation potential of human MSCs [title, abstract], such that α2-6 sialic acid- specific lectins show stronger binding to early passage adipose-derived hMSCs (with differentiation ability) than late passage cells (without the ability to differentiate) [abstract]. Tateno further teaches that human somatic stem cells (hSSCs), such as MSCs, are attractive cell sources for stem cell-based therapy due to their ability to differentiate into lineages of mesodermal origin, but safety and efficacy concerns have been raised in terms of their overall quality, since the properties of hSSCs vary depending on donors, passage, cell culture conditions, and derived tissues [column 13 ¶ 2]. Tateno also teaches that the differentiation potential of hSSCs is one of the most important properties for their therapeutic effect, and as such, a marker to evaluate the differentiation potential of hSSCs, such as α2-6 sialylation, is important for their use in stem cell-based therapy [column 13 ¶ 2]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to measure an amount of α2-6 sialic acid in a culture comprising stem cells to assess the differentiation potential / growth state of the stem cells in the culture to ensure overall quality for safety and efficacy in cell-based therapies.
Regarding claim 19, Mizuno further teaches that the cartilage tissue is isolated from a subject who has osteoarthritis [0134, 0186, 0330-0331, 0337]. Mizuno also teaches specifically isolating chondrocytes from osteoarthritic patients which are 40 years old [0402], that the only available treatment of severe osteoarthritis of the knee is a total knee replacement in elderly patients [0330], and that damage to the articular cartilage which occurs in active individuals and older generation adults as a result of either acute or repetitive traumatic injury or aging is quite common and leads to pain, affects mobility, and results in debilitating disability. Mizuno additionally teaches that their method is used to treat damaged, diseased, or aged cartilage [0128], and that trauma such as osteoarthritis causes disruption of the contiguous surface of the cartilage resulting in cracks, tears, ruptures, holes, or shredded surfaces requiring repair [0121-0122]. Mizuno further teaches that older inactive chondrocytes could be activated from static non-dividing stage to an active stage where they divide, multiply, promote growth of the extracellular matrix and develop into new cartilage (e.g., neo cartilage), which contains chondrocytes which are rejuvenated and surrounded by newly synthesized extracellular-matrix macromolecules [0125]. Mizuno also teaches that existing treatment options can lead to greater deterioration of the joint cartilage and may eventually lead to a total knee joint replacement, which generally lasts only 10 to 15 years and is therefore not recommended for people under the age of 50 years [0006]. Mizuno also teaches the use autologous chondrocytes in a method of preparing neocartilage for transplantation into a subject with osteoarthritis [0019-0021, 00121, 0328-0331, 0338-0339]. As such, Mizuno teaches the need for therapeutic treatments of osteoarthritic subjects to relieve symptoms associated with osteoarthritis (e.g., pain and limited mobility), including the treatment of “elderly”, “older generation”, and people not under the age of 50 years.
Additionally, Nazempour teaches to collect osteoarthritic hACHs (oahAChs) from donors between 60 and 75 years old, and that the freshly isolated oahAChs induced chondrogenesis in MSCs better than TGF-β1 supplementation [column 16 ¶ 2].
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to collect cartilage tissue from a subject who is 50 years of age or older and has osteoarthritis for production of a chondrocyte culture for cell-based regenerative therapy.
Regarding claim 20, Mizuno also teaches adding growth factors consisting of growth factors contained in serum to the thermoreversible gelation polymer, such that there are no added growth factor other than those contained in serum (i.e., FBS) [0134, 0359, 0382, 0402].
Regarding claim 21, Mizuno teaches that the polymers are high molecule weight [0090]. Mizuno also teaches that if the molecular weight of the PEG chains is several thousand Daltons (500 to 10,000 Daltons along any linear chain segment), the network will be open, swellable by water, and compatible with living cells [0145].
Additionally, Mori teaches that the thermoreversible hydrogel-forming polymers are prepared by polymerization followed by concentration by ultrafiltration with a molecular weight cutoff of 10x104, followed by additional dilution and ultrafiltration cycles so as to eliminate products with a molecular weight of 10x104 or lower, thereby teaching the removal of contaminants having molecular weights that are less than 100,000, such as unpolymerized monomers and polymers which are less than 100 kDa [0193-0194, 0196-0197, 199-200].
Regarding claim 22, as described above, Mizuno teaches that the sol-gel temperature of the thermoreversible gelation polymer is from about 15oC to about 30oC (e.g., higher than 10oC and not higher than 33oC) [0027, 0033-0036, 0089, 0139, 0144]. Additionally, as described above, Mori teaches that the thermoreversible hydrogel polymers obtained by polymerizing N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate in an aqueous solution have sol-gel transition temperatures of 8-20 oC and 37 oC for TGP-5 and TGP-7, respectively [0193-0201]. Therefore, Mori teaches that the specific hydrogels as claimed have sol-gel temperatures which are higher than 10 oC and not higher than 33 oC.
Regarding claim 23, as described above, Mizuno, Mori, Caron, and Kumar teach all the limitations of independent claim 13 of a method of producing a chondrocyte culture, and as such also teach a chondrocyte culture produced by the method.
Regarding claim 25, as discussed above, Mizuno teaches that the cartilage tissue is isolated from a subject who has osteoarthritis [0134, 0186, 0330-0331, 0337]. Mizuno also teaches specifically isolating chondrocytes from osteoarthritic patients which are 40 years old [0402]. Therefore, Mizuno teaches wherein a disease is osteoarthritis.
Given the motivation taught by Mori to use the thermoreversible gelation polymer which is a product of a polymerization reaction of N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate in an aqueous solution for 3D culturing human cells to allow for a gelled state at both room temperature and at cell incubation temperature (i.e., 37 oC) while allowing for a liquid state at a refrigerated temperature (i.e., 4 oC), which is commonly used and readily achievable for common laboratory procedures such as centrifugation, thereby facilitating the collection of cells from the hydrogel for downstream applications; the expectation taught by Caron and Kumar that culturing a chondrocyte cell population in the presence of a thermoreversible gelation polymer, wherein the thermoreversible gelation polymer is in a gelled state to effect a 3D culture, would stimulate a higher SOX9 expression level compared to culturing a chondrocyte cell population merely in basal media without a gelation polymer, thereby effecting a 2D culture; the motivation taught by Nazempour to maintain and/or expand stem cells, such as mesenchymal stem cells, in a chondrocyte culture for tissue-regeneration to promote chondrogenesis and prevent cartilage degeneration; the expectation taught by Nazempour that culturing a first chondrocyte cell population comprising stem cells (i.e., MSCs) in the presence of a thermoreversible gelation polymer, wherein the thermoreversible gelation polymer is in a gelled state to effect a 3D culture, would maintain an increased level of stem cells compared to culturing a chondrocyte cell population comprising stem cells merely in basal media without a gelation polymer (i.e., in a 2D culture); ; the motivation taught by Tateno to measure an amount of α2-6 sialic acid in a culture comprising stem cells to assess the differentiation potential / growth state of the stem cells in the culture to ensure overall quality for safety and efficacy in cell-based therapies; the teachings and motivations taught by Mizuno and Nazempour to collect cartilage tissue from a subject who is 50 years of age or older and has osteoarthritis for production of a chondrocyte culture for cell-based regenerative therapy; it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to modify the method of Mizuno to use a thermoreversible gelation polymer which is a product of a polymerization reaction of N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate in an aqueous solution, to measure α2-6 sialic acid levels as a marker to determine the differentiation capacity/ growth state of the stem cells, to collect cartilage tissue from a subject who is at least 50 years old and has osteoarthritis, wherein culturing the chondrocyte cell population comprising stem cells with the thermoreversible gelation polymer in a gelled state would stimulate an increased expression of a SOX9 gene and promote the maintenance and differentiation of stem cells within the cell population with a reasonable expectation of success.
Insofar as applicant’s arguments apply to this new grounds of rejection, Applicant argues that:
Mizuno does not teach nor suggest each and every limit of claim 13;
the compilation of references is an elaborate, detailed review of technologies in a landscape surrounding the instant claims, but the instant claims have carved out a place of their own such that none of the references, alone or in combination, teaches, suggests, or provides the motivation needed to create instant claim 13 and dependent new claims 14-25; specifically in that there is a high level of skill in this art in particular, and a high degree of uncertainty in the chemical and biological arts in general, such that the activity of chemical compositions is at least as diverse as their plethora of possible configurations, and that the behavior of living tissue within such chemical compositions makes the interactions between the chemicals and the living tissue even more unpredictable.
However, this is not agreed.
In response to Applicant’s arguments against the references individually, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Further, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, Regarding Applicant’s argument 1), note that Mizuno was not relied on to teach each and every limitation of claim 13. As discussed above, the combination of Mizuno, Mori, Caron, and Kumar provide the teachings and motivation for the ordinarily skilled artisan to arrive at the invention recited in instant claim 13. Mori was cited for teaching a thermoreversible gelation polymer (i.e., TGP-5, TGP-6, and TGP-7) for use in 3D human cell culture, wherein the thermoreversible gelation polymer is a product of a polymerization reaction of N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate in an aqueous solution. Additionally, Caron and Kumar were cited for teaching that Sox9 expression is increased in chondrocytes cultured in a 3D gel compared to chondrocytes cultured in a 2D monolayer.
Regarding Applicant’s argument 2), note that the combination of Mizuno, Mori, Caron, Kumar, Nazempour, and Tateno, as presented above, provides the teachings and motivations for each element of the claimed invention recited in claims 13-15, 17-23, and 25 as written. Although the interactions between living cells and chemical environments are complex, Mizuno teaches a method of producing a chondrocyte culture having an increased tissue-regenerating ability by separating a chondrocyte cell population from a cartilage tissue and culturing the chondrocyte cell population dispersed within a gelled thermoreversible gelation polymer. Mori teaches specific thermoreversible gelation polymers as recited in claim 13, wherein the polymers are products of polymerization reactions of N-isopropyl acrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate in an aqueous solution and have sol-gel transition temperatures which are in useful ranges for both culturing cells in a 3D hydrogel scaffold at optimal culturing temperatures and for collecting the cells following culture at a workable refrigerated temperature. Additionally, Caron and Kumar teach that increased Sox9 expression is a consequence of culturing chondrocytes in 3D culture compared to culturing chondrocytes in 2D culture. Nazempour and Tateno teach further dependent limitations. Therefore, given the teachings and motivations of the cited references, it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to combine the cited references to arrive at the instantly claimed invention with a reasonable expectation of success.
As such, Applicant’s arguments do not overcome the rejection over Mizuno, Mori, Caron, Kumar, Nazempour, and Tateno under 35 U.S.C. 103.
Double Patenting
The rejection of amended and cancelled claims 1-2, 4-8, and 11-13 on the ground of nonstatutory double patenting as being unpatentable over claims 11-22 of copending Application No. 18/027,892, hereafter referred to as the ‘892 application, is withdrawn in view of Applicant’s amendments to the ‘892 application claims, wherein the broadest independent claim, claim 23, now recites:
“A method of modifying a cell population to have an increased tissue regeneration capacity for use in a cell therapy, comprising: separating a first cell population from a biological tissue, the cell population having a combination of healthy cells and senescent cells; and, providing a second cell population having an increased tissue regeneration capacity; wherein, the providing includes culturing the first cell population in a 3D thermoreversible polymer gel culture for at least 24 days before the cell therapy to create a supply of cells enriched with the healthy cells that function normally from the second cell population for use in the cell therapy; wherein the culturing for at least 24 days includes removing the senescent cells from the first cell population; extending the average telomere length of the second cell population over the first cell population; and, obtaining a normal cell morphology in the 3D thermoreversible polymer gel culture as compared to creating an abnormal cell morphology in a 2D culture, the normal cell morphology selected from the group consisting of a shape of the cells constituting the cell population, nuclear localization, and size ration between the cell and the nucleus.”
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
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