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 .
Continued Examination Under 37 CFR 1.114
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. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 9/29/2025 has been entered.
Claim Status
As of the Final Office Action mailed 6/27/2025, claims 1-10, 13, and 17-21 were pending.
In Applicant's Response filed on 9/29/2025, claims 1-2, 4, and 9-10 were amended.
As such, claims 1-10, 13, and 17-21 are pending and have been examined herein.
Withdrawn Objections/Rejections
The rejection of record of claims 9 and 21 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) have been withdrawn in view of Applicant’s amendment to claim 9.
The rejection of record of claim 20 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) and further in view of Lin et al (Stem Cells and Dev, Feb 2005) have been withdrawn in view of Applicant’s amendment to claim 9.
Maintained/Modified Rejections
Applicant's arguments regarding the rejection of record of claims 1-2, 5-6, and 13 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) have been fully considered but they are not persuasive. Thus, the rejection has been maintained and recast below to account for amendments to the claims. Response to arguments will follow the recast rejection.
Applicant's arguments regarding the rejection of record of claims 3 and 17-18 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) and further in view of Liu et al (Int J Mol Med 29 Oct 2018; 43(1):199-208) have been fully considered but they are not persuasive. Thus, the rejection has been maintained and recast below to account for amendments to the claims. Response to arguments will follow the recast rejection.
Applicant's arguments regarding the rejection of record of claims 4 and 10 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) and further in view of Lin et al (Stem Cells and Dev, Feb 2005) have been fully considered but they are not persuasive. Thus, the rejection has been maintained and recast below to account for amendments to the claims. Response to arguments will follow the recast rejection.
Applicant's arguments regarding the rejection of record of claim 7 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) and further in view of Austen (US 201120128641 A1, 20 July 2012; previously cited) have been fully considered but they are not persuasive. Thus, the rejection has been maintained and recast below to account for amendments to the claims. Response to arguments will follow the recast rejection.
Applicant's arguments regarding the rejection of record of claim 8 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) and further in view of Gunetti et al (Exp Hematol. Feb 2008; 36(2):235-43; previously cited) have been fully considered but they are not persuasive. Thus, the rejection has been maintained and recast below to account for amendments to the claims. Response to arguments will follow the recast rejection.
Applicant's arguments regarding the rejection of record of claim 19 under 35 USC § 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN 109221082 A, 14 Sept 2018) and Liu et al (Int J Mol Med 29 Oct 2018; 43(1):199-208) and further in view of Austen (US 201120128641 A1, 20 July 2012; previously cited) have been fully considered but they are not persuasive. Thus, the rejection has been maintained and recast below to account for amendments to the claims. Response to arguments will follow the recast rejection.
Claim Rejections - 35 USC § 112(b) – New Grounds of Rejection
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.
Claim 21 is 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 21 recites the limitation "the ASCs" in line 10. There is insufficient antecedent basis for this limitation in the claim as there is no previous recitation of any ASCs in the claim or in claim 9, which this claim depends from. Thus, the claim is indefinite.
Claim Rejections - 35 USC § 102/103 – New Grounds of Rejections
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 9 and 21 is/are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Holman et al (WO 2015195758A1, 6/17/2015; published 12/18/2016).
Holman teaches a cell culture method comprising providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma (see claim 1 of Holman). The reference teaches that the cells are thawed from a frozen stock (see claim 5 of Holman) (“A method for . . . cell cryopreservation, the method comprising the steps of:(a) freezing a population of . . . cells to obtain a frozen population of . . . cells;(b) thawing the frozen population of . . . cells to obtain a thawed population of . . . cells; and (c) culturing the thawed population of . . . cells in the presence NAC to obtain an expanded population of . . . cells” as in instant claim 9). It also teaches that the cell culture medium comprises N- acetylcysteine at a concentration of from about 0.25 mM to about 3 mM (see claim 17 of Holman) (overlaps with “wherein the culturing step comprises adding NAC to an initial concentration of 2 mM” as in instant claim 9). The reference also teaches that others have suggested adding N-acetyl cysteine to cell culture media as a reducing agent to support the growth of, for example, neuronal progenitor/stem cells, or muscle progenitor/stems cells (para 3) (“stem cells” in instant claim 9). While the method itself does not utilize stem cells, the reference clearly shows that the use of NAC to culture stem cells is known in the art.
Instant claim 21 recites language that intends to express the intended result of a process step positively recited in claim 9 (see MPEP 2111.04). Absent evidence to the contrary, the resulting “number of viable cells following thaw is increased…, etc.” as instantly claimed flow from the method step of claim 9.
Thus, claim 9 and 21 are anticipated by or, in the alternative, prima facie obvious over the teachings of Holman.
Response to Arguments
Applicant’s arguments with respect to claim(s) 9 and 21 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Claim Rejections - 35 USC § 103 – Maintained/Modified Rejections
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-2, 5-6, and 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al (CN 102228016 B, 18 Apr 2011; previously cited) in view of Yan et al (CN109221082A, 14 Sept 2018; Published 18 Jan 2019; Ref. 1 of Foreign Patent Documents in IDS filed 31 March 2025; previously cited).
Jin teaches a method of cryopreserving neural stem cells by adding cryopreservation liquid containing 0.8-1.2 mM N-acetylcysteine to a neural stem cell suspension, freezing in liquid nitrogen, then recovering the frozen cells (i.e., thawing) (see claim 1 of Jin) (“method for stem cell cryopreservation, the method comprising the steps of: a. treating a population of stem cells with N-acetylcysteine (NAC) to obtain a treated population of stem cells; and b. freezing the treated population of stem cells to obtain a frozen population of stem cells” as in instant claim 1 in-part; “thawing the frozen population of stem cells to obtain a thawed population of stem cells” as in instant claim 2). Then, the cells are washed, put in cell culture medium, and cultivated (para 10 of “Summary of Invention”) (“culturing the thawed population of stem cells to obtain an expanded population of stem cells” as in instant claim 5). The reference also teaches that after freezing, the cells are put in medium and recovered by using resuscitation fluid containing DMEM or DMEM/F12 culture fluid, comprises following composition: 1 * B27 additive, 1 * N2 additive, L-glutaminate; Final concentration is 1.6-2.4mM; Sodium Pyruvate, final concentration are 0.8-1.2mM, NAC; Final concentration 0.8-1.2mM and BSA, final concentration 0.008-0.012g/ml and cultured (same para) (“culturing the thawed population of stem cells in the presence of NAC to obtain an expanded population of stem cells, optionally wherein the culturing step comprises adding NAC to an initial concentration in the range of around 0.5-5 mM” as in instant claim 6).
Jin differs from the instant invention in that it does not teach the addition of 6 mM NAC (related to instant claim 1 in-part).
Yan teaches a cryopreservation solution that contains reducing agent n-acetylcysteine (see claims 1 and 11-12 of Yan). The reference also teaches that the content of the NAC is 1-5 mM (see claim 13 of Yan). While the reference does not teach “6 mM NAC” as recited in instant claim 1 in-part, it does teach that the reducing agent (NAC) help to stabilize the cell membrane structure, save cell activity, and prevent cell aging (para 12 of “Summary of the Invention”). This shows that NAC can be used in cryopreservation of cells with beneficial effects.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to cryopreserve stem cells with NAC as taught by Jin, where 5 mM of NAC is used as taught by Yan, to arrive at the instantly claimed invention. As Yan shows 1-5 mM NAC can be used in cryopreservation of cells, one of ordinary skill would have been motivated to modify the method of Jin to include any amount of NAC, including at least 5 mM, as taught by Yan with a reasonable expectation of advantageously stabilizing the cell membrane structure, save cell activity, and prevent cell aging as taught by the prior art.
Instant claim 13 recites language that intends to express the intended result of a process step positively recited in claim 1 (see MPEP 2111.04). Absent evidence to the contrary, the resulting “number of viable cells following thaw is increased…, etc.” as instantly claimed flow from the method step of claim 1. Thus, the combination of Jin and Yan render claim 13 prima facie obvious.
Response to Arguments
Applicant’s arguments have been fully considered but are not persuasive.
On p. 8-11 of Remarks, Applicant argues that none of the cited references teach contacting stem cells with 6 mM NAC and that a person of ordinary skill would have no reason to modify the methods of the references in a way to arrive at the instantly claimed method. Applicant argues that the Jin and Yan references both disclose concentrations lower than 6mM and one of ordinary skill would have no expectation that 6mM of NAC would be suitable in cryopreservation methods. Applicant also points to Fig. 4 of the instant specification that demonstrates the beneficial effect of pretreatment with 6mM NAC.
In response, the examiner disagrees. First, it has been repeatedly established that a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985) (Court held as proper a rejection of a claim directed to an alloy of "having 0.8% nickel, 0.3% molybdenum, up to 0.1% iron, balance titanium" as obvious over a reference disclosing alloys of 0.75% nickel, 0.25% molybdenum, balance titanium and 0.94% nickel, 0.31% molybdenum, balance titanium. "The proportions are so close that prima facie one skilled in the art would have expected them to have the same properties."). See also Warner-Jenkinson Co., Inc. v. Hilton Davis Chemical Co., 520 U.S. 17, 41 USPQ2d 1865 (1997) (under the doctrine of equivalents, a purification process using a pH of 5.0 could infringe a patented purification process requiring a pH of 6.0-9.0); In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%) (see MPEP 2144.05(I)). Second, "[A] prior art reference that discloses a range encompassing a somewhat narrower claimed range is sufficient to establish a prima facie case of obviousness." In re Peterson, 315 F.3d 1325, 1330, 65 USPQ2d 1379, 1382-83 (Fed. Cir. 2003). See also In re Harris, 409 F.3d 1339, 74 USPQ2d 1951 (Fed. Cir. 2005) (claimed alloy held obvious over prior art alloy that taught ranges of weight percentages overlapping, and in most instances completely encompassing, claimed ranges; furthermore, narrower ranges taught by reference overlapped all but one range in claimed invention) (see MPEP 2144.05(I)).
Applicant has not provided any empirical evidence on the record that there are any patentably distinct characteristics imparted to the method when 6 mM of NAC is utilized instead of 5 mM. The Patent and Trademark Office is not equipped to conduct experimentation in order to determine to what extent 5 mM of NAC as taught by the prior art differs from applicant' s instantly claimed 6 mM NAC. The prior art discloses 5 mM which is similar to applicant' s 6 MM for these reasons: NAC helps to stabilize the cell membrane structure, save cell activity, and prevent cell aging during cryopreservation. Where an examiner cannot determine whether or not the reference inherently possesses properties which anticipate, or render obvious, the claimed invention a rejection under §§102/103 is appropriate. See MPEP §§ 2112-2112.02.
The cited art taken as a whole demonstrates a reasonable probability that 5mM of NAC is either identical or sufficiently similar to the claimed 6 mM that whatever differences exist, they are not patentably significant. Therefore, the burden of establishing novelty or unobviousness by objective evidence is shifted to applicants. See MPEP § 2112(v). Clear evidence that the 5 mM NAC of the cited prior art does not possess a critical characteristic that is possessed by the claimed 6 mM NAC would advance prosecution and might permit allowance of claims to applicant' s instantly claimed method. Applicant is requested to specifically point out the support for any amendments made to the disclosure and arguments in response to this Office Action, including the claims. See MPEP §§ 714.02 and 2163.06. Applicant is also requested to refer to pages and line numbers in the as-filed specification. It is noted that other art may be applicable under 35 U.S.C. § 102 or 35 U.S.C. § 103(a) once the aforementioned issue(s) is/are addressed. Thus, the rejection is proper absent evidence to the contrary.
Claim(s) 3 and 17-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al in view of Yan et al as applied to claims 1-2, 5-6, and 13 above, and further in view of Liu et al (Int J Mol Med. 29 Oct 2018;43(1):199-208; previously cited).
Regarding claim 3, Jin teaches a method of cryopreserving neural stem cells by adding cryopreservation liquid containing 0.8-1.2 mM N-acetylcysteine to a neural stem cell suspension, freezing in liquid nitrogen, then recovering the frozen cells (i.e., thawing) (see claim 1 of Jin) (“treating the population of stem cells with NAC to obtain a treated population of stem cells, . . . thawing the frozen population of stem cells to obtain a thawed population of stem cells” as in instant claim 3 in-part).
Regarding instant claim 17 and 18, Jin also teaches that after freezing, neural stem cells are washed, put in cell culture medium, and cultivated by using resuscitation fluid containing DMEM or DMEM/F12 culture fluid, comprises following composition: 1 * B27 additive, 1 * N2 additive, L-glutaminate; Final concentration is 1.6-2.4mM; Sodium Pyruvate, final concentration are 0.8-1.2mM, NAC; Final concentration 0.8-1.2mM and BSA, final concentration 0.008-0.012g/ml and cultured (para 10 of “Summary of Invention”) (“culturing the thawed population of stem cells to obtain an expanded population of stem cells” as in instant claim 17; “culturing the thawed population of stem cells in the presence of NAC to obtain an expanded population of stem cells, optionally wherein the culturing step comprises adding NAC to an initial concentration in the range of around 0.5-5 mM” as in instant claim 18).
The teachings of Jin and Yan in combination differ from the instant claims in that they do not teach washing the treated cells to remove the NAC before freezing (related to instant claim 3 in-part).
Liu teaches that NAC is a thiol-containing antioxidant that modulates the intracellular redox state. NAC can scavenge reactive oxygen species (ROS) and maintain reduced glutathione (GSH) levels (abstract). Results revealed that NAC pretreatment increased cell viability and inhibited the activation of caspase-3, -8 and -9 during hydrogen peroxide (H2O2)-induced oxidative stress in H9c2 cells (abstract). Furthermore, decreased ROS levels, and increased total and reduced GSH levels were detected in response to NAC pretreatment (abstract). Following incubation with 4 mM NAC for 1 hr at 37°C, the cells (3-5×106) were treated with 0.75 mM H2O2 for 0, 15 and 30 min, and NEM-alkylated redox western blotting was performed to detect the redox state of mito-GFP, Prx1, Trx1, GSR and PTEN and the cells were washed twice with ice-cold PBS immediately after treatment (“NEM-alkylated redox western blotting” para 2) (“washing the treated population of . . . cells to remove the NAC and to obtain a washed population” as in instant claim 3 in-part). This shows that NAC pretreatment alleviates oxidation of intracellular antioxidant proteins and that cells can be washed after pre-treatment with NAC.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to treat a population of stem cells with NAC and freeze them as taught by Jin and Yan in combination, where the cells washed to remove the NAC as taught by Liu, to arrive at the instantly claimed invention. As Liu shows that cells can be pretreated with NAC then washed, one of ordinary skill would have been motivated to modify the method of Jin and Yan in combination with a reasonable expectation of advantageously maintaining the antioxidant effects of NAC even after washing it off as taught by the prior art.
Response to Arguments
On p. 11 of Remarks, Applicant argues that the Liu reference describes NAC in a concentration of 4 mM, which is lower than the concentration recited by claim 1. Applicant also argues that Liu does not teach or suggest that the NAC can be removed before oxidative stress is applied and that the cells used in Liu are not the same as the pending claims.
In response, the examiner notes that the Liu reference was not cited to render obvious the NAC concentration utilized in the method. Rather, it was cited to render obvious the washing step claimed in claim 3. Moreover, the cited reference explicitly teaches the “following incubation with 4 mM NAC for 1 hr at 37°C, the cells (3-5×106) were treated with 0.75 mM H2O2 for 0, 15 and 30 min, and NEM-alkylated redox western blotting was performed to detect the redox state of mito-GFP, Prx1, Trx1, GSR and PTEN and the cells were washed twice with ice-cold PBS immediately after treatment” (i.e., pretreated and washed). Finally, while the cells of Liu are different from the instantly claimed cells, the teachings of the reference of using washing NAC off of cells after treatment still applies and is reasonably pertinent to the instant claims. It has been held that a prior art reference must either be in the field of the inventor' s endeavor or, if not, then be reasonably pertinent to the particular problem with which the inventor was concerned, in order to be relied upon as a basis for rejection of the claimed invention. See In re Oetiker, 977 F.2d 1443, 24 USPQ2d 1443 (Fed. Cir. 1992). Thus, the rejection is proper.
Claim(s) 4 and 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al in view of Yan et al as applied to claims 1-2, 5-6, 9, and 21 above, and further in view of Lin et al (Stem Cells and Dev, Feb 2005; 14:92-102; previously cited).
The teachings of Jin and Yan were recited in the above 35 U.S.C. 103 rejection as applied to claim 1 of which claims 4 and 10 depend. The teachings will not be repeated here.
The difference between the teachings and the invention as instantly claimed is that they do not teach incubating the population of stem cells with NAC for at least about 1 hour prior to freezing the population of stem cells.
Lin teaches the accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in mediums using reduced calcium and antioxidants (title). The reference teaches that MSCs from adipose tissue can be cultures using a method that accelerates greatly the growth rate and prolongs the lifespan of adipose MSCs by culturing in low calcium conditions and supplementing with N-acetyl-L-cysteine (abstract) (“wherein the stem cells are mesenchymal stems and/or wherein the stem cells are adipose-derived stem cells” as in instant claim 10). The reference teaches that after centrifugation of adipose tissue, it was weighed and distributed into several 50-ml tubes (8 gram/tube) containing 40 ml of Dulbecco’s modified Eagle medium(DMEM) with collagenase (1 mg/ml), NAC (2 mM), and ascorbic acid 2-phosphate (0.2 mM) (p. 96, col 1 top para). After incubation overnight at 37°C, tubes were centrifuged to remove the collagenase solution and the pellet was washed and then incubated in DMEM with 10% fetal bovine serum (FBS), 2 mM NAC, and 0.2 mM ascorbic acid 2-phosphate in a 5% CO 2 incubator. On the next day, the unattached cells were removed by washing with PBS (same para). Then, 5 ml of K-NAC medium with 5% FBS was added to each 25-cm 2 flask. Media was changed every other day and the cells were allowed to grow until near confluence (same para). After 1 week, sufficient cells were generated for trypsinization and storage in liquid nitrogen or for subculturing (same para) (“wherein the treatment step comprises incubating the population of stem cells for at least about 1 hour prior to freezing the population of stem cells” as in instant claim 4).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to treat a population of stem cells with NAC and freeze them as taught by Jin and Yan in combination, where the cells are incubated with NAC before freezing as taught by Lin, to arrive at the instantly claimed invention. As Lin shows cells can be incubated with NAC before freezing, one of ordinary skill would have been motivated to modify the method of Jin and Yan in combination to include incubation with NAC prior to freezing with a reasonable expectation of advantageously having adipose-derived MSCs with accelerated growth and prolonged lifespan as taught by the prior art.
Response to Arguments
On p. 12 of Remarks, Applicant argues that the method of Lin is different from that of claim 1, which is focused on short-term pretreatment. Applicant argues that one of ordinary skill would not extrapolate the long-term culture in Lin to that of the instant claims.
In response, the examiner disagrees. First, Applicant’s instant claims are drawn to, inter alia, “incubating the population of stem cells with NAC between 1 and 48 hours.” Lin explicitly teaches incubation with NAC for 1 day (24 hours) (see above rejection). While it does discuss a further culturing steps wherein the medium contains NAC, the initial incubation renders obvious the instant claims. Second, the transitional term "comprising", which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. See, e.g., Mars Inc. v. H.J. Heinz Co., 377 F.3d 1369, 1376, 71 USPQ2d 1837, 1843 (Fed. Cir. 2004) ("[L]ike the term ‘comprising,’ the terms ‘containing’ and ‘mixture’ are open-ended."). Invitrogen Corp. v. Biocrest Manufacturing, L.P., 327 F.3d 1364, 1368, 66 USPQ2d 1631, 1634 (Fed. Cir. 2003) ("The transition ‘comprising’ in a method claim indicates that the claim is open-ended and allows for additional steps."); Genentech, Inc. v. Chiron Corp., 112 F.3d 495, 501, 42 USPQ2d 1608, 1613 (Fed. Cir. 1997) ("Comprising" is a term of art used in claim language which means that the named elements are essential, but other elements may be added and still form a construct within the scope of the claim.) (see MPEP 2111.03(I)). Finally, 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).
Claim(s) 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al in view of Yan et al as applied to claims 1-2, 5-6, and 13 above, and further in view of Austen (US 20120128641 A1, 20 July 2010; published 24 May 2012; previously cited).
The teachings of Jin and Yan were recited in the above 35 U.S.C. 103 rejection as applied to claim 2 of which claim 7 depends. The teachings will not be repeated here.
Regarding claim 7, Jin teaches that after freezing, the neural stem cells are washed, put in cell culture medium, and cultivated (para 10 of “Summary of Invention”) (“further comprises a step of washing the thawed population of stem cells” as in instant claim 7 in-part).
The difference between the teachings and the invention as instantly claimed is that they do not teach resuspending the washed cells in a pharmaceutically acceptable carrier.
Austen teaches methods for increasing the viability of cryopreserved cells after thawing (abstract). The reference teaches thawing the cryopreserved cells in the presence of a polyether (see claim 1 of Austen) and that after thawing, the cells in the presence of the polymer can be combined with pharmaceutically acceptable excipients and antioxidant to form a composition useful for adding to cells to be transplanted and can be added during or following thawing to prevent cell death (para 0065-0066). The antioxidant includes N-acetylcysteine (para 0066).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to treat stem cells with NAC and freeze them as taught by Jin and Yan in combination, where the cells are resuspended in a pharmaceutically acceptable excipient as taught by Austen, to arrive at the instantly claimed invention. As Austen shows that stem cells can be cryopreserved then mixed with a pharmaceutically acceptable excipient post-thaw, one of ordinary skill would have been motivated to modify the method as taught by Jin and Yan in combination to include mixing the cells in a pharmaceutically acceptable excipient post-thaw with a reasonable expectation of advantageously prevent post-thaw cell death as taught by the prior art.
Response to Arguments
Applicant has not provided any arguments challenging the specific teachings of cited reference Austen related to instant claim 7 nor has Applicant attempted to distinguish the teachings of Austen and the instantly claimed invention.
Claim(s) 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al in view of Yan et al as applied to claims 1-2, 5-6, and 13 above, and further in view of Gunetti et al (Exp Hematol. Feb 2008; 36(2):235-43; previously cited).
The teachings of Jin and Yan in combination were recited in the above 35 U.S.C. 103 rejection as applied to claim 6 of which claim 8 depends. The teachings will not be repeated here.
The difference between the teachings and the invention as instantly claimed is that they do not teach freezing expanded population of stem cells after thawing.
Gunetti teaches that several requirements need to be fulfilled for clinical use of expanded hematopoietic stem cells (abstract). Successive freeze-thaw procedures do not significantly affect the clonogenic potential of cord blood hematopoietic stem cells (p. 236, first full para) when quickly refrozen within 15 minutes after re-thaw (same page, col 2, top para). 25% of the total volume of cells was drawn through an inserted injection coupler in 150-mL collection bags, slowly mixed with the same volume of sterile washing solution and centrifuged once (400g/10 min) (p. 236 col 2 top para). The remaining 75% of the thawed unit was quickly refrozen within 15 minutes, cryopreserving immediately the remaining volume of the thawed CB units, contained in the Haemofreeze DF700 bags, by a standard method (1C/min programmed cooling rate, stored in liquid nitrogen) (same para). They demonstrated that successive freeze-thaw procedures do not significantly affect the clonogenic potential and human long-term engraftment in NOD/SCID mice (same para).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to treat stem cells with NAC and freeze them as taught by Jin, where the stem cells are frozen after expansion as taught by Gunetti, to arrive at the instantly claimed invention. As Gunetti shows that hematopoietic stem cells can be re-frozen after culture, one of ordinary skill would have been motivated to refreeze the expanded stem cells of Jin with a reasonable expectation of advantageously having stem cells that maintain their clonogenic potential after refreezing as taught by the prior art.
Response to Arguments
Applicant has not provided any arguments challenging the specific teachings of cited reference Gunetti related to instant claim 8 nor has Applicant attempted to distinguish the specific teachings of Gunetti and the instantly claimed invention.
Claim(s) 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al (in view of Yan et al and Liu et al as applied to claims 3 and 17-18 above, and further in view of Austen et al (US 20120128641 A1, 20 July 2010; published 24 May 2012; previously cited).
The teachings of Jin, Yan, and Liu in combination were recited in the above 35 U.S.C. 103 rejection as applied to claim 3 of which claim 19 depends. The teachings will not be repeated here.
Regarding claim 19, Jin teaches that after freezing, the neural stem cells are washed, put in cell culture medium, and cultivated (para 10 of “Summary of Invention”) (“further comprises a step of washing the thawed population of stem cells” as in instant claim 19 in-part).
The difference between the combined teachings and the invention as instantly claimed is that they do not teach resuspending the washed cells in a pharmaceutically acceptable carrier.
Austen teaches methods for increasing the viability of cryopreserved cells after thawing (abstract). The reference teaches thawing the cryopreserved cells in the presence of a polyether (see claim 1 of Austen) and that after thawing, the cells in the presence of the polymer can be combined with pharmaceutically acceptable excipients and antioxidant to form a composition useful for adding to cells to be transplanted and can be added during or following thawing to prevent cell death (para 0065-0066). The antioxidant includes N-acetylcysteine (para 0066).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to treat stem cells with NAC and freeze them as taught by Jin, Yan, and Liu in combination, where the cells are resuspended in a pharmaceutically acceptable excipient as taught by Austen, to arrive at the instantly claimed invention. As Austen shows that stem cells can be cryopreserved then mixed with a pharmaceutically acceptable excipient post-thaw, one of ordinary skill would have been motivated to modify the method as taught by Jin, Yan, and Liu in combination to include mixing the cells in a pharmaceutically acceptable excipient post-thaw with a reasonable expectation of advantageously prevent post-thaw cell death as taught by the prior art.
Response to Arguments
Applicant has not provided any arguments challenging the specific teachings of cited reference Austen related to instant claim 7 nor has Applicant attempted to distinguish the teachings of Austen and the instantly claimed invention.
Claim Rejections - 35 USC § 103 – New Grounds of Rejections
Claim(s) 20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Holman et al as applied to claims 9 and 21 above, and further in view of Lin et al (Stem Cells and Dev, Feb 2005; 14:92-102; previously cited).
The teachings of Holman was recited in the above 35 U.S.C. 102/103 rejection as applied to claim 9 of which claims 20 depend. The teachings will not be repeated here.
The difference between the teachings and the invention as instantly claimed is that they do not teach incubating the population of stem cells with NAC for at least about 1 hour prior to freezing the population of stem cells.
Lin teaches the accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in mediums using reduced calcium and antioxidants (title). The reference teaches that MSCs from adipose tissue can be cultures using a method that accelerates greatly the growth rate and prolongs the lifespan of adipose MSCs by culturing in low calcium conditions and supplementing with N-acetyl-L-cysteine (abstract) (“wherein the stem cells are mesenchymal stems and/or wherein the stem cells are adipose-derived stem cells” as in instant claim 20). The reference teaches that after centrifugation of adipose tissue, it was weighed and distributed into several 50-ml tubes (8 gram/tube) containing 40 ml of Dulbecco’s modified Eagle medium(DMEM) with collagenase (1 mg/ml), NAC (2 mM), and ascorbic acid 2-phosphate (0.2 mM) (p. 96, col 1 top para). After incubation overnight at 37°C, tubes were centrifuged to remove the collagenase solution and the pellet was washed and then incubated in DMEM with 10% fetal bovine serum (FBS), 2 mM NAC, and 0.2 mM ascorbic acid 2-phosphate in a 5% CO 2 incubator. On the next day, the unattached cells were removed by washing with PBS (same para). Then, 5 ml of K-NAC medium with 5% FBS was added to each 25-cm 2 flask. Media was changed every other day and the cells were allowed to grow until near confluence (same para). After 1 week, sufficient cells were generated for trypsinization and storage in liquid nitrogen or for subculturing (same para).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to culture stem cells in NAC after freezing and thawing as taught by Holman, where the cells are adipose MSCs as taught by Lin, to arrive at the instantly claimed invention. As Lin shows adipose MSCs cells can be cultured with NAC, one of ordinary skill would have been motivated to simply substitute the cells of Holman with the adipose MSCs of Lin with a reasonable expectation of advantageously having adipose-derived MSCs with accelerated growth and prolonged lifespan as taught by the prior art.
Conclusion
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/G.R./Examiner, Art Unit 1632
/KARA D JOHNSON/Primary Examiner, Art Unit 1632