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 .
Detailed Action
The examiner for this Application has changed. Please direct all future correspondence to Patent Examiner, Katriel B Kasayan, AU 1634. Additional contact information can be found at the end of this paper.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office 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. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 February 4th, 2026 has been entered.
Claims 1-13 and 25 are currently pending. No claims were amended by Applicants’ amendment filed on February 4, 2026.
Therefore, claims 1-13 and 26 are currently under examination to which the following grounds of rejection are applicable.
Priority
The present application is a CON of International Application No. PCT/IN2020/050623, filed on July 18th, 2020, which claims priority to Indian applications 201941029039, 201941029042, 201941029040, and 201941029041 all of them published on July 18, 2019.
. Filing of a certified copies of the Indian Applications 201941029039 07/18/2019 201941029040, 201941029041 and 201941029042, filed February 17, 2022 is acknowledged.
Thus, the earliest possible priority for the instant application is July 18th, 2019.
Information Disclosure Statement
The information disclosure statements submitted on 02/04/2026 was filed. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
RESPONSE TO ARGUMENTS
Maintained objections/ Rejections in response to Applicants’ arguments or amendments
Claim Rejections - 35 USC § 112- First paragraph- New Matter
Claims 1-13 and 26 remain rejected under 35 U.S.C. 112, first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter, which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP § 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims” and “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112, para. 1, as lacking adequate written description”. According to MPEP § 2163.I.B, “While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure” and “The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117”.
Claim 1 (from which all other claims depend) recites “ wherein the population of mesenchymal stem cells does not comprise the corneal limbal stem cells” The response dated 7/7/2025 indicates that support for the amendments regarding a population of mesenchymal stem cells that do not comprise the corneal limbal stem cells could be found throughout the application as originally filed. There is not specific indication however for culture and expansion of MSCs where the population of mesenchymal stem cell does not comprise the corneal limbal stem cells (e.g. Corneal Stromal Stem Cells (CSSC) and limbal epithelial stem cells (LESC)) in a culture medium comprising corneal stromal stem cell (CSSC) derived condition medium (CSSC-Cm) to prime and expand a population of mesenchymal stem.
The Specification teaches in Examples 2, 3 and 4 isolation and expansion of populations of mesenchymal stem cells comprising Primary Human Bone Marrow-Mesenchymal Stromal Cells (hBM-MSC), Adipose-Derived Mesenchymal Stem Cells (ADMSC), Umbilical Cord Derived Mesenchymal Stromal Cells (UCMSC), respectively. The Specification contemplates in paragraph [0153] that any source of MSCs can be used , e.g, “The MSCs derived from the sources such as, bone marrow, umbilical cord, adipose tissue, dental pulp, wharton's jelly) and these MSCs can be primed with the conditioned media derived from CSSCs. …. It can be contemplated that the same process is applied for priming the MSCs derived from other sources also, and in obtaining the conditioned media-derived from MSCs.”
Example 1 discloses the isolation and culture of Corneal Stromal Stem Cells (CSSC) from excised limbal ring using the combination I, i.e. Digestion with Liberase (LIB)+Minimum Essential Medium (MEM) media (LIB_MEM) relative to other three combinations (paraphs [0113]-[0114]0 which resulted in high-quality yield of CSSCs , where “the combination of LIB_MEM (combination 1) was found to be most suitable for the maintenance of stemness markers in CSSCs, as compared to processes using the combination II, III, and IV respectively” (para. [0119]). The Specification further discloses that expanded high quality CSSCs obtained at P1 and P2 were then characterized using markers for Limbal epithelial stem cells (LESC) and Corneal stromal stem cells (CSSC) with the goal of obtaining a high-quality yield of CSSCs and enrichment of CSSCs over limbal epithelial stem cells (LESCs). Passage 2 specifically resulted in “The enrichment of CSSCs over LESCs resulted in a pure stromal stem cell population. The yield of pure stromal stem cell population obtained at passage 3 was in the range of 4-6 million.” (paragraph [0121]). Therefore, the “high population of CSSC and CSSC-derived secretomes and exosomes can be then used individually and in combination thereof, as a final product for various clinical applications from Passage 2-3.”. Thus the Specification clearly provides support for the use of CSSC-derived secretomes and exosome from Passages 2-3 isolated using the combination I (LIB_MEM). Finaly, Example 5 discloses that the CSSC-conditioned media (CSSC-CM) obtained by the culturing the CSSCs isolated from a single cornea, by following the steps as described in the Example 1 (e.g., CSSC-derived secretomes and exosome from Passages 2-3) was cultured with BMMSCs (e.g., In particular, the BMMSCs were cultured in the presence of CSSC-CM at a concentration of 10% and 20%. ) (paragraph [0154] of the published application) “ . The Specification further states, to demonstrate the benefits of the priming of the MSCS (BMMSCs) with the CSSCs conditioned media, characterization of CSSC-CM primed BMMSCs was done (paragraphs [0157]-[0162]). Example 8 in paragraphs [0245]-[249] describes isolation and purification of secretome and exosomes from the Cell Culture using three methods: (i) Single step ultracentrifugation; (ii) Sucrose based cushion density ultracentrifugation and (iii) Iodixanol density gradient ultracentrifugation, wherein the (i) “Single-Step Ultracentrifugation” allows for the collection of supernatant (e.g, “conditioned medium was collected from the CSSC and hBMMSC”) to isolate exosomes after filtered through a 0.45-micron filter. [0256] The media was further filtered through a 0.22-micron filter (para [0248])
The Specification is silent about culture and expansion of MSCs where the population of mesenchymal stem cell that is obtained for the steps of b) priming and c) expanding excludes a distinct species of cultured population of mesenchymal stem cells, corneal limbal stem cells (e.g., LESC and CSSC).
The therapeutic effect of Conditioned Medium Derived from Corneal Mesenchymal Stromal Cells was known in the art before the effective filing date of the claimed invention. For example, Jabbehdari, et al. (May 2020, CURRENT EYE RESEARCH 2020, VOL. 45, NO. 12, 1490-1496of record IDS filed on 7/7/2025) studied the therapeutic effect of MSC-derived conditioned-medium using in vitro and in vivo models of corneal epithelial wound healing (abstract). Jabbehdari, also refers to the prior art of Roddy et al. (page 1490, col. 2) and the benefits of MSC- conditioned-medium derived from their secreted bioactive factors. Moreover, the prior art of Cha et al., ( (US 20190144830; of record ) teaches cultures of expanded primed mesenchymal stem cell population selected from corneal stroma and umbilical cord blood, for example (paragraph [0024] of the published application]). However, Cha et al., does not teach or provide any reason as to why a population of corneal limbal stem cells would be excluded from mesenchymal stem cell populations that need to be primed and expanded .
Though the applicants provide support for co-cultures of mesenchymal stem cells from various sources with conditioned medium obtained from expanded high quality CSSCs obtained at P1 and P2 , it is not clear that the Applicant was in possession of a genus of undefined of co-cultures of mesenchymal stem cells for priming and expansion where corneal limbal stem cells were excluded before the effective filing date of the claimed invention
Response to Applicants’ Arguments as they apply to rejection of claims 1-13 and 26 rejected under 35 U.S.C. 112, first paragraph
At pages 6 and 7, Applicants’ argue that the limitation of the claimed population of MSCs “does not comprise the corneal limbal stem cells”, 1) meets the written description requirement as paragraph 0162 specifies the population of MSCs to be primed as being derived from non-ocular sources, 2) a person with ordinary skill in the art would understand that the population of MSCs as described in the specification is necessarily distinct from corneal limbal cells and 3) “paragraph [0162] of the present application, which specifies the population of MSCs to be primed as being derived from non-ocular sources”. Applicants’ arguments received on February 04, 2026, have been fully considered but are not persuasive.
Regarding 1) as stated in page 5 of the Final Rejection filed September 4th, 2025, there is no teaching within the Specification where priming and expansion of MSCs excludes a distinct population of corneal limbal stem cells Although it is noted in paragraphs [0246]-[0249] that the CSSC-CM was purified (supernatant was collected) through filtration, the limitation only provides support in the Specification in the context of isolating exosomes with therapeutic value after filtering and where conditioned medium was collected from the CSSC and hBMMSC . There is no evidence nor reason provided by Applicants or disclosed in the Specification supporting exclusion corneal limbal stem cells from cultured population of mesenchymal stem cells.
Regarding 2) Applicants’ contend that a person with ordinary skill in the art would know to exclude the population of corneal limbal stem cells, suggesting that limbal stem cells require a strict limbal niche and not expected to proliferate in CSSC-CM. However, Yoon et al. (Published: 2014. World J Stem Cells;6(4):391–403) teaches that limbal stem cells are likely to survive outside of their niche (“The Wnt/β-catenin signaling system has been shown to be responsible for preventing apoptosis of limbal cells in vitro[131]. The authors suggested that as long as survival factors are present, limbal stem cells are likely to survive outside their niche. Indeed, in a mouse model, LSCD (limbal stem cell deficiency) was successfully treated with human limbal fibroblast-conditioned culture medium but not with skin fibroblast-conditioned medium, again emphasizing the importance of chemical signals produced in the limbus”). Therefore, if limbal stem cells are capable of living outside of their own niche, then there would be no biological barrier preventing them from being cultured in a CSSC-CM. As such, a skilled artisan would not assume the exclusion of limbal stem cells form the population of MSCs.
Regarding 3), paragraph [0162], merely teaches that priming MSCs derived from non-ocular sources e.g., bone-marrow MSCs (BM-MSCs) with CSSC-CM (where secretomes have been isolated) , appear to exhibited many features resembling exosomes derived from corneal MSCs. In fact, paragraph [0065] teaches that “The yield of secretory proteins, extracellular vesicle (EV), such as, exosomes derived from the enriched population of CSSCs is a limiting factor for large-scale production for stem cell therapies.” Figure 29 illustrates higher yield of corneal MSC-like exosomes through priming than exosomes derived from corneal limbal stem cells. Thus, there is nothing in paragraph [0162] leading one of ordinary skill in the art to consider exclusion of corneal limbal stem cells from a culture of MSCs.
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Withdrawn objections/ Rejections in response to Applicants’ arguments or amendments
Claim Rejections - 35 USC § 103
In view of Applicants’ arguments, the rejection of claims 1-4 and 13 under 35 USC 103 in view of Cha et al. (US Pub 20190144830) in view of Zhu et al (Published: 2016. Int J Opthalmol, 332-339) and Then et al (Asian K Opthalmol;15:224-33) has been withdrawn.
Although Zhu discloses a corneal stromal cell conditioned medium, it does not explicitly recite that corneal limbal cells are excluded.
Applicants’ arguments are moot in view of the withdrawn rejection.
In view of Applicants’ arguments, the rejection of claims 1-8 and 13 under 35 USC 103 in view of Cha et al. (US Pub 20190144830) in view of Zhu et al (Published: 2016. Int J Opthalmol, 332-339) and Then et al (Asian K Opthalmol;15:224-33) and Bartosh et al. (Published: 2014. Current Protocols in Stem Cell Biology, 32 pages; of record) has been withdrawn.
Applicants’ arguments are moot in view of the withdrawn rejection.
New rejections necessitated by amendment of the claims in the response filed on February 4th, 2026
Claim Rejections - 35 USC § 112
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 10 and 12 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.
Regarding claim 10, it is indefinite in its recitation of the phrase” wherein the microcarriers are in a size ranging from 50-500 micrometers”. It is unclear whether the “size” is directed to diameter, radius, circumference, etc.
Regarding claim 12, step (f), it is unclear how the passaging of corneal limbal stem cells results in expanded corneal stromal cells and a corneal stromal cell conditioned medium. Claim 12 is directed to a product by process limitation wherein the corneal stromal stem cell derived-conditioned medium is obtained through steps a) -f). However, step f) requires passaging the corneal limbal stem cells of step ( e) to obtain both expanded corneal stromal stem cells, and a corneal stromal stem cell derived conditioned medium. It is noted in the Specification details a method of obtaining the conditioned media through filtration. Thus, it is unclear if the corneal stromal stem cell derived-conditioned medium contains corneal limbal stem cells or the corneal limbal stem cells / exosomes are isolated from the culture medium via filtration.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-8 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Cha et al. (US Pub 20190144830) in view of Morales et al. (World J Stem Cells 2019; 11(2): 84-99).
Regarding claim 1 and 2, Cha teaches a method for obtaining a mesenchymal stem cells (para [0024], “In one embodiment, the adult stem cells may be mesenchymal stem cells, for example, human mesenchymal stem cells”) and culturing the mesenchymal stem cells (para [0033], “In the present disclosure, as suggested above, when stem cells are cultured by the three-dimensional cell culture method, three-dimensional cell aggregates are produced by self-aggregation of cells”), culturing the population of mesenchymal stem cells in a culture medium comprising a corneal stromal stem cell derived conditioned medium (para [0024], “The mesenchymal stem cells may be selected from multipotent cells derived from non-marrow tissues such as placenta, umbilical cord blood, adipose tissues, adult muscle, corneal stroma, dental pulp of baby teeth, etc.), to obtain primed mesenchymal stem cells (para [0034], “] Accordingly, the method of producing the stem cell-derived extracellular vesicles may include producing three-dimensional cell aggregates by culturing stem cells by three-dimensional cell culture.”); expanding the primed mesenchymal stem cells obtained in a culture medium (para [0034]), to obtain the claimed population of expanded primed mesenchymal stem cells (para [0060], “Therefore, the culture of the cell aggregates of stem cells including the stem cell-derived extracellular vesicles may be usefully applied to various therapies”).
However, Cha fails to teach the population of mesenchymal stem cells does not comprise the corneal limbal stem cells.
Morales et al. teaches a corneal stromal stem cell-derived conditioned medium excluding corneal limbal stem cells (page 88, “Conditioned medium was collected from CSSC at P4 cultured in SCM and SCM supplemented with 1 ng/mL IL-1β. CSSC were seeded at 10000 cells/cm2 in T75 cm2 flasks. Conditioned medium was produced by adding 12 mL appropriate media and culturing for 72 h, before collection and filtering through a 0.22 µm filter.) Furthermore, Morales teaches that the CSSC-derived conditioned medium “demonstrate some positive effects by reducing cytotoxicity and IL-6/IL-8 production, demonstrating that CSSC secrete factors into the media that have a positive anti-inflammatory effect” (page 85).
A person with ordinary skill in the art would have been motivated to incorporate the CSSC conditioned media to exclude corneal limbal cells, as taught by Morales, into the invention described in Cha in order to promote a positive anti-inflammatory effect, reduce overall cytotoxicity and decrease IL-6/IL-8 production within claimed mesenchymal cells in culture. There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results in relation to producing the stem cell-derived extracellular vesicles for various therapies.
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Regarding claim 3, the combined teachings of Cha and Morales render obvious claim 1. Moreover, Morales teaches that the population of cells is done in a culture medium comprising the CSSC-derived conditioned medium comprising a volume percentage in a range of 5-50% with respect to the culture medium (Figure 5A).
The adjustment of particular conventional working conditioned therein (e.g. determining the various effective amount/ranges of each claimed active ingredient within the claimed method and the substitution of one system for another to obtained the desired result) is deemed merely a matter of judicious selection and routine optimization which is well within the purview of the skilled artisan.
Regarding claim 4, the combined teachings of Cha and Morales render obvious claim 1. Moreover, Cha teaches that expanding the mesenchymal stem cells is done in a spheroid-based system (para [0035], “The three-dimensional cell aggregate produced by three-dimensional cell culture may have, for example, a spheroidal structure, but is not limited thereto”).
Regarding claims 5-8, wherein the expanded primed mesenchymal stem cells are claimed as a product by process of obtaining said expanded primed mesenchymal stem cells, it is noted that Cha teaches that expanding the mesenchymal stem cells is done in a spheroid-based system, therefore the expanded mesenchymal stem cells of Cha have the same structural limitations of the claimed expanded primed mesenchymal stem cells of claim 4, absent any evidence to the contrary. It is noted that In re Best (195 USPQ 430) and In re Fitzgerald (205 USPQ 594) discuss the support of rejections wherein the prior art discloses subject matter which there is reason to believe inherently includes functions that are newly cited or is identical to a product instantly claimed. In such a situation the burden is shifted to the applicants to "prove that subject matter shown to be in the prior art does not possess characteristic relied on" (205 USPQ 594, second column, first full paragraph). It is noted that, if the prior art discloses identical chemical structure, the properties applicant discloses and/or claims are necessarily present, In re Spada, 911 F.2d 705, 709, 15 USPQ2d.
Regarding claim 13, the combined teachings of Cha and Morales render obvious claim 1. Moreover, Cha teaches that the population of mesenchymal stem cells are bone marrow derived (para [0023], “The adult stem cells are stem cells extracted from the cord blood (umbilical cord blood) or adult bone marrow, blood, nerve, etc., and refer to primitive cells immediately before they are differentiated into cells of specific organs”).
***
To the extent that the process of obtaining an expanded primed mesenchymal stem cells in a spheroid-based system as recited in claims 5-8 is given patentable weight , the following rejection applies.
Claim(s) 1, 4 and 5-8 are rejected under 35 U.S.C. 103 as being unpatentable over Cha et al. (US Pub 20190144830) in view of Morales et al. (World J Stem Cells 2019; 11(2): 84-99), and in further view of Bartosh et al (Published: 2014. Current Protocols in Stem Cell Biology, 32 pages. Cited in IDS filed June 14 2022) and Matak et al. (Published: 2017. Cytotechnology. 2017 Aug;69(4):565-578).
Regarding claim 1 and 4, the cited references of Cha and Morales render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated herein, in its entirety. . Cha teaches that expanding the mesenchymal stem cells is done in a spheroid-based system (para [0035], “The three-dimensional cell aggregate produced by three-dimensional cell culture may have, for example, a spheroidal structure, but is not limited thereto.”). Moreover, Cha teaches that the spheroids have a cell density of mesenchymal stem cells of 600 stem cells per spheroid, as required by claim 5 (para [0078], “Human bone marrow-derived mesenchymal stem cells cultured in a laboratory were seeded on the PEG microwell array (600-700 cells/microwell)”; para [0036], “The number of the stem cells included in the three-dimensional cell aggregate may be about 10 cells to about 2500 cells”).
However, the combined teachings of Cha and Morales do no expressly teach the overall claimed steps in the process of expanding primed mesenchymal stem cells performed by a spheroid-based system to obtain the claimed population of expanded primed mesenchymal stem cells.
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Bartosh described obtaining a population of expanded stem cells and pelleting the primed mesenchymal stem cells obtained in step (b) of claim 1 to obtain a stem cell pellet (page 5-6, Steps 1-16, describes culturing and expanding a population of mesenchymal stem cells, as well as pelleting the population of stem cells into a single tube in preparation for spheroid formation.); resuspending the primed mesenchymal stem cell pellet in an MSC basal medium (page 6, Step 10, “Aspirate the supernatant and resuspend cell pellet in up to 1 ml of CCM.”; page 19, Recipe for Complete Culture Medium (CCM), comprising Minimal Essential Medium, a basal medium); processing the primed mesenchymal stem cells to obtain mesenchymal stem cell spheroids with a cell density (page 2, Hanging Drop Culture Technique for the Development of MSC Spheres, Step 3, bottom of page, “For hanging drops consisting of 25,000 cells per drop, dilute the MSC cell suspension to 714 cells/μl with CCM and transfer the suspension to a sterile reagent reservoir.”) and culturing the primed mesenchymal stem cell spheroids in a culture medium comprising MSC (page 1, “Three-dimensional (3-D) culture techniques, such as hanging-drop cultures, have been used in studies to mimic more closely the microenvironments and cellular interactions occurring in vivo”; Figure 2B6.2).
A person with ordinary skill in the art would have been motivated to expand the primed mesenchymal stem cells in a three-dimensional culture taught by Cha and Morales, while incorporating the spheroid-based culture method taught by Bartosh to promote cell-to-cell and extracellular matrix interactions that cannot be achieved in 2D adherent cultures, and permit activation with various signalling pathways or basic cell function. There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results.
However, the combined teachings of Cha, Morales and Bartosh do not teach that the culture medium contains methylcellulose, as recited in claims 6-8.
Matak discloses a method for aggregating stem cells using a 3D stem cell culture comprising of methylcellulose within a concentration range of 0.2-2% with respect to the culture medium (page 567 col 2 “Cells after the passage were collected as described and used for 3D culture with methylcellulose (MC). Frozen methylcellulose containing medium (3% stock solution, R&D Systems, Minneapolis, MN, USA) was thawed at 4 °C. Working concentrations ranging from 0.01 to 1% of MC were prepared by dilution with RPMI with varying amounts of FBS”). Furthermore, Matak teaches that the incorporation of methylcellulose in culture generally improved aggregation rates of stem cells, claiming “Cell lines formed aggregates at a higher rate with the methylcellulose method than with the hanging drop assay. Cells that did not create solid structures in the hanging drop assay could aggregate in MC [methylcellulose]” (page 575 col 2).
It would have been obvious for one with ordinary skill in the art to modify the hanging-drop culture for developing spheroids, as taught by the combined teaching of Bartosh, Cha and Morales, and have methylcellulose in the mixture to promote the aggregation of stem cells at a faster rate than a generic hanging drop assay. Additionally, incorporation of methylcellulose in the culture may circumvent the issue of cells not developing solid structures through the hanging drop assay, as recited by Matak. Such modification would allow the improvement of the spheroid-based system taught by the combined teachings of Cha, Bartosh, and Morales, and yield predictable results to one of ordinary skill in the art.
***
Claim(s) 1, 4, 9-11, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Cha et al. (US Pub 20190144830) in view of Morales et al. (World J Stem Cells 2019; 11(2): 84-99), as applied to claims 1 and 4 above, and further in view of Xu et al. (Published: June 2019. Journal of Orthopaedic Translation, vol. 18, pp. 128–141).
Regarding claim 1 and 4, the cited references of Cha, Morales render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated herein, in its entirety. Moreover, Cha teaches that expanding the mesenchymal stem cells is done in a spheroid-based system (para [0035], “The three-dimensional cell aggregate produced by three-dimensional cell culture may have, for example, a spheroidal structure, but is not limited thereto.”).
However, the combined teachings of Cha and Morales do no expressly teach the overall claimed process performed by a microcarrier-based system to obtain the claimed population of expanded primed mesenchymal stem cells.
Xu teaches the microcarrier based system (page 128, Abstract “Alg-hBMSC microspheres cultured in the 2D well plate (Alg-hBMSCsþ2D) group”) comprising the steps of obtaining microcarriers with alginate and gelatin (page 128, abstract, “Our study reports the optimization of electrospray human bone marrow stromal cell (hBMSCs) embedded alginate gelatin (Alg-Gel, same as following) microspheres for the purpose of their assembly in 3D-printed poly(ε-caprolactone) (PCL) scaffold for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct.”), suspending the microcarriers in a culture medium (page 128, Abstract). Furthermore, it is noted that the use of the alginate-gelatin microspheres improved proliferation of stem cells (page 140, “Alg-Gel microspheres produced by electrospray had an excellent cytocompatibility and promoted the stem cell proliferation. The Alg-Gel composition selected maintained a higher level of hBMSC viability in comparison to alginate microspheres and supported chondrogenesis in vitro.”).
A person with ordinary skill in the art would have been motivated alternatively to choose a microcarrier based system taught by Xu rather than the three-dimensional culture taught by Cha and Morales, particularly to promote and improve cell viability within mesenchymal stem cells as disclosed by Xu. Such modification would allow the improvement of the microcarrier-based system taught by the combined teachings of Cha, Morales and Xu , and yield predictable results to one of ordinary skill in the art.
Regarding claims 9-11, and 26 wherein the expanded primed mesenchymal stem cells are claimed as a product by process of obtaining said expanded primed mesenchymal stem cells, it is noted that Xu teaches that expanding the mesenchymal stem cells is done in a microcarrier-based system, therefore the expanded mesenchymal stem cells of Xu have the same structural limitations of the claimed expanded primed mesenchymal stem cells of claim 4, absent any evidence to the contrary. It is noted that In re Best (195 USPQ 430) and In re Fitzgerald (205 USPQ 594) discuss the support of rejections wherein the prior art discloses subject matter which there is reason to believe inherently includes functions that are newly cited or is identical to a product instantly claimed. In such a situation the burden is shifted to the applicants to "prove that subject matter shown to be in the prior art does not possess characteristic relied on" (205 USPQ 594, second column, first full paragraph). It is noted that, if the prior art discloses identical chemical structure, the properties applicant discloses and/or claims are necessarily present, In re Spada, 911 F.2d 705, 709, 15 USPQ2d.
***
To the extent that the process of obtaining an expanded primed mesenchymal stem cells in a microcarrier-based system as recited in claims 5-8 is given patentable weight, the following rejection applies.
Claim(s) 1, 4, 9-11 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Cha et al. (US Pub 20190144830) in view of Morales et al. (World J Stem Cells 2019; 11(2): 84-99), as applied to claims 1 and 4 above, and further in view of Xu et al. (Published: June 2019. Journal of Orthopaedic Translation, vol. 18, pp. 128–141) and Thermofisher (Wayback Machine Publication Date: 2017. “Isolation of Viable Spheroids from AlgiMatrix Reagent | Thermo Fisher Scientific - US”).
Regarding claim 1 and 4, the cited references of Cha, Morales and Xu render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated herein, in its entirety. Moreover, Xu teaches the microcarrier based system (page 128, Abstract)
Regarding claim 9, Xu teaches the microcarrier based system (page 128, Abstract “Alg-hBMSC microspheres cultured in the 2D well plate (Alg-hBMSCsþ2D) group”) comprising the steps of obtaining microcarriers with alginate and gelatin (page 128, abstract, “Our study reports the optimization of electrospray human bone marrow stromal cell (hBMSCs) embedded alginate gelatin (Alg-Gel, same as following) microspheres for the purpose of their assembly in 3D-printed poly(ε-caprolactone) (PCL) scaffold for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct.”), suspending the microcarriers in a culture medium (page 128, Abstract). Furthermore, it is noted that the use of the alginate-gelatin microspheres improved proliferation of stem cells (page 140, “Alg-Gel microspheres produced by electrospray had an excellent cytocompatibility and promoted the stem cell proliferation. The Alg-Gel composition selected maintained a higher level of hBMSC viability in comparison to alginate microspheres and supported chondrogenesis in vitro.”).
Regarding claim 10, the combined teachings of Cha, Morales, and Xu render obvious the claimed invention. Moreover, Xu teaches that the microcarriers are in a size ranging from 50-500 micrometers (page 128, “Nonaggregated, low polydispersity and almost spherical microspheres of average diameter of 200e300 mm were produced with alginate”).
Regarding claim 11, the combined teachings of Cha, Morales, and Xu render obvious the claimed invention claim 9. Moreover, Xu teaches that the microcarriers a comprise sodium alginate at a concentration range of 0.2%-20% and gelatin at a concentration range of 0.2%-20% (page 130, “Sodium alginate (Alg) (powder, from brown algae, cat. n. 71238, Sigma, USA) was dissolved in distilled water at room temperature at a concentration of 1.5% w/v.”; Gelatin (powder, from bovine skin, Type B, cat. n. G9391, Sigma, USA) was dis solved in distilled water at 40 C at a concentration of 0.5, 1.5 and 2.5% w/w.”).
However, the combined teachings of Cha, Morales and Xu do not teach a dissolution buffer containing sodium chloride and trisodium citrate, as required by claim 26.
Thermofisher teaches a method of isolating spheroids from an Algimatrix using a dissolution buffer comprising trisodium citrate and sodium chloride (Preparation of iso-osmolar tri-sodium citrate solution, Steps 1-3 describe a solution containing 55mM of trisodium citrate from a 1M stock and adding NaCl).
It would have been obvious for someone with ordinary skill in the art to utilize the method of isolating spheroids using a dissolution buffer comprising trisodium citrate and sodium chloride as taught by Thermofisher for breaking down the alginate-gelatin matrix. There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results.
Conclusion
No claims are allowed.
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/KATRIEL BARCELLANO KASAYAN/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634