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 .
Priority
Acknowledgment is made of Applicant's claim for foreign priority based on an application filed in the Socialist Republic of Vietnam on 12 October 2022. It is noted, however, that Applicant has not filed a certified copy of the VN1-2022-06603 application as required by 37 CFR 1.55.
Therefore, the effective filing date of the instant application is 16 February 2023, which is the filing date of the instant application.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The drawings filed 16 February 2023 are objected to for the following minor informalities:
Part 101 in Figure 1 should instead recite “OBTAIN MESENCHYMAL STEM CELL (MSC) SHEETS EXPRESSING PD-L1”. In addition, Figure 6B spells “chondroblast” incorrectly.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The abstract of the disclosure is objected to for comprising grammatical errors and unclear language. More specifically, the abstract filed 16 February 2023 should be amended as shown below, or similarly, to obviate this objection:
“A method of manufacturing [[a]] stem cell sheets is provided which includes: (a) obtaining mesenchymal stem cells; (b) proliferating mesenchymal stem cells that comprise programmed death ligand one (PD-L1); (c) selecting only PD-L1 positive mesenchymal stem cells (PD-L1+ MSCs) from the proliferated mesenchymal stem cells forming the stem cell sheets by mixing the PD-L1+ MSCs with platelet rich plasma solution and CaCl2; and (e) differentiating the PD-L1+ MSCs sheets into osteoblasts and chondroblasts in a predetermined activation condition”.
A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Claim Objections
Claims 1-18 are objected to because of the following informalities:
Regarding claim 1: The instant claim recites “programmed death ligands one” instead of “programmed death ligand[[s]] one” in Line 1 of step (b).
The instant claim also recites “PD-L1+ MSCs” in Lines 5-6 showing a superscript “+”, but recites “PD-L1+ MSCs” without a superscript “+” in Line 8 of the instant claim. Applicant must select either “PD-L1+ MSCs” or “PD-L1+ MSCs” to use consistently throughout the claim language.
Appropriate correction is required.
Regarding claim 2: The instant claim recites “comprising” instead of “comprises” in Line 2.
Appropriate correction is required.
Regarding claim 3: The instant claim recites the limitation “wherein said step (a) further comprises washing said human umbilical cord tissues twice with a washing buffer of antibiotic mycotic.” Instead, the claim should recite the limitation as “wherein said step (a) further comprises washing said human umbilical cord tissues twice with a washing buffer, wherein the washing buffer comprises an antibiotic-mycotic substance”, or similar language, to improve the claim language and match the language within the instant Specification.
Appropriate correction is required.
Regarding claim 4: The instant claim recites “fragments of 1-2 mm2” instead of “fragments of 1-2 mm3” in Line 2. This amendment is necessary to denote that the umbilical cord fragments are three-dimensional, rather than planar fragments that are not physically possible to produce.
Appropriate correction is required.
Regarding claim 5: The instant claim recites “programmed death ligands one” instead of “programmed death ligand[[s]] one” in Line 2.
The instant claim also recites “fragements” instead of “fragments” in Line 3.
The instant claim further requires the word “and” after the recitation of “with a mesenchymal stem cell culture medium for five days; and” in Line 3.
Appropriate correction is required.
Regarding claim 6: The instant claim requires the removal of an extraneous “of” in Line 1.
The instant claim further recites “programmed death ligands one” instead of “programmed death ligand[[s]] one” in Line 2.
The instant claim also recites the limitation “and repeating said steps of replenishing and removing until said mesenchymal stem cells for five more times” in Lines 3-4. The recitation of “until” and “for” in Line 4 should be removed to make the claim language grammatically correct.
Appropriate correction is required.
Regarding claim 7: The instant claim requires the removal of an extraneous “of” in Line 1.
The instant claim also recites “programmed death ligands one” instead of “programmed death ligand[[s]] one” in Line 2.
Appropriate correction is required.
Regarding claim 8: The instant claim recites the limitation “wherein step (c) of selecting only PD-
L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises staining said PD-L1 MScs anti programmed death ligand one antibody conjugated with Phycoerythrin (PE).” Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises staining said PD-L1 MSCs with anti-programmed death ligand one antibody conjugated with Phycoerythrin (PE)” to improve the claim language such that the claim language matches the previously established abbreviations, as well as make the recitation grammatically correct.
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 of the instant claim.
Appropriate correction is required.
Regarding claim 9: The instant claim recites the limitation “wherein step (c) of selecting only PD-L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises performing flow cytometry using a fluorescent activated cell sorting (FACS) tests…”. Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises performing flow cytometry using [[a]] fluorescent activated cell sorting (FACS) tests…” to improve the claim language such that the claim language matches the previously established abbreviations, as well as make the recitation grammatically correct.
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 of the instant claim.
Appropriate correction is required.
Regarding claim 10: The instant claim does not require the recitation of “further” in Line 1. Instead, the limitation should be recited as “wherein said plurality of CDs comprises: …”.
Appropriate correction is required.
Regarding claim 11: The instant claim recites the limitation “wherein step (c) of selecting only PD-L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises...”. Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises…”.
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 of the instant claim.
Appropriate correction is required.
Regarding claim 12: The instant claim recites the limitation “wherein step (c) of selecting only PD-L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises...”. Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises…”.
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 and 3 of the instant claim.
Appropriate correction is required.
Regarding claim 13: The instant claim recites the limitation “wherein step (c) of selecting only PD-L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises...”. Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises…”.
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 of the instant claim.
Applicant must also amend the recitation of “antibiotic mycotic substance” in Line 3 to instead recite “antibiotic-mycotic substance”, as that matches the recitation of instant claim 1 as suggested above.
Appropriate correction is required.
Regarding claim 14: The instant claim recites the limitation “wherein step (c) of selecting only PD-L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises...”. Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises…”.
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 of the instant claim.
Appropriate correction is required.
Regarding claim 15: The instant claim recites the limitation “wherein step (c) of selecting only PD-L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises...”. Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises…”.
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 of the instant claim.
Applicant must also amend the recitation of “antibiotic mycotic substance” in Line 3 to instead recite “antibiotic-mycotic substance”, as that matches the recitation of instant claim 1 as suggested above.
Appropriate correction is required.
Regarding claim 16: The instant claim recites the limitation “wherein step (c) of selecting only PD-L1 positive (PD-L1+ MSCs) from said PD-L1 further comprises repeating said step replenishing unti …”. Instead, the limitation should be recited as “wherein step (c) of selecting only MSCs further comprises repeating said step until
In addition, Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Lines 1-2 and 2-3 of the instant claim.
Appropriate correction is required.
Regarding claim 17: The instant claim recites the limitation “wherein said step (d) of forming PD-L1+ MSCs sheets by mixing said PD-L1+ MSCs with platelet rich plasma solution and CaCl2 further comprises said CaCl2 comprises 5 μl to 20 μl of CaCl2 solution with a concentration is from 0.3 M to 1.8 M per ml of said platelet rich plasma solution”. Instead, the limitation should be recited as “wherein said CaCl2 in said step (d) 2 solution with a concentration
Appropriate correction is required.
Regarding claim 18: Applicant must ensure the selected recitation of either “PD-L1+ MSCs” or “PD-L1+ MSCs” is utilized within Line 1 of the instant claim.
Appropriate correction is required.
Claim Interpretation
The transitional term “contain” recited in instant claim 1 is synonymous with "comprise", which is inclusive or open-ended and allows for additional, unrecited elements or method steps. See MPEP § 2111.03(I).
Furthermore, Applicant has not defined “stem cell sheets”, as recited in instant claim 1 in either the claims or the Specification. Therefore, under broadest reasonable interpretation, the “stem cell sheets” of the instant claims also read on a confluent layer of stem cells. See MPEP § 2111.
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.
Claims 8-20 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 claims 8-20: Instant claim 8 fails to define a claim with which it is dependent upon. Therefore, the ordinary artisan cannot readily determined the metes and bounds of the claim, thus rendering the scope of the claim indefinite. See MPEP § 2173.02.
Instant claims 9-20 either directly depend upon and/or fully incorporate the limitations of instant claim 8, and are thus rendered indefinite as well.
Appropriate correction is required.
For the sake of compact prosecution, the Examiner will interpret instant claim 8 as being dependent upon instant claim 1.
Regarding claims 12-20: Instant claim 12 recites the limitation "said HLA-DR" in Line 3. There is insufficient antecedent basis for this limitation in the claim, as there is no prior recitation of “HLA-DR” within instant claim 12, nor any of the parent claims. See MPEP § 2173.05(e).
Instant claims 13-20 either directly depend upon and/or fully incorporate the limitations of instant claim 12, and are thus rendered indefinite as well.
Appropriate correction is required.
Regarding claims 15-20: Instant claim 15 recites the limitation "said culture medium supplemented with said 2X antibiotic mycotic substance" in Lines 2-3. There is insufficient antecedent basis for this limitation in the claim, as there is no prior recitation of a “culture medium supplemented with said 2X antibiotic mycotic substance” within instant claim 15, nor any of the parent claims. It is of note that instant claim 15 is dependent upon instant claim 14, which is dependent upon instant claim 12. The recitation of a “culture medium supplemented with a 2X antibiotic mycotic substance” occurs in instant claim 13. See MPEP § 2173.05(e).
Instant claims 16-20 either directly depend upon and/or fully incorporate the limitations of instant claim 15, and are thus rendered indefinite as well.
Appropriate correction is required.
For the sake of compact prosecution, the Examiner will interpret the claim as requiring the replenishing of any culture medium inside said container every 24 hours.
Regarding claim 20: The instant claim recites the limitation “wherein said step (e) further comprises adding 5% to 20% insulin and growth factors.” The scope of the claim is indefinite, as the ordinary artisan cannot readily determine to which the 5% to 20% insulin and growth factors are added to, as step (e) requires two separate mediums and the PD-L1+ MSCs sheets – all of which the ordinary artisan could add or otherwise incorporate insulin and growth factors to. Therefore, the metes and bounds of the claim cannot be determined. See MPEP § 2173.02.
Appropriate correction is required.
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.
Claims 1-2, 8-12, and 14-16 are rejected under 35 U.S.C. 103 as being unpatentable over Diao et al (CN 112553155 A, as translated by Espacenet) in view of Womba et al (WO 2018/081514 A1) and Hoogduijn (US 2019/0255115 A1).
Diao et al is considered prior art under 35 USC 102(a)(1). Womba et al and Hoogduijn are each considered prior art under 35 USC 102(a)(1) and 35 USC 102(a)(2).
Regarding claim 1: Diao et al disclose the culture of umbilical cord mesenchymal stem cells (MSCs) in a transwell system, wherein the MSCs are cultured in complete culture medium that comprises platelet-rich plasma that has been activated with added calcium chloride (Abstract; Pages 2-4). Diao et al further disclose that the MSCs cultured in the complete medium form a confluent layer on the bottom of the well (Pages 4-5). It is of note that this confluent layer of MSCs reads on the “sheet” of the instant claims. See Claim Interpretation section above.
Diao et al further disclose that the MSCs can be differentiated into osteoblasts via the culturing of the MSCs in osteoblast differentiation medium (Page 5).
Diao et al do not disclose the sorting of MSCs that express PD-L1, nor the differentiation of the PD-L1+ MSCs layer into chondroblasts, as required by instant claim 1.
Womba et al, however, disclose the pre-treatment of MSCs with IFN-γ, which results in the expression of PD-L1 within the MSCs, and sorting of the PD-L1+ MSCs using FACS (Paragraphs [0087], [0111], [0118], [0128]-[0129], [0144], [0147], [0168]).
Womba et al further disclose that MSCs have the capacity to differentiate into osteoblasts and cells of chondrogenic lineage (Paragraphs [0053], [0102]; Figure 1A). It is of note that such cells of chondrogenic lineage include chondroblasts (see Paragraph [0029] of Hoogduijn).
Therefore, it would have been prima facie obvious to modify the culture method of Diao et al such that the MSCs are pre-treated with IFN-γ and subsequently sorted, as detailed in Womba et al. One of ordinary skill before the effective filing date of the invention would have been motivated to pre-treat the MSCs, as it allows for the MSCs to have an anti-inflammatory, pro-survival phenotype (Womba et al: Paragraph [0091]), and would have had a reasonable expectation based on the disclosure of Womba et al and ease of adaptability into the methods of Diao et al, especially since both teach the differentiation of MSCs into at least osteoblasts. Furthermore, the ordinary artisan would have recognized that the PD-L1+ MSCs can also be differentiated into chondroblasts given the combined disclosures of Womba et al and Hoogduijn. See MPEP § 2143(I)(G).
Consequently, Diao et al as modified by Womba et al and Hoogduijn render obvious a method of culturing MSCs, wherein umbilical cord MSCs are cultured with IFN-γ (reads on step (a)), proliferated (reads on step (b)), sorted via FACS to select PD-L1+ MSCs (reads on step (c)), cultured in complete medium comprising platelet-rich plasma that has been activated with CaCl2 until a confluent layer is formed (reads on step (d)), and differentiated into osteoblasts or chondroblasts using two respective differentiation mediums (reads on step (e)). This therefore renders obvious the method of instant claim 1.
Regarding claim 2: Following the discussion of claim 1, Womba et al further disclose that the mesenchymal stem cells are isolated from umbilical cord tissue (Paragraphs [0038], [0095). This therefore renders obvious the method of instant claim 2 for the same reasons as discussed in the rejection of instant claim 1.
Regarding claim 8: As aforementioned in the discussion of claim 1, Womba et al disclose the sorting of PD-L1+ MSCs via FACS (Paragraph [0109]-[0111]).
Hoogduijn further discloses the sorting of MSCs via FACS using PDL1-PE antibodies, or an anti-PD-L1 antibody that is conjugated with Phycoerythrin (Paragraph [0071]).
This therefore renders obvious the method of claim 8 for the same reasons as discussed in the rejection of instant claim 1.
Regarding claim 9: Following the discussion of claim 8, Womba et al further disclose performing flow cytometry using FACS on the PD-L1+ MSCs against CD45, CD73, CD90, CD105, and HLA-G (Paragraph [0109]-[0111]). This therefore renders obvious the method of claim 9 for the same reasons as discussed in the rejection of instant claim 1.
Regarding claim 10: As aforementioned in the discussion of claim 9, Womba et al disclose performing flow cytometry using FACS on the PD-L1+ MSCs against CD45, CD73, CD90, CD105, and HLA-G (Paragraph [0109]-[0111]).
Diao et al further disclose analyzing the MSCs by flow cytometry against CD44, CD45, CD34, and CD105 (Page 5).
Hoogduijn further discloses analyzing MSCs by flow cytometry against CD73, CD90, and PD-L1 (Paragraph [0071]). Hoogduijn further discloses that CD14 is a marker for monocytes (Paragraphs [0021]-[0022], [0027], [0073], [0081], [0098]).
Therefore, it would have been prima facie obvious to modified the method of Diao et al in view of Womba et al and Hoogduijn to have also performed FACS analysis on the PD-L1+ MSCs against CD14. One of ordinary skill in the art before the effective filing date of the invention would have been motivated to differentiate between monocytes and MSCs within a cell sample, and would have had a reasonable expectation of success given that the disclosures of Diao et al, Womba et al, and Hoogduijn all teach the flow cytometry analysis of MSCs. See MPEP § 2143(I)(G).
Consequently, Diao et al as modified by Womba et al and Hoogduijn render obvious a method of culturing MSCs, wherein the PD-L1+ MSCs are analyzed via FACS against CD14, CD34, CD44, CD45, CD73, CD90, and CD105. This therefore renders obvious the method of instant claim 10.
Regarding claim 11: Following the discussion of claim 10, Womba et al disclose that the PD-L1+ MSCs have positive expression of CD73, CD90, and CD105 (Paragraphs [0102], [0118]; Figure 1B).
Diao et al further disclose that the MSCs have positive expression of CD44 (Page 5).
This therefore renders obvious the method of claim 11 for the same reasons as discussed in the rejection of instant claim 1.
Regarding claim 12: Following the discussion of claim 11, Diao et al further disclose that the MSCs do not express CD34, CD45, and HLA-DR (Page 5).
With that, Hoogduijn reasonably suggests that the MSCs will not express CD14 (Paragraphs [0021]-[0022], [0027], [0073], [0081], [0098]).
This therefore renders obvious the method of claim 12 for the same reasons as discussed in the rejection of instant claim 10.
Regarding claim 14: Following the discussion of claim 12, Diao et al further disclose transferring the MSCs from the transwell system to a six-well culture plate (Page 5). This therefore reads on the method of instant claim 14.
Regarding claim 15: Following the discussion of claim 14, Diao et al further disclose that the culture medium in the six-well plate is changed after one to two days (Page 5). The embodiment wherein the culture medium is changed after one day reads on the method of instant claim 15. See MPEP § 2131.03 and MPEP § 2144.05.
Regarding claim 16: Following the discussion of claim 15, Diao et al further disclose that the MSCs reach 100% confluence in one to two days within the six-well plate (Page 5). The embodiment wherein the MSCs reach 100% confluence within one day – and one culture medium change within 24 hours – reads on the method of instant claim 16.
Claims 1-16 are rejected under 35 U.S.C. 103 as being unpatentable over Diao et al (CN 112553155 A, as translated by Espacenet) in view of Womba et al (WO 2018/081514 A1) and Hoogduijn (US 2019/0255115 A1), and further in view of Skubis et al (Int J Mol Sci, 2017).
The discussion of Diao et al as modified by Womba et al and Hoogduijn regarding claims 1-2, 8-12, and 14-16 can be observed above and is relied upon herein, the content of which is incorporated in its entirety. Diao et al as modified by Womba et al and Hoogduijn render obvious claims 1-2, 8-12, and 14-16. Skubis et al is considered prior art under 35 USC 102(a)(1).
Regarding claim 3: Following the discussion of claim 2, Diao et al further disclose washing the source of the cells with a cleaning solution twice, in a PBS solution comprising human serum albumin (Pages 2-3).
The combination of Diao et al, Womba et al, and Hoogduijn fail to teach the washing of the umbilical cord tissue with a washing buffer comprising an antibiotic-mycotic substance, as required by instant claim 3.
Skubis et al, however, disclose that antibiotics are extremely important in transplantation and regenerative medicine procedures, including the isolation and cultivation of the MSCs (Page 2). Skubis et al further disclose that MSCs are routinely cultured with a combination of Pen/Strep and amphotericin B (AmB) – which is an antimycotic agent (Abstract; Pages 2, 8, 10-11; Figures 1, 8-9).
Therefore, it would have been prima facie obvious to modify the culture method of Diao et al in view of Womba et al and Hoogduijn such that the umbilical cord tissue is initially washed with a PBS solution comprising Pen/Strep and AmB, as suggested in Skubis et al. One of ordinary skill in the art before the effective filing date of the invention would have been motivated to further include Pen/Strep and AmB within the washing solution, as they are the most routinely used antibiotics and the most effective antifungal compound, respectively (Skubis et al: Page 2), and would have had a reasonable expectation of success since the combination of Pen/Strep and AmB within culture does not have any negative effects upon the resulting MSCs in culture. See MPEP § 2143(I)(G).
Consequently, Diao et al as modified by Womba et al, Hoogduijn, and Skubis et al render obvious a method of culturing MSCs, wherein the umbilical cord tissue is washed twice in a PBS solution comprising Pen/Strep-AmB – or an antibiotic-mycotic substance. This therefore renders obvious the method of instant claim 3.
Regarding claim 13: Following the discussion of claim 12, Womba et al further disclose that the PD-L1+ MSCs are maintained at a temperature of 37°C in a culture medium that comprises Pen/Strep (Paragraphs [0102]-[0103], [0113]). It is of note that Pen/Strep are antibiotics.
The combination of Diao et al, Womba et al, and Hoogduijn fail to teach the supplementation of the culture medium with a 2X antibiotic mycotic substance, as required by instant claim 13.
Skubis et al, however, disclose MSCs that have been cultured with a combination of Pen/Strep and either amphotericin B (AmB) or AmB copper ions – which are antimycotic agents – wherein the viability of MSCs cultured with Pen/Strep and Pen/Strep with AmB is comparable for 48-72 hours of culture and does not impact the ability of the MSCs to differentiate into osteoblasts (Abstract; Pages 2, 8, 10-11; Figures 1, 8-9). It is of note that the concentration of Pen/Strep is roughly two times that of AmB within solution (Page 12). See MPEP § 2144.05.
Therefore, it would have been prima facie obvious to modify the culture method of Diao et al in view of Womba et al and Hoogduijn such that the PD-L1+ MSCs are cultured in a culture medium that has been supplemented with Pen/Strep and AmB, as detailed in Skubis et al. One of ordinary skill in the art before the effective filing date of the invention would have been motivated to further include AmB within the culture medium supplemented with Pen/Strep, as it is one of the most effective antifungal compounds (Skubis et al: Page 2), and would have had a reasonable expectation of success since the combination of Pen/Strep and AmB within culture medium effects the MSCs in a similar manner to the culture of MSCs in culture medium supplemented with just Pen/Strep. See MPEP § 2143(I)(G).
Consequently, Diao et al as modified by Womba et al, Hoogduijn, and Skubis et al render obvious a method of culturing MSCs, wherein the PD-L1+ MSCs are cultured in a culture medium supplemented with a 2X Pen/Strep-AmB – or antibiotic mycotic substance. This therefore renders obvious the method of instant claim 13.
Claims 1-2, 4-12, and 14-17 are rejected under 35 U.S.C. 103 as being unpatentable over Diao et al (CN 112553155 A, as translated by Espacenet) in view of Womba et al (WO 2018/081514 A1), Hoogduijn (US 2019/0255115 A1), and further in view of Nagamura-Inoue et al (World J Stem Cells, 2014).
The discussion of Diao et al as modified by Womba et al and Hoogduijn regarding claims 1-2, 8-12, and 14-16 can be observed above and is relied upon herein, the content of which is incorporated in its entirety. Diao et al as modified by Womba et al and Hoogduijn render obvious claims 1-2, 8-12, and 14-16. Nagamura-Inoue et al is considered prior art under 35 USC 102(a)(1).
Regarding claim 4: As aforementioned in the discussion of claim 2, Womba et al disclose that the mesenchymal stem cells are isolated from umbilical cord tissue.
The combination of Diao et al, Womba et al, and Hoogduijn fail to teach the dissection of the umbilical cord tissue into fragments of 1-2 mm3, as required by instant claim 4.
Nagamura-Inoue et al, however, disclose the mincing of umbilical cord tissue into small fragments of 1-2 mm3 to ultimately obtain isolated MSCs via the explant method (Page 196).
Therefore, it would have been prima facie obvious to modify the culture method of Diao et al in view of Womba et al and Hoogduijn such that the umbilical cord tissue is dissected into fragments of 1-2 mm3, as detailed in Nagamura-Inoue et al. One of ordinary skill in the art before the effective filing date of the invention would have been motivated to handle the umbilical cord tissue using a known technique to isolate MSCs, and would have had a reasonable expectation of success since the disclosures of Womba et al and Nagamura-Inoue et al are both concerned with the isolation of MSCs from umbilical cord tissue. See MPEP § 2143(I)(G).
Consequently, Diao et al as modified by Womba et al, Hoogduijn, and Nagamura-Inoue et al render obvious a method of culturing MSCs, wherein the umbilical cord tissue is dissected into fragments of 1-2 mm3. This therefore renders obvious the method of instant claim 4.
Regarding claim 5: Following the discussion of claim 4, Nagamura-Inoue et al further disclose that the umbilical cord tissue fragments are placed onto tissue culture-treated dishes in mesenchymal stem cell culture medium, wherein the culture medium is replaced every 3-7 days – or every five days, including after the initial placement of the umbilical cord tissue fragments on the culture dish – for 2-4 weeks until the mesenchymal stem cells reach 80-90% confluence (Page 196). See MPEP § 2144.05.
Hoogduijn, however, discloses the culture of mesenchymal stem cells from tissue in T75-cm2 culture flasks (Paragraphs [0063], [0065]).
This therefore renders obvious the method of instant claim 5 for the same reasons as discussed in the rejection of instant claim 4.
Regarding claim 6: Following the discussion of claim 5, Diao et al further disclose the trypsinization – or use of a detachment reagent – and replating of the MSCs (Pages 4-5). Diao et al disclose that the MSCs are at least passaged two times (Page 5).
Womba et al, however, disclose passaging the MSCs at least five times (Paragraphs [0102], [0165]).
This therefore renders obvious the method of instant claim 6 for the same reasons as discussed in the rejection of instant claim 1.
Regarding claim 7: Following the discussion of claim 6, Womba et al disclose washing the PD-L1 MSCs with PBS (Paragraph [0120]). This therefore renders obvious the method of claim 7 for the same reasons as discussed in the rejection of instant claim 1.
Claims 1-2, 8-12, and 14-17 are rejected under 35 U.S.C. 103 as being unpatentable over Diao et al (CN 112553155 A, as translated by Espacenet) in view of Womba et al (WO 2018/081514 A1), Hoogduijn (US 2019/0255115 A1), and further in view of Carroll (WO 2022/250969 A1).
The discussion of Diao et al as modified by Womba et al and Hoogduijn regarding claims 1-2, 8-12, and 14-16 can be observed above and is relied upon herein, the content of which is incorporated in its entirety. Diao et al as modified by Womba et al and Hoogduijn render obvious claims 1-2, 8-12, and 14-16. Carroll is considered prior art under 35 USC 102(a)(2), with an effective filing date of 27 May 2021.
Regarding claim 17: Following the discussion of claim 16, Diao et al further disclose that the fraction of calcium chloride within the platelet-rich plasma solution is 3-5% (Pages 2-4). The embodiment wherein the fraction of calcium chloride is 3% is merely close to the instantly claimed ratio of 20 μl calcium chloride solution per ml of platelet-rich plasma solution. See MPEP § 2144.05.
The combination of Diao et al, Womba et al, and Hoogduijn fail to teach the that the calcium chloride has a concentration of 0.3 M to 1.8 M, as required by instant claim 17.
Carroll, however, disclose the activation and coagulation of platelet-rich plasma using calcium chloride that has a concentration of 0.3 M (Paragraphs [0030], [0037], [0064], [0082], [0084]).
Therefore, it would have been prima facie obvious to have substituted the calcium chloride within the complete medium of Diao et al as modified by Womba et al and Hoogduijn for the 0.3 M calcium chloride of Carroll, as doing so would have been a simple substitution of one calcium chloride for another. See MPEP § 2143(I)(B). One of ordinary skill in the art before the effective filing date of the invention would have recognized that the calcium chlorides are functionally comparable, as both are capable of activating platelet-rich plasma, and would have thereby been able to substitute the two calcium chlorides with predictable results.
Consequently, Diao et al as modified by Womba et al, Hoogduijn, and Carroll render obvious a method of culturing MSCs, wherein the PD-L1+ MSCs are cultured in complete medium comprising 3% – which is merely close to 20 μl calcium chloride solution per ml of platelet-rich plasma solution, or 2% – calcium chloride having a concentration of 0.3 M. This therefore renders obvious the method of instant claim 17.
Claims 1-2, 8-12, and 14-19 are rejected under 35 U.S.C. 103 as being unpatentable over Diao et al (CN 112553155 A, as translated by Espacenet) in view of Womba et al (WO 2018/081514 A1), Hoogduijn (US 2019/0255115 A1), and Carroll (WO 2022/250969 A1), and further in view of Jalowiec et al (Tissue Eng Part C Methods, 2015).
The discussion of Diao et al as modified by Womba et al, Hoogduijn, and Carroll regarding claims 1-2, 8-12, and 14-17 can be observed above and is relied upon herein, the content of which is incorporated in its entirety. Diao et al as modified by Womba et al and Hoogduijn render obvious claims 1-2, 8-12, and 14-16. Diao et al as modified by Womba et al, Hoogduijn, and Carroll render obvious claims 1-2, 8-12, and 14-17. Jalowiec et al is considered prior art under 35 USC 102(a)(1).
Regarding claim 18: Following the discussion of claim 17, Diao et al as modified by Womba et al and Hoogduijn render obvious a method of culturing MSCs, wherein PD-L1+ MSCs are cultured in complete medium comprising platelet-rich plasma that has been activated with CaCl2 until a confluent layer is formed.
The combination of Diao et al, Womba et al, Hoogduijn, and Carroll fail to teach the that the layer of PD-L1+ MSCs has a thickness of 100 μm to 3 mm, as required by instant claim 18.
Jalowiec et al, however, disclose the culture of MSCs within platelet-rich plasma gels, wherein platelet-rich plasma has been activated with thrombin (Page 50, Preparation of PRP and PRP-gels; Page 51, MSC isolation, culture, and encapsulation in PRP-gels; Page 54). It is of note that Jalowiec et al disclose that it is well known in the art that the platelet-rich plasma can also be activated with calcium chloride (Page 50, Column 1).
Jalowiec et al further disclose that the MSCs and platelet-rich plasma gel form a layer that is 0.2 mm thick (Page 50, Rheology).
Therefore, it would have been prima facie obvious to modify the culture method of Diao et al in view of Womba et al, Hoogduijn, and Carroll such that the culture of PD-L1+ MSCs with platelet-rich plasma and calcium chloride forms a layer that is 0.2 mm thick, as detailed in Jalowiec et al. One of ordinary skill in the art before the effective filing date of the invention would have been motivated to have a thicker layer, as it is more stable (Jalowiec et al: Page 56), and would have had a reasonable expectation of success since the disclosures of Diao et al and Jalowiec et al are both concerned with the culture of MSCs with activated platelet-rich plasma. See MPEP § 2143(I)(G).
Consequently, Diao et al as modified by Womba et al, Hoogduijn, Carroll, and Jalowiec et al render obvious a method of culturing MSCs, wherein the culturing of PD-L1+ MSCs with platelet-rich plasma and calcium chloride results in a layer that has a thickness of 0.2 mm. This therefore renders obvious the method of instant claim 18.
Regarding claim 19: Following the discussion of claim 18, Diao et al as modified by Womba et al and Hoogduijn render obvious the differentiation of the PD-L1+ MSC layer into osteoblasts or chondroblasts using two respective differentiation mediums. This therefore renders obvious the method of instant claim 19 for the same reasons as discussed in the rejection of instant claim 1.
Claims 1-2, 8-12, and 14-20 are rejected under 35 U.S.C. 103 as being unpatentable over Diao et al (CN 112553155 A, as translated by Espacenet) in view of Womba et al (WO 2018/081514 A1), Hoogduijn (US 2019/0255115 A1), Carroll (WO 2022/250969 A1), and Jalowiec et al (Tissue Eng Part C Methods, 2015), and further in view of Muller et al (Int Orthop, 2013).
The discussion of Diao et al as modified by Womba et al, Hoogduijn, Carroll, and Jalowiec et al regarding claims 1-2, 8-12, and 14-19 can be observed above and is relied upon herein, the content of which is incorporated in its entirety. Diao et al as modified by Womba et al and Hoogduijn render obvious claims 1-2, 8-12, and 14-16. Diao et al as modified by Womba et al, Hoogduijn, and Carroll render obvious claims 1-2, 8-12, and 14-17. Diao et al as modified by Womba et al, Hoogduijn, Carroll, and Jalowiec et al render obvious claims 1-2, 8-12, and 14-19. Mueller et al is considered prior art under 35 USC 102(a)(1).
Regarding claim 20: Following the discussion of claim 19, Diao et al as modified by Womba et al and Hoogduijn render obvious the differentiation of the PD-L1+ MSC layer into osteoblasts or chondroblasts using two respective differentiation mediums.
The combination of Diao et al, Womba et al, Hoogduijn, Carroll, and Jalowiec et al fail to teach the that the differentiation of PD-L1+ MSCs to osteoblasts or chondroblasts to include added growth factors and 5-20% insulin, as required by instant claim 20.
Mueller et al, however, disclose the in vitro chondrogenesis of MSCs via the addition of 50 μg/ml – or 5% – and transforming growth factor beta (TGF-β) within the differentiation medium (Page 154, Cell differentiation; Pages 155, 157).
Therefore, it would have been prima facie obvious to modify the culture method of Diao et al in view of Womba et al, Hoogduijn, Carroll, and Jalowiec et al such that chondroblast differentiation medium comprises added TGF-β and 5% insulin, as detailed in Mueller et al. One of ordinary skill in the art before the effective filing date of the invention would have been motivated to utilize a differentiation medium that is known to successfully induce the chondrogenic differentiation of MSCs, and would have had a reasonable expectation of success since the disclosures of Diao et al and Mueller et al are both concerned with the culture and differentiation of MSCs. See MPEP § 2143(I)(G).
Consequently, Diao et al as modified by Womba et al, Hoogduijn, Carroll, Jalowiec et al, and Mueller et al render obvious a method of culturing MSCs, wherein the PD-L1+ MSCs layer is differentiated into chondroblasts using differentiation medium comprising added TGF-β and 5% insulin. This therefore renders obvious the method of instant claim 20.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ALYSSA G WESTON whose telephone number is (571)272-0337. The examiner can normally be reached Monday-Thursday 8AM - 4PM (CT); Friday 8AM - 11AM (CT).
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Christopher Babic can be reached at (571) 272-8507. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ALYSSA G WESTON/Examiner, Art Unit 1633
/CHRISTOPHER M BABIC/Supervisory Patent Examiner, Art Unit 1633