DETAILED ACTION
Claims 1-10 are pending.
Priority
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
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.
1.Claims 1-10 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.
Instant claims 1 and 8 recite the use of a “re-staining solution”. However, there is no recitation of a first staining, thus it is unclear how the cells can be re-stained, if they had not been stained prior to step 3 of claim 1. Further, it is unclear what a “re-staining solution” is and thus it is unknown what solution is being used and when. The metes and bounds of the “re-staining solution” are unknown and one would not know what is being referred to.
For the purpose of compact prosecution, the limitation of “a re-staining solution” is interpreted to encompass a staining solution.
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.
2.Claims 1-10 are rejected under 35 U.S.C. 103 as being unpatentable over Song et al. “Dual growth factor-modified microspheres nesting human-derived umbilical cord mesenchymal stem cells for bone regeneration.” Journal of biological engineering vol. 17,1 43. 10 Jul. 2023, doi:10.1186/s13036-023-00360-w, in view of Lu et al., “Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.” Haematologica 91 8 (2006): 1017-26, and in view of Ma et al., “Immunomodulatory effect of human umbilical cord mesenchymal stem cells on T lymphocytes in rheumatoid arthritis.” International Immunopharmacology Vol. 74, 2019 https://doi.org/10.1016/j.intimp.2019.105687.
Instant claim 1 recites “A detection method for immune protein of human umbilical cord mesenchymal stem cells (hUC-MSCs), comprising the following steps: 1) inoculating hUC-MSCs in a 24-well plate, and adding a medium for incubation; 2) when the hUC-MSCs grow to 60%-80%, discarding the medium, conducting immobilization, and obtaining immobilized cells; 3) adding a substrate application solution into the immobilized cells obtained in step 2) for incubation away from light, mixing the immobilized cells with a re-staining solution for staining, and conducting microscopic observation; when no brown or dark-brown particle is found in cytoplasm, no immune protein is secreted by the hUC-MSCs; and when brown or dark-brown particles are found in cytoplasm, immune protein is secreted by the hUC-MSCs”.
Song teaches a detection method for immune protein of human umbilical cord mesenchymal stem cells (hUC-MSCs) (see page 16 “The osteogenic differentiation of HUMSCs was directly assessed by measuring the expression levels of related genes, including Runx-2, COL-1 and OCN, by RT‒qPCR analysis (Fig. 9).”), comprising the following steps:
1) inoculating hUC-MSCs in a 24 well plate (see abstract “we constructed a micromodule for MTE and developed engineered osteon-like microunits by inoculating human-derived umbilical cord mesenchymal stem cells (HUMSCs) onto nHA/PLGA microspheres with surface modification of dual growth factors (BMP2/bFGF).”, see page 4 “HUMSCs were seeded on different microspheres in 24-well plates for 4 days”),
adding a medium for incubation (see page 4 “HUMSCs were cultured in F12 medium containing 10% fetal bovine serum, in a 5% CO2, 37 °C cell culture incubator, with fluid changes every two days…After 2 h of incubation, 200 μl of medium was transferred into a 96-well plate for measurement”);
2) growing the hUC-MSCs (see page 4 “pDA/nHA/PLGA nanocomposite microsphere samples were sterilized with 75% ethanol for 30 min before loading growth factors and growing HUMSCs, followed by sterilization in a UV sterilizer for 2–3 h”), discarding the medium (see page 4 “The culture medium was aspirated and discarded”), conducting immobilization, and obtaining immobilized cells (see page 9 “With increasing culture time, the outer surface of the nHA/PLGA-pDA microspheres was covered by a fusion layer of HUMSCs. In particular, the outer surface of the micromodules with growth factors immobilized on the surface showed more green sites than the group without cytokine immobilization, indicating that the presence of cytokines led to a more significant promotion of the adhesion of the micromodules with growth factors immobilized on the surface showed more green sites than the group without cytokine immobilization, indicating that the presence of cytokines led to a more significant promotion of the adhesion, proliferation…”);
3) adding a substrate application solution into the immobilized cells obtained in step 2) for incubation away from light (see page 3 “For the loading of BMP2/ bFGF growth factor, different ratios (2:8, 5:5, 8:2) (Table1) of 500 μl mixed solutions as well as two kinds of single composition solutions were injected into 48-well plates.”, see page 4 “First, the BCA working solution was prepared by mixing the A and B solutions in a 50:1 volume ratio, and 200 μl of BCA working solution was added to each well of a 96-well plate. Then, 20 μl of the abovementioned cell lysate was added to each well. The plate was incubated at 37 °C for 30 min. A Tecan Infinite M200 was used to read the absorbance at 562 nm (OD562)”), mixing the immobilized cells with a re-staining solution for staining, and conducting microscopic observation; (see page 4 under “cell adhesion assay”, see page 5 under “histological staining”) (instant claim 1).
Song teaches the use of serum free media (see page 3 “Analytical grade (99.99% purity) N-Methylpyrrolidone (NMP) was purchased from Sigma − Aldrich. Dulbecco’s modified Eagle medium (DMEM),Dulbecco’s modified Eagle medium nutrient mixture F-12 (DMEM F-12)… Briefly, nHA/ PLGA microsphere samples were placed in 48-well cell culture plates (Corning Costar, USA), and 1 ml of preconfigured pDA solution (2 mg/ml in 10 mM Tris–HCL, pH = 8.5) was added to each well and placed on a constant temperature shaker at 37 °C for 4 h.”, see page 4 under “construction of MTE units”) (instant claim 4), and the conditions for incubation include incubation being conducted at 37ºc and 5% CO2 (see page 4 “HUMSCs were cultured in F12 medium containing 10% fetal bovine serum, in a 5% CO2, 37 °C cell culture incubator, with fluid changes every two days”) (instant claim 5). Song teaches using a paraformaldehyde solution with a mass percentage of 4% (see page 4 “HUMSCs were seeded on different microspheres in 24-well plates for 4 days. The cells were fixed in 4% PFA for 30 min” (instant claim 6). Song teaches wherein the addition amount of the substrate application solution in step 3) is 200 µl/well (see page 4 “After 2 h of incubation, 200 μl of medium was transferred into a 96-well plate for measurement”) (instant claim 7).
Song does not teach the hUC-MSCs growing to 60-80%, brown particles in the cytoplasm, the concentration of hUC-MSCs being 1x105 cells/well, nor the generation of the hUC-MSCs being P3 to P6.
Lu teaches wherein the hUC-MSCs grow to 60%-80% (see page 1018 “Once 60%-80% confluence had been reached, adherent cells were replated at a density of 1×104/cm2 in UC-GM for expansion”) (instant claim 1). Lu teaches the concentration of hUC-MSCs being 1x105 cells/well (see page 1019 “…from UC (n=16) or BM (n=6) were seeded in UC or BMGM in six-well plates in six dilution steps. UC: 1×105…”) (instant claim 2). Lu teaches wherein the hUC-MSCs in step 1) belong to any generation from P3 to P6 (see page 1019 under “cell cycle analysis”) (instant claim 3).
Ma teaches when there are no brown or dark-brown particle found in cytoplasm, no immune protein is secreted by the hUC-MSCs; and when brown or dark-brown particles are found in cytoplasm, immune protein is secreted by the hUC-MSCs (see page 3 “When brown staining appeared in the cytoplasm, the reaction was stopped by tap water. Then, the tissue was re-dyed with hematoxylin, dehydrated, clarified, and finally sealed with gum. Image-Pro Plus 5.1 Graphic Analysis System (Media Cybernetics, Rockville, MD, USA) was used to capture histochemical images through optical microscopy (Olympus BX41 microscope).”, see figure 4) (instant claim 1).
It would have been obvious to one of ordinary skill in the art to routinely optimize the amount of time the immobilization and paraformaldehyde interact, the amount of time the stain is in contact with the cells, the amount of time the cells are incubated, the temperature of the incubation, and the amount of staining solution in a well, depending on the cells being cultured. Absent evidence of unexpected results, it would have been prima facie obvious to have arrived at the claimed times (3, 4, and 15 minutes), the incubation temperature (20-25ºC) and the amount of staining solution being used (200 µl/well) as an obvious matter of routine optimization, namely performing routine experimentation to determine the optimum and workable times, temperature, and amount of solution needed for the assay and cell culture (instant claims 6 and 8- 10).
In general, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See also MPEP §2144.05.
It would have been obvious to one of ordinary skill in the art at the time of the instant application to modify the method of culturing cells taught by Song with the methods of isolating hUCMSC’s taught by Lu, and with the method of detecting immune cells in hUCMSC’s taught by Ma. One of ordinary skill in the art would have been motivated to grow the cells 60-80% because Lu teaches growing the cells to 60-80% confluence is a common technique in the art, in the art it is known that growing the cells 60-80% of the culture disk, maintains the cells health and avoiding inhibiting contact. One of ordinary skill in the art would have been motivated to detect immune particles in hUCMSC’s because Ma teaches that hUCMSCs have the ability to inhibit the proliferation of immune cells and the secretion of inflammatory factors (see page 2). Ma further teaches hUCMSC’s compared to other mesenchymal stem cells have many more advantages such as a wide source, easy access to materials, strong proliferation ability, low immunogenicity, and great differentiation potential (see page 2). The artisan would have reasonable expectation of success based on the cumulative disclosures of these prior art references.
Conclusion
No claim is allowed.
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/MCKENZIE A DUNN/ Examiner, Art Unit 1678
/GREGORY S EMCH/ Supervisory Patent Examiner, Art Unit 1678
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