DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant’s response filed December 8, 2025 has been received and entered into the application file. All arguments have been fully considered. Claims 1, 4, 6-7, 9, 11-12 and 14 are currently pending. Claims 1 and 4 are currently amended. Claims 9, 11-12 and 14 are withdrawn. Claims 2-3, 5, 8, 10 and 13 are cancelled.
REJECTION(S) WITHDRAWN
Claim Rejections - 35 USC § 112
RE: Rejection of Claim 4 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite:
Applicant’s amendment submitted 12/8/2025 has removed the phrase “the extracellular matrix” from claim 4, thus obviating the previous rejection of record. The rejection is withdrawn.
Claim Rejections - 35 USC § 103
RE: Rejection of Claim(s) 1 and 4 under 35 U.S.C. 103 as being unpatentable over Noh et al, in view of Sun:
Applicant’s amendment submitted 12/8/2025 has amended claim 1 to now recite the limitation “…wherein the nanofiber composed of chitin is obtained by pulverizing the chitin…”
Noh, in view of Sun did not further teach obtaining the chitin nanofibers by pulverizing the chitin. Therefore, the previous rejection of record is withdrawn. However, Applicant’s amendment has necessitated a new ground of rejection as set forth below.
RE: Rejection of Claim(s) 6-7 under 35 U.S.C. 103 as being unpatentable over Noh, in view of Sun, and further in view of Hayashi:
For the reasons discussed above, the rejection of claims 1 and 4 over Noh et al, in view of Sun, is withdrawn, and thus the rejection of claims 6-7 that is based on the same basis is likewise withdrawn. However, the amendment submitted 12/8/2025 has necessitated new grounds of rejection, as set forth below.
NEW GROUND(S) OF REJECTION, NECESSITATED BY AMENDMENT
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, 4 and 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Noh et al, Noh et al., (Biomaterials, 27(21): 3934-3944 (2006); IDS 8/30/2022, previously cited) (“Noh”), in view of Sun et al., (Rev. Adv. Mater. Sci. 44(2016) 160-167; previously cited) (“Sun”) and Hayashi et al (US 2017/0009201; IDS 3/14/2022, previously cited) (“Hayashi”).
Noh is directed to the degradation behavior and cellular response of chitin nanofibers (i.e., water-insoluble polysaccharide) employed in cell culturing of normal human keratinocytes and fibroblasts (Abstract).
Regarding claim 1, Noh specifically teaches preparing a cell culture medium by placing 14 mm diameter (o.1 mm thick) chitin nanofibrous matrices in 24-well culture plates for the purpose of conducting cell attachment and spreading assays. Noh teaches that, prior to combining the chitin nanofibers with cells, the chitin nanofibers are subjected to pretreating wherein one type of pretreatment is coating the chitin nanofibers with ECM proteins, such as collagen, fibronectin or laminin (200 µl/well at 0.1-50µg/ml) for overnight absorption. Concentrations of 50 μg/ml type I collagen, 1 μg/ml fibronectin, and 10 μg/ml laminin showed the approximately maximal effect on NHOK (2.7 Cell attachment and spreading assays, page 3936).
Although Noh teaches producing a cell culture medium by coating (carried) various extracellular matrices, such as collagen, fibronectin or laminin, on a nanofiber composed of chitin and adding the ECM-coated nanofiber to the culture medium, Noh does not further exemplify coating the chitin nanofibers with vitronectin.
However, Sun is directed to nanofiber design for use in stem cell cultures. Sun teaches that nanofibers (e.g., chitosan) have been successfully used in cell culture, including ECM-modified nanofibers (extracellular matrix-modified nanofibers) (Abstract; 2.2.3 Chitosan, page 162). Sun further teaches that nanofiber surface modifications include incorporation of ECM molecules, e.g., vitronectin, into the nanofibers by physical adsorption or covalent surface modification (3. SURFACE MODIFICATION OF NANOFIBERS, 3.1 Natural ECM modified nanofibers, page 163).
Thus, Sun has established it was well known in the art that vitronectin is a suitable extracellular matrix for modifying nanofibers used for cell culture.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute vitronectin, as the extracellular matrix component in the method of Noh.
The person of ordinary skill in the art would have been motivated to use vitronectin, as taught by Sun, for the predictable result of providing a cell adherent ECM coating to the nanofiber scaffolds thus promoting efficient cell proliferation.
The skilled artisan would have had a reasonable expectation of success in substituting the vitronectin extracellular matrix, for the collagen, fibronectin or laminin ECMs of Noh because Sun has shown that vitronectin is a suitable extracellular matrix for coating or covalent modification of nanofibers used in cell culture.
Further regarding claim 1 and the newly amended limitation “wherein the nanofiber composed of chitin is obtained by pulverizing the chitin”, it is first noted Applicant’s specification ([0028]-[0029]) discloses the chitin starting material is pulverized by various techniques including a high-pressure homogenizer, a grinder (stone mill), a bead mill, or wet grinding as disclosed in JP-A-2005-270891.
It is further noted that although Noh teaches the nanofibers composed of chitin are obtained from chitin powder (2.1 Reagents, page 3935), Noh does not further comment on the chitin powder being prepared by being pulverized. However, Hayashi is directed to cell culture methods since said cultures can be utilized for pharmaceutical products and regenerative medicine to supplement damaged organs or tissues, wherein the cell culture medium compositions comprise nanofibers prepared from chitin (Abstract and [0032]-[0034]). Hayashi teaches the nanofibers are prepared by pulverization, wherein pulverization is carried out by a high-pressure homogenizer, a grinder (stone mill), a beadmill or by the wet pulverization method method disclosed in JP-A-2005-270891 ([0033] and [0115]-[0116]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to obtain the chitin powder by pulverizing the chitin starting material for preparation of the chitin nanofibers.
The person of ordinary skill in the art would have been motivated to modify the method of Noh to obtain the chitin powder by pulverizing the chitin starting material for preparation of the chitin nanofibers, as taught by Hayashi, for the predictable result of successfully obtaining the particulated chitin powder to allow dispersion and dissolving in solution for subsequent electrospinning of the nanofibers, thus meeting the limitation of claim 1.
The skilled artisan would have had a reasonable expectation of success in combining the teachings of Noh and Hayashi because each of these teachings are directed at using chitin nanofibers in cell culture medium.
Regarding claim 4 and the limitation directed to the quantity of vitronectin extracellular matrix per gram of the nanofiber, specifically 0.01-50 mg/gram of nanofiber (0.01-50 µg/mg), it is noted that Noh specifically teaches preparing a cell culture medium by placing 14 mm diameter (o.1 mm thick) chitin nanofibrous matrices in 24-well culture plates for the purpose of conducting cell attachment and spreading assays. Noh teaches that, prior to combining the chitin nanofibers with cells, the chitin nanofibers are pre-treated by coating the chitin nanofibers with ECM proteins using concentrations ranging from 0.1-50µg/ml, wherein 200 µl of the ECM solutions are combined with the 14 mm diameter chitin nanofiber matrix (correlates to 0.02µg to 10µg of ECM per 14 mm nanofiber matrix) (2.7 Cell attachment and spreading assays, page 3936).
Although Noh teaches the 14 mm nanofiber matrices are combined with 0.02µg to 10µg (prior art mass overlaps claimed mass) of the ECM proteins, Noh does not further indicate the weight of the 14 mm chitin nanofiber matrices prior to coating with the ECM proteins. Thus, the only difference between Noh’s teaching and the claimed method is Noh does not further indicate the weight of the 14 mm chitin nanofiber matrices prior to coating with the ECM proteins.
It is noted the claimed range comprises only the routine optimization of the ECM proteins. Said optimization would have been obvious and well-within the purview of the ordinarily skilled artisan at the time of filing as these proteins promote cell attachment.
Note that the optimization of doses/concentrations would have been prima facie obvious to one of ordinary skill in the art at the time of filing:
“[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 MPEP 2144.05).
As set forth at MPEP 2144.05 II. A:
“Generally, 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.”
Regarding claims 6-7 and the limitations directed to the addition of a chitosan nanofiber to the culture medium (claim 6), and further content ratio of chitin nanofiber carrying vitronectin to chitosan nanofiber = 1: 0.5-20 (claim 7), it is noted that Noh does not further teach the additional chitosan nanofiber matrix. However, Hayashi is likewise directed to adherent cell culture methods comprising the use of a cell culture medium comprising nanofiber matrices (e.g., chitin, chitosan, cellulose) and thus retaining more cells on the matrices and preventing the sedimentation of said cells (Abstract; [0033]). Hayashi teaches the use of the culture medium comprising the nanofiber matrices permits culturing the adherent cells at high density with efficient proliferation ([0167]). Hayashi, at Experimental Example 18 ([0312]) specifically exemplifies preparing serum-free cell culture mediums wherein chitosan nanofiber or chitin nanofiber is added at a final concentration of 0.001% (w/v), the medium is added to 96-well plates and 100,000 cells/mL are added and cultured for 12 days. The culture mediums comprising chitosan and chitin nanofibers are compared to culture medium comprising deacylated gellan gum. Hayashi, at [0313], teaches that cell proliferation was higher for the culture mediums comprising chitin and chitosan nanofiber matrices, as compared to the culture medium comprising deacylated gellan gum. The chitin nanofiber showed high proliferative capacity even at 0.001% concentration, and the chitosan nanofiber showed high proliferative capacity from 0.01% concentration. Thus, Hayashi has shown that cell culture mediums comprising chitin and chitosan nanofibers promote higher cell proliferation.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further add a chitosan nanofiber matrix to the culture medium of Noh, in view of Sun.
The person of ordinary skill in the art would have been motivated to modify the method of Noh, in view of Sun, to include a chitosan nanofiber matrix, as taught by Hayashi, for the predictable result of successfully promoting higher cell proliferation resulting in culturing the adherent cells at high density with efficient proliferation, thus meeting the limitation of claim 6.
The skilled artisan would have had a reasonable expectation of success in combining the teachings of the cited prior art and Hayashi because each of these teachings are directed at preparing cell culture mediums comprising nanofiber matrices prepared from water-insoluble polysaccharides.
Regarding claim 7 and the claimed content ratio of chitin nanofiber and chitosan nanofiber, it is noted that Hayashi does not further teach a culture medium comprising both chitin and chitosan nanofibers at the claimed content ratio. However, Hayashi, at [0313], teaches the chitin nanofiber showed high proliferative capacity even at 0.001% (w/v) concentration, and the chitosan nanofiber showed high proliferative capacity from 0.01% (w/v) concentration. Thus, Hayashi has demonstrated using 10-fold higher content of chitosan, as compared to chitin nanofiber, which correlates to a chitin: chitosan content ratio of 1:10 (claimed range overlaps the prior art range).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to prepare a cell culture medium wherein a content ratio of chitin nanofiber to chitosan nanofiber is 1:10.
The person of ordinary skill in the art would have been motivated to modify the method of the cited prior art to use a content ratio of 1:10 for chitin nanofiber to chitosan nanofiber, as taught by Hayashi, for the predictable result of successfully promoting higher cell proliferation resulting in culturing the adherent cells at high density with efficient proliferation, thus meeting the limitation of claim 7.
In the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05
Claim(s) 1 and 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Hayashi et al (US 2017/0009201; IDS 3/14/2022, previously cited) (“Hayashi”), in view of Sun et al., (Rev. Adv. Mater. Sci. 44(2016) 160-167; previously cited) (“Sun”).
Regarding claim 1, Hayashi is directed to cell culture methods since said cultures can be utilized for pharmaceutical products and regenerative medicine to supplement damaged organs or tissues, wherein the cell culture medium compositions comprise nanofibers prepared from chitin (i.e., nanofiber is added to a medium) and the medium composition comprising the nanofibers is for culturing adherent cells in a suspended state (Abstract; [0032]-[0034] and [0167]). Hayashi teaches the chitin nanofibers are prepared by pulverization, wherein pulverization is carried out by a high-pressure homogenizer, a grinder (stone mill), a beadmill or by the wet pulverization method disclosed in JP-A-2005-270891 (i.e., wherein nanofiber composed of chitin is obtained by pulverizing the chitin) ([0033] and [0115]-[0116]).
Although Hayashi does not further teach coating the nanofibers with vitronectin, Hayashi teaches extracellular matrices and cell adhesion molecules (e.g., vitronectin, fibronectin, laminin, collagen) can be added to the culture ([0142], [0146]). Hayashi teaches that culture tools, such as various culture substrates to which cells attach, including flasks, bags, multi-well plates, may be coated with an extracellular matrix or adhesion molecule, e.g., vitronectin ([0168]). Hayashi teaches that adherent cells are attached to the chitin nanofibers ([0033]).
Furthermore, Sun is directed to nanofiber design for use in stem cell cultures. Sun teaches that nanofibers (e.g., chitosan) have been successfully used in cell culture, including ECM-modified nanofibers (extracellular matrix-modified nanofibers) (Abstract; 2.2.3 Chitosan, page 162). Sun further teaches that nanofiber surface modifications include incorporation of ECM molecules, e.g., vitronectin, into the nanofibers by physical adsorption or covalent surface modification (3. SURFACE MODIFICATION OF NANOFIBERS, 3.1 Natural ECM modified nanofibers, page 163).
Thus, Sun has established it was well known in the art that vitronectin is a suitable extracellular matrix for modifying nanofibers used for cell culture.
Therefore, taking into hand the teaching of Sun, and given that Hayashi teaches that a variety of culture substrates may be coated with an extracellular matrix or adhesion molecule, e.g., vitronectin ([0168]) and further teaches that adherent cells are attached to the chitin nanofibers ([0033]), it would be obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add vitronectin to the nanofiber cell substrates, in the method of Hayashi.
The person of ordinary skill in the art would have been motivated to use vitronectin, as taught by Sun, for the predictable result of providing a cell adherent ECM coating to the nanofiber scaffolds thus promoting efficient cell proliferation.
The skilled artisan would have had a reasonable expectation of success in adding vitronectin extracellular matrix because both Hayashi and Sun have shown that vitronectin is a suitable extracellular matrix for coating of nanofibers used in adherent cell culture.
Regarding claims 6-7 and the limitations directed to the addition of a chitosan nanofiber to the culture medium (claim 6), and further content ratio of chitin nanofiber carrying vitronectin to chitosan nanofiber = 1: 0.5-20 (claim 7), it is noted that Hayashi teaches the use of the culture medium comprising the nanofiber matrices permits culturing the adherent cells at high density with efficient proliferation ([0167]). Hayashi, at Experimental Example 18 ([0312]) specifically exemplifies preparing serum-free cell culture mediums wherein chitosan nanofiber or chitin nanofiber is added at a final concentration of 0.001% (w/v), the medium is added to 96-well plates and 100,000 cells/mL are added and cultured for 12 days. The culture mediums comprising chitosan and chitin nanofibers are compared to culture medium comprising deacylated gellan gum. Hayashi, at [0313], teaches that cell proliferation was higher for the culture mediums comprising chitin and chitosan nanofiber matrices, as compared to the culture medium comprising deacylated gellan gum. The chitin nanofiber showed high proliferative capacity even at 0.001% concentration, and the chitosan nanofiber showed high proliferative capacity from 0.01% concentration. Thus, Hayashi has shown that cell culture mediums comprising chitin and chitosan nanofibers promote higher cell proliferation.
Thus, Hayashi does render obvious the addition of a chitosan nanofiber to the culture medium, that is, Hayashi teaches the limitation required by claim 6 and therefore it is held that the addition of a chitosan nanofiber to the culture medium is within the scope of the teachings of Hayashi, and thus renders the invention of claim 6 prima facie obvious. The rationale to support this conclusion of obviousness is that Hayashi provides the teachings and suggestion to the add chitosan nanofiber to the culture medium. Furthermore, there is no evidence on the record that shows that the claimed limitation has any greater or unexpected results than that exemplified by Hayashi.
Regarding claim 7 and the claimed content ratio of chitin nanofiber and chitosan nanofiber, it is noted that Hayashi does not further teach a culture medium comprising both chitin and chitosan nanofibers at the claimed content ratio. However, Hayashi, at [0313], teaches the chitin nanofiber showed high proliferative capacity even at 0.001% (w/v) concentration, and the chitosan nanofiber showed high proliferative capacity from 0.01% (w/v) concentration. Thus, Hayashi has demonstrated using 10-fold higher content of chitosan, as compared to chitin nanofiber, which correlates to a chitin: chitosan content ratio of 1:10 (claimed range overlaps the prior art range).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to prepare a cell culture medium wherein a content ratio of chitin nanofiber to chitosan nanofiber is 1:10.
The person of ordinary skill in the art would have been motivated to modify the method of the cited prior art to use a content ratio of 1:10 for chitin nanofiber to chitosan nanofiber, as taught by Hayashi, for the predictable result of successfully promoting higher cell proliferation resulting in culturing the adherent cells at high density with efficient proliferation, thus meeting the limitation of claim 7.
In the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Hayashi, in view of Sun, as applied to claims 1 and 6-7 above, and further in Li et al., (Biointerphases, 5(3), September 2010, FA132-FA142; see PTO-892) (“Li”).
The teaching of Hayashi and Sun is set forth above.
Regarding claim 4, it is noted Hayashi and Sun do not further comment on the concentration of the vitronectin extracellular matrix, specifically 0.01-50 mg/g of the nanofiber.
However, Li is directed to the assessing vitronectin concentrations and their impact on the propagation of human embryonic stem cells, as compared to Matrigel (Abstract). Li teaches that vitronectin is a known cell adhesion molecule (FA133, right col, first full paragraph). Li teaches preparing the cell culture substrates using vitronectin at 1.25, 2.5, 5 and 10 µg/ml for coating 60 mm culture dishes (1. Coating tissue culture polystyrene with varying vitronectin surface density, pages FA133-FA134). Fig. 1 of Li illustrates the effect of the varying vitronectin concentrations on cell proliferation over a 7-day time period. Li’s data clearly shows the increasing concentrations of vitronectin result in increasing cell densities.
Thus, although Li employs a polystyrene culture substrate and not a nanofiber substrate, Li has clearly shown that the concentration of vitronectin is a results effective variable. Thus, the claimed range comprises only the routine optimization of the ECM concentration. Said optimization would have been obvious and well-within the purview of the ordinarily skilled artisan at the time of filing as these ECM proteins promote cell attachment.
Note that the optimization of doses/concentrations would have been prima facie obvious to one of ordinary skill in the art at the time of filing:
“[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 MPEP 2144.05).
As set forth at MPEP 2144.05 II. A:
“Generally, 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.”
Response to Applicant’s Remarks
Claim Rejections - 35 USC § 103:
Applicant has traversed the rejection of record on the grounds that the cited references to Noh and Sun fail to disclose or suggest the chitin nanofiber is obtained by pulverizing the chitin, as discussed at Applicant’s remarks (pages 6-8).
Applicant’s remarks have been carefully considered, but are not found persuasive since the claims as currently written do not exclude electrospinning of chitin to prepare the nanofibers. Claim 1 as currently written employs the transitional phrase “comprising”. The transitional term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. See, e.g., Mars Inc. v. H.J. Heinz Co., 377 F.3d 1369, 1376, 71 USPQ2d 1837, 1843 (Fed. Cir. 2004) (“[L]ike the term ‘comprising,’ the terms ‘containing’ and ‘mixture’ are open-ended.”). “The word ‘comprising’ transitioning from the preamble to the body signals that the entire claim is presumptively open-ended.” Id. MPEP 2111.03
Although claim 1 has been amended to recite “wherein the nanofiber composed of chitin is obtained by pulverizing the chitin”, the claim does not exclude that the pulverized chitin (powder) is subsequently dispersed in a solution for electrospinning of nanofibers.
As to Applicant’s remarks regarding the cited reference to Hayashi, as discussed at Applicant’s remarks (page 8), Applicants rely on the arguments used against Noh and Sun. However, as explained above, Applicant’s arguments regarding Noh and Sun are not found persuasive. Therefore, the response set forth above to arguments also applies to Hayashi.
Conclusion
No claim is allowed. No claim is free of the prior art.
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E. YVONNE PYLA
Primary Examiner
Art Unit 1633
/EVELYN Y PYLA/ Primary Examiner, Art Unit 1633