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 .
This action is in reply to papers filed 10/02/2025.
Claims 1-2 and 4 are pending and examined herein. Claims 3 and 5 are cancelled.
Withdrawn and Moot Objections/Rejections
The objections to claim 2 is regarding informalities is withdrawn in light of clarifying amendments to the claim.
The rejection of claim 1 under 35 U.S.C. 103 over Topouzi, in view of Freshney, as evidenced by Miranda, is withdrawn in light of the amendment to claim 1 to incorporate the limitations regarding the size of chopped hair bulbs (previously in claim 3), passage number (previously in claim 5), and medium components in step (C) (previously in claim 2).
The rejection of claim 2 under 35 U.S.C. 112(b) is withdrawn in light of clarifying amendments to the claim.
The cancellation of claim 3 renders the rejection thereof under 35 U.S.C. 112(b) and 35 U.S.C. 103 over Topouzi, in view of Freshney, as evidenced by Blume-Peytavi, moot.
The cancellation of claim 5 renders the rejection thereof under 35 U.S.C. 103 over Topouzi, in view of Freshney, Li, and R&D Systems, moot.
Maintained Rejections
Claim 4 remains rejected under both 35 U.S.C. 112(b) and 35 U.S.C. 103 over Topouzi, in view of Freshney, Li, and R&D Systems. Applicant’s arguments are addressed following the maintained rejections.
Claim Interpretation
The term “hair bulb” is defined in the specification as “a thick club-like structure which forms the lower part of the hair root surrounded by the hair follicle and in which capillaries, dermal papilla cells, keratinocytes, etc. are located” (p 6, para 21). This definition is used for the purposes of examination.
Claim 1 recites the phrase “chopping the hair bulbs,” which is neither defined in the specification nor commonly used in the art. For purposes of examination, this phrase is be interpreted to mean “dissecting the hair bulbs.”
Claim 4 recites the phrase “next-step culture dish” (step (c5)), which is not a term recognized in the art. For purposes of examination, given the context of this phrase in Example 3 on page 11, para 45 of the specification, “next-step culture dish” is interpreted to mean “a new culture dish,” which is distinct from the culture dish used in a previous step of the protocol.
Claim 4 recites the phrase, expanding the cells “until the culture dish is full of cells” (step (c5)). For purposes of examination, this phrase is interpreted to mean “until the culture reaches confluence.”
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 4 remains 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 4: The amended claim recites the phrase “determining a culture dish to be passaged depending on the number of cells collected in the cell culture dish” (step (c1)). It is unclear what is meant by this limitation. First, there is a lack of antecedent basis for the phrase “cells collected in the cell culture dish,” so it is unclear what this phrase refers to. Claim 1 does not recite a step of establishing a primary culture, and although this step is implied in claim 1, “the number of cells collected in the cell culture dish” could refer to the number of cells collected and seeded in the culture dish, or to the number of cells that grow over the course of culture. Importantly, this limitation is critical to step (c1) of claim 4, as the determination of a culture dish to be passaged depends on “the number of cells collected in the cell culture dish.”
Claim Rejections - 35 USC § 103
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.
Claims 1-2 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Topouzi (Experimental Dermatology, 2017, 26(6): 491-496; cited in IDS dated 11/28/2024), in view of Freshney (Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 2010), Li (CN 103497930, 2014; Clarivate Analytics English translation), and R&D Systems (Amphotericin B Product Datasheet, 2019), as evidenced by Miranda (British Journal of Dermatology, 2010, 163(2): 287-295).
Regarding claim 1: Topouzi teaches a method for the isolation and culture of dermal papilla cells from a skin biopsy, including scalp tissue (p 492, col 1, section 2.1; step (A)). Topouzi teaches isolating end bulbs (reads on hair bulbs) from the tissue (p 492, col 2, section 2.1.1; step (A)), isolating dermal papilla cells from the hair bulbs (p 493, col 1, section 2.1.2; step (B)), and culturing and passaging the dermal papilla cells 3 times (p 493, col 2, section 2.1.3.L; p 494, section 2.2; step (C)). Topouzi teaches placing tissue comprising the hair bulbs in a petri dish with DMEM (p 492, col 2, section 2.1.1.B), and using Noyles spring scissors (p 492, col 2, section 2.1.1.C) to transect the hair follicle to isolate the end bulb (p 493, col 1, section 2.1.1.D) and inverting the end bulb step to expose the dermal papilla (p 493, section 2.1.2) (reads on chopping the hair bulb), then further transecting the dermal papilla (p 493, col 2, section 2.1.3.J) (step (b1)).
Topouzi does not teach collecting the dissected hair bulbs in a tube and centrifuging the hair bulbs (step (b2)).
Freshney teaches that centrifugation can increase the concentration of cells in cell suspensions (p 47, col 1, section 4.2.7). It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of isolating dermal papilla cells taught in Topouzi by centrifuging the chopped hair bulbs before plating on a culture dish. One of ordinary skill in the art would have been motivated to make this modification because Freshney teaches that centrifugation can increase the concentration of cells in cell suspensions. One of ordinary skill in the art would have had a reasonable expectation of making this modification because Freshney teaches that cell suspensions, such as the hair bulbs in DMEM taught in the method of Topouzi, can be centrifuged.
Topouzi is silent regarding the size of the dissected hair bulbs in step (b2). The method of dissecting hair bulbs, as taught in Topuzi, results in isolation of the dermal papilla (p 493, section 2.1.2; Fig 1 A-F).
Miranda teaches that the mean diameter of a terminal hair dermal papilla is 0.119 mm (119 µm), whereas the mean diameter of an intermediate hair dermal papilla is 0.063 mm (63 µm) (p 289, col 2, para 2; Fig 2). Therefore, the diameter of the dermal papillae (reads on chopped hair bulbs) isolated following the method of Topouzi would have a diameter between approximately 63 µm and 119 µm.
Topouzi does not teach the use of a medium comprising MEM alpha, basic FGF, 10 % fetal bovine serum, penicillin-streptomycin and amphotericin B in step (C). Instead of MEME alpha, Topouzi teaches using DMEM as the culture medium (Section 2.1.1.A).
Li teaches a method for culturing dermal papilla cells from hair follicles, wherein the culture medium comprises alpha-MEM, 10% fetal calf serum, penicillin-streptomycin, and basic fibroblast growth factor (p 1, Description, section C1).
Given the teachings of Topouzi and Li, there was a reasonable expectation that DMEM and alpha-MEM would work equivalently as a culture medium for papilla cells. Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention have substituted the DMEM-based culture medium, as taught in Topouzi, with alpha-MEM based culture medium comprising basic FGF, 10 % fetal bovine serum, penicillin-streptomycin, as taught in Li, with predictable results. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
The culture medium taught in Li does not comprise amphotericin B. R&D Systems teaches that amphotericin B is a strong antimycotic agent that can effectively inhibit or eradicate fungus and/or yeast contamination in cell cultures, and is especially useful in establishing contamination-free primary cell cultures (p 1, Product Description, para 2). It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have added amphotericin B to the culture medium taught in Li, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to make this modification because R&D Systems teaches that amphotericin B can inhibit or eradicate fungus and/or yeast contamination in cell cultures. One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because R&D Systems teaches that amphotericin B can be added to cell culture media.
Regarding claim 2: Following the discussion of claim 1, Topouzi teaches washing the skin biopsy in a petri dish containing Dulbecco’s minimal essential medium (DMEM), then transferring the skin biopsy to a new petri dish containing DMEM (p 492, col 2, section 2.1.1.A; step (a1)). Topuozi teaches placing eight small drops and one larger drop of DMEM onto the inverted lid of a petri dish, to be used to hold hair bulbs in a later step (p 492, col 2, section 2.1.1.B; Fig 1B; step (a2)).
Topuozi teaches using Noyles spring scissors and forceps to trim adherent adipose or connective tissue surrounding the lower section of the follicle to expose and isolate the end bulb (reads on hair bulb) located at the base of the follicle (p 492, col 2, section 2.1.1.C), then transferring the end bulb to a small drop of DMEM formed on the petri dish lid (p 493, col 1, section 2.1.1.D; Fig 1B; step (a2)).
Topouzi teaches using a fine needle to invert the end bulb and expose the dermal papilla, then transferring the inverted end bulb with the exposed and cleaned dermal papilla to the large DMEM drop on the inverted petri dish lid (p 493, col 2, section 2.1). Topouzi teaches that the dermal papilla can be identified using a stereomicroscope at a high magnification (p 492, col 1, section 2.1.1.C; step (a3)).
Topouzi does not teach the use of a MEM alpha medium.
Li teaches a method for culturing dermal papilla cells from hair follicles, wherein the culture medium comprises alpha-MEM, 10% fetal calf serum, penicillin-streptomycin, and basic fibroblast growth factor (p 1, Description, section C1).
Given the teachings of Topouzi and Li, there was a reasonable expectation that DMEM and alpha-MEM would work equivalently as a culture medium for papilla cells. Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention have substituted the DMEM-based culture medium, as taught in Topouzi, with alpha-MEM based culture medium, as taught in Li, with predictable results. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
Regarding claim 4: Topouzi teaches that dermal papilla cells are maintained in an incubator set at 37℃ and 5% CO2 (p 493, 2.1.2.L). Topouzi teaches that the dermal papilla cells can be passaged using standard cell culture techniques, using a culture medium comprising DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (p 493-494, section 2.1.3.L). Topouzi teaches that passaging comprises removing the media and washing cells with PBS (p 494, section 2.2.1.A, section 2.2.2.C; step (c1)); treating the cells with 0.5% trypsin-EDTA, followed by incubation for approximately 5 minutes in a 37℃ incubator (p 494, section 2.2.2.C; step (c2)); adding DMEM containing 10% FBS to inhibit the trypsin, then transferring the cells in suspension to a 50-mL centrifuge tube and centrifuging for 4 minutes to pellet the cells (p 494, section 2.2.2.C; step (c3)); removing the supernatant and resuspending the pellet in 10mL DMEM/10% FBS, then counting the cells (p 494, section 2.2.2.D; step (c4)); then resuspending the cells at a concentration of 300 cells per microliter (p 494, section 2.2.2.D).
Topouzi does not teach 1) 0.25% trypsin/EDTA in step (c2); 2) MEM alpha medium containing 1% FBS in step (c3); 3) “expansion medium 2” in step (c4); or 4) seeding the cells at a density of 1,500 cells/cm2, then replacing the medium every 3 days while the cells grow to confluency (c5).
Regarding differences 1-2: Freshney teaches a method for subculturing (reads on passaging) monolayer cells (p 196-204). Freshney teaches that the procedure for dissociating an adherent cell line varies depending on cell type (p 196, section 12.4), and teaches variations of the protocol, including one wherein 0.25% EDTA in trypsin is used (p 198, Table 12.5; p 199, section 12.4.2).
Given the teachings of Freshney, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the conditions for dissociating cells, as taught by Freshney, to arrive at the claimed invention. See MPEP 2144.05(II)(A). As noted in In re Aller, 105 USPQ 233 at 235, where 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.
Regarding difference 3: Li teaches a method for culturing and passaging dermal papilla cells from hair follicles, wherein the culture medium comprises alpha-MEM, 10% fetal calf serum, penicillin-streptomycin, and basic fibroblast growth factor (p 1, Description, section C1; p 2, claims 8-9).
Given the teachings of Topouzi and Li, there was a reasonable expectation that the DMEM-based culture medium of Topouzi and the alpha-MEM-based culture medium of Li would work equivalently as a culture medium for papilla cells. Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention have substituted the DMEM-based culture medium comprising 10% FBS and 1% penicillin-streptomycin, as taught in Topouzi, with alpha-MEM based culture medium comprising 10% fetal calf serum, penicillin-streptomycin, and basic fibroblast growth factor, as taught in Li, with predictable results. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
The culture medium taught in Li does not comprise amphotericin B. R&D Systems teaches that amphotericin B is a strong antimyotic agent that can effectively inhibit or eradicate fungus and/or yeast contamination in cell cultures, and is especially useful in establishing contamination-free primary cell cultures (p 1, Product Description, para 2). It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have added amphotericin B to the culture medium taught in Li. One of ordinary skill in the art would have been motivated to make this modification because R&D Systems teaches that amphotericin B can inhibit or eradicate fungus and/or yeast contamination in cell cultures. One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because R&D Systems teaches that amphotericin B can be added to cell culture media.
Regarding difference 4: Freshney teaches that after counting, cell suspensions should be diluted to the appropriate seeding concentration (p 200, steps 12-13), then placed in a 5% CO2 incubator (p 201, steps 14-16). Freshney further teaches that a cell culture, whether it is a primary culture or a subculture, needs a periodic medium change, followed by subculture if the cells are proliferating (reads on until the culture dish is full of cells) (p 193, section 12.3). Freshney teaches that the intervals between medium changes vary among cell lines, depending on the rate of growth on metabolism (p 193, section 12.3).
Given the teachings of Freshney, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the seeding concentration and intervals between media changes, as taught by Freshney, to arrive at the claimed invention. See MPEP 2144.05(II)(A). As noted in In re Aller, 105 USPQ 233 at 235, where 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.
Response to Arguments
RE: Claim Rejections - 35 USC § 112
Regarding claim 4, Applicant argues: The Examiner indicated that the phrase "determining a culture dish to be passaged depending on cell count" is indefinite, noting that in commonly used protocols cells are counted after dissociation with trypsin, not before. In this regard, Applicant respectfully submits that the specification provides clear support for this step and clarifies its meaning. Specifically, the specification describes "[f]irst, a culture dish to be passaged was determined depending on the number of cells collected in a 35-mm cell culture dish, and then the medium was discarded from the 35-mm cell culture dish, followed by washing once with PBS." (see paragraph [0035] of the specification). To clarify this, Applicant has amended claim 4, step (cl) to recite "(cl) determining a culture dish to be passaged depending on the number of cells collected in the cell culture dish, and then discarding medium from the cell culture dish, followed by washing with PBS." This amendment clarifies the meaning of the step (c1).
In response: Applicant’s arguments have been fully considered, but are not persuasive. As set forth in the claim rejection above, the amended phrase “the number of cells collected in the cell culture dish” could refer to the number of cells collected and seeded in the culture dish, or to the number of cells that grow over the course of culture. Examiner notes the importance of establishing clear metes and bounds regarding this phrase because this limitation is critical to step (c1) of claim 4, as the determination of a culture dish to be passaged depends on “the number of cells collected in the cell culture dish.”
RE: Claim Rejections - 35 USC § 103
Applicant argues: Blume-Peytavi teaches terminal hair diameter, not chopped hair bulb diameter.
In response: Applicant’s arguments have been fully considered and are persuasive. A new evidentiary reference, Miranda, has been applied to the rejection, which addresses hair bulb diameter.
Applicant argues: Blume-Peytavi teaches diameters of intact, unchopped hair shafts. Blume-Peytavi provides no teaching regarding how to chop hair bulbs, what size fragments would result
from chopping, or what size fragments would be optimal for dermal papilla cell isolation. The
act of chopping tissue creates fragments whose dimensions depend on the chopping technique,
number of chopping steps, and characteristics of the tissue being chopped-none of which are
addressed by Blume-Peytavi's teaching about intact hair shaft diameter.
In response: Applicant’s arguments have been fully considered, but are not found persuasive. Blume-Peytavi was used as an evidentiary reference to show the size of hair bulbs (which is now shown in the new evidentiary reference, Miranda), and was not relied upon for the limitation of chopping hair bulbs. This limitation is taught in Topouzi, as set forth in the rejection above.
Applicant argues: While Topouzi broadly discloses that cells at "passage 3-5" can be used, there is no teaching or suggestion in Topouzi that exactly three passages would be optimal, particularly when combined with the hair bulbs chopped having a diameter of 15-120 μm, as claimed. The claimed invention requires that the specific combination of 15-120 μm chopping diameter and three passages using the claimed medium produces optimal results-a finding not taught or suggested by the broad disclosure of "passage 3-5" in Topouzi.
In response: Applicant’s arguments have been fully considered, but are not found persuasive. First, the claimed limitation regarding three passages is taught in Topouzi, which teaches passaging cells three to five times. The new evidentiary reference Miranda shows that the hair bulbs dissected following the method of Topouzi have a diameter between approximately 63 µm and 119 µm. Furthermore, Applicant argues that “The claimed invention requires that the specific combination of 15-120 μm chopping diameter and three passages using the claimed medium produces optimal results.” However, the claims do not recite an unexpected property resulting from the combination of 15-120 μm chopping diameter and three passages. The preamble of claim 1 recites “A method for isolation and expansion of dermal papilla cells.” This effect is achieved using methods disclosed in the prior art, which teaches the combination of 15-120 μm chopping diameter, three passages, and the claimed medium. That is, any properties arising from the criticality of the combination of 15-120 μm chopping diameter, three passages, and the claimed medium, is not recited in the claims.
Applicant argues: The specification demonstrates through extensive experimental data that the specific combination of (1) chopping size range of 15 μm to 120 μm, (2) the specific medium composition, and (3) three passages produces remarkable and unexpected effects in dermal papilla cell expansion.
In response: Applicant’s arguments have been fully considered, but are not found persuasive. The claims do not recite “remarkable and unexpected effects in dermal papilla cell expansion” as a result of the claimed method, but merely a preamble for “A method for isolation and expansion of dermal papilla cells” in claim 1. Although Applicant presents data on page 9-13 of the Remarks and state, for example, “Case 2 shows well-maintained cell morphology and significantly higher proliferation rates of dermal papilla cells throughout all passages (Pl-P5) compared to cases 1, 3, and 4” (p 9, para 2 of Remarks), properties such as cell morphology and higher proliferation rates are not recited in the claims.
Applicant argues: As demonstrated above, the claimed invention achieves remarkable effects in dermal papilla cell expansion by combining the specific limitations of: (1) chopping size range of 15 μm to 120 μm, (2) medium composition comprising MEM alpha, basic FGF, 10% fetal bovine serum, penicillin-streptomycin and amphotericin B, and (3) three passages. These superior results were obtained through extensive repeated experimentation and could not have been easily achieved by one of ordinary skill in the art.
In Response: Applicant’s arguments have been fully considered, but are not found persuasive. As outlined in the rejection above, the prior art teaches each of the limitations set forth by Applicant, and therefore, the argument that the results could not have been easily achieved by one of ordinary skill in the art not persuasive.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Risa Takenaka whose telephone number is (571)272-0149. The examiner can normally be reached M-F, 12-7 EST.
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/RISA TAKENAKA/Examiner, Art Unit 1632
/TITILAYO MOLOYE/Primary Examiner, Art Unit 1632