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
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 08/05/2026 has been entered.
Claim 1 has been amended.
Claims 1-10 are pending and under examination.
Claim Objections
2. Claim 1 is objected to because of the recitations “a supernatant” and “a pellet” in line 6. Correction to “the supernatant” and “the pellet” is required.
Claim Rejections - 35 USC § 103
3. 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.
4. Claims 1-9 are rejected under 35 U.S.C. 103 as being unpatentable over Marret et al. (Reprod. Nutr. Dev., 2000, 40: 305-319), in view of both Guan et al. (Nature Protocols, 2009, 4: 143-157) and Moraveji et al. (J. Cell. Biochem., 2019, 120: 613-621).
Marret et al. teach a method for obtaining spermatogonial stem cells (SSCs), the method comprising subjecting porcine testicular tissue to partial digestion with collagenase and DNase, in a water bath at 33º C to obtain a dispersion of seminiferous tubules, collecting and washing the seminiferous tubules, followed by culturing the seminiferous tubules in DMEM/F12 until the SSCs migrate out; the steps of digestion and culturing are conducted in the absence of trypsin (claim 1) (see p. 308, column 2, first full paragraph; p. 312, column 1, last two paragraphs; p. 313, Fig. 3a and 3b).
Marret et al. do not specifically teach collecting the migrated SSCs, followed by their enriching and purification via Percoll discontinued gradient centrifugation (claims 1, 8, and 9). However, Marret et al. teach that the isolated and purified SSCs are useful as an in vitro model for defining the mechanisms regulating their survival and proliferation (see p. 306). Marret et al. also teach that cell dispersions obtained via the full digestion of the testicular tissue could be enriched for SSCs by Percoll discontinuous gradient centrifugation, which comprises sequentially layering a 40% Percoll solution, a 20% Percoll solution, and the cell suspension in a centrifuge tube, collecting the SSCs at the interface between the 40 and 20% Percoll layers, and culturing the collected SSCs in DMEM/F12 (see p. 307). One of skill in the art would
readily understood that the migrated cells constitute a source of SSCs and would have found obvious to collect the migrated cells (claim 1), subject them to Percoll discontinuous gradient centrifugation, and collect the cells at the interface between the 40 and 20% Percoll layers to achieve the predictable result of obtaining SSCs suitable to be used as an in vitro model (claims 8 and 9).
Marret et al. do not specifically teach that collagenase and DNase are collagenase IV and DNase I, nor do they specifically teach digesting at 36-38º C, washing in DPBS and collecting the tubules by centrifugation (claims 1 and 6). Guan et al. teach that digestion with collagenase IV and DNase I at 37º C could be used to obtain dispersions of seminiferous tubules, which are washed in DPBS and collected by centrifugation (see p. 145, column 1, first two lines; p. 146, Procedure, steps 1-4). One of skill in the art would have found obvious to modify Marret et al. by digesting with collagenase IV and DNase I at 37º C, washing with DPBS, and collecting by centrifugation, to achieve the predictable result of obtaining SSCs suitable to be used as an in vitro model.
Marret et al. and Guan et al. do not teach frozen testicular tissue and thawing in a 36-38º C water bath (claims 1 and 2). Moraveji et al. teach that SSCs could be isolated from frozen human testicular tissue, which is thawed in a 37º C water bath (see Abstract; p. 614, paragraph bridging columns 1 and 2). Practicing the method of Marret et al. and Guan et al. by using frozen tissue thawed in a 37º C water bath would have been obvious to one of skill in the art to achieve the predictable result of obtaining SSCs suitable to be used as an in vitro model.
With respect to claim 3, Marret et al. teach porcine SSCs, Guan et al. teach mouse SSCs, and Moraveji et al. teach human SSCs. Thus, one of skill in the art would have reasonably concluded that the method could be practiced with frozen testicular tissue of any origin. One found obvious to practice the method of Marret et al, Guan et al., and Moraveji et al. by starting with frozen bovine testicular tissue, when obtaining bovine SSCs was desired.
With respect to claim 5, Guan et al. teaches digesting with 1 mg/ml collagenase IV and 0.3 mg/ml DNase I in DMEM (see p. 145, column 2, Reagent Setup section). While DMEM is not DPBS, there is no evidence of record that specifically using DPBS leads to unexpected results compared to DMEM. Specifically using DPBS is not significant if it does not provide a novel feature. With respect to claim 4, there is nothing of record indicating that sequentially adding DMEM, collagenase IV, and DNase I to the tissue leads to unexpected results over adding them in a solution at the same time as taught by Guan et al. Sequentially adding DMEM, the collagenase IV solution, and the DNase I solution is not significant if it does not provide a novel feature. Furthermore, the recitation of adding the solutions in equal volumes is not given patentable weight; adding equal volumes is irrelevant because it does not inform one of skill in the art of the enzyme concentrations used for digestion, which is the important parameter for the claimed method. The essential parameter (i.e., the enzyme concentration) is taught by the prior art.
With respect to claim 7, Marret et al. teach cultivating in 5% CO2 in DMEM/F12 (not DFS), and at 33º C (not 36-38º C). However, as per MPEP § 716.02, [a]ny differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected. In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Marret et al. teach that DMEM/F12 is DMEM comprising Ham’s F12 nutrient mixture and FBS (see paragraph bridging p. 307 and 308). The specification defines DFS as being DMEM with FBS (see [0013]). There is no evidence of record indicating that the absence of F12 results in cells exhibiting unexpected properties over using DMEM comprising F12 as taught by the prior art. Similar considerations apply to the claimed range 36-38º C. Furthermore, the prior art teaches that SSCs can be successfully cultivated at 37º C (see Guan et al., p. 147, Culture of SSCs). One of skill in the art would have found obvious to use 37º C, to achieve the predictable result of obtaining SSCs suitable to be used as an in vitro model.
Thus, the claimed invention was prima facie obvious at the time of its effective filing date.
5. Claims 1-10 are rejected under 35 U.S.C. 103 as being unpatentable over Marret et al. taken with both Guan et al. and Moraveji et al., in further view of both Kim et al. (Cryobiology, 2015, 70: 175-183) and Haslett et al. (Am. J. Pathol., 1985, 19: 101-110).
The teachings of Marret et al., Guan et al., and Moraveji et al., are applied as above for claims 1-9. Marret et al., Guan et al., and Moraveji et al. do not specifically teach a 1:1:2 volume ratio for the Percoll discontinuous gradient centrifugation (claim 10). However, the recited volume ratio is irrelevant because it does not inform one of skill in the art of the number of cells subjected to Percoll gradient centrifugation, which is the important parameter for successful separation (see Haslett et al., p. 102, column 2).
Furthermore, Kim et al. teach enriching SSCs via Percoll centrifugation using 2 ml of 40% Percoll, 2 ml of 20% Percoll, and 2 ml of a suspension containing 5x106 SSCs (volume ratio of 1:1:1; see p. 176, column 2, last paragraph). Haslett et al. teach that cell concentration is a result-effective variable with respect to separation on Percoll gradients; the gradients can be optimized by varying the number of cells and also the volume to be layered on top of the gradient (see p. 102, column 2). One of skill in the art would have found obvious to optimize the gradient by starting with the 1:1:1 ratio taught by the prior art and further varying the volume and cell density of the overlaying cell suspension, with the reasonable expectation that doing so would identify the conditions for optimal separation.
Thus, the claimed invention was prima facie obvious at the time of its effective filing date.
Response to Arguments
6. The arguments related Moraveji are not found persuasive because they do not address the basis for rejection. The teachings in Moraveji indicated by the applicant pertain to obtaining SSCs by the enzymatic digestion of previously frozen seminiferous tubules (see paragraph bridging p. 614 and 615; p. 618, Fig. 3A), which is not the basis for rejection. The rejection is based on culturing previously frozen seminiferous tubules in the absence of enzymatic digestion until the SSCs migrate out of the tissue, and further collect the migrated SSCs. The applicant did not provide any evidence indicating that SSCs would not be able to migrate out of previously frozen seminiferous tubules. There is no teaching in Moraveji to this effect.
For these reasons, the following arguments are not found persuasive: (1) the invention takes an approach which is opposite to Moraveji; and (2) the prior art does not teach the exclusion of trypsin in the context of frozen tissue.
The argument that neither Marret nor Moraveji teach centrifugation and collecting the pellet comprising seminiferous tubules is not found persuasive. Neither reference has to teach centrifugation. It is also noted that, although not specifically disclosing centrifugation, Marret teaches collecting and washing the seminiferous tubules. Using centrifugation is rendered obvious by Guan.
The applicant argues that retaining tubule architecture and relying on migration achieves the unobvious result of successful SSC isolation without adding trypsin.
This is not found persuasive. Marret’s method of culturing the seminiferous tubules retains tubule architecture and results in SSC migrating out, i.e., culturing results in SSC isolation without adding trypsin, as claimed. Practicing Marret’s method with frozen seminiferous tubules would have been obvious and there is no evidence of record showing that using frozen tissue would change the outcome.
Conclusion
7. All claims are identical to or patentably indistinct from, or have unity of invention with claims in the application prior to the entry of the submission under 37 CFR 1.114 (that is, restriction (including a lack of unity of invention) would not be proper) and all claims could have been finally rejected on the grounds and art of record in the next Office action if they had been entered in the application prior to entry under 37 CFR 1.114. Accordingly, THIS ACTION IS MADE FINAL even though it is a first action after the filing of a request for continued examination and the submission under 37 CFR 1.114. See MPEP § 706.07(b). 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.
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/ILEANA POPA/Primary Examiner, Art Unit 1633
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