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 .
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 23-29, 33, 34-36 are rejected under 35 U.S.C. 103 as being unpatentable over Shen et al. (US 20170152486 A1) in view of Kümper et al. (Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis, eLife, 2016), Saito et al. (The role of HGF/MET and FGF/FGFR in fibroblast-derived growth stimulation and lapatinib-resistance of esophageal squamous cell carcinoma, 2015) Oottamasathien et al. (Bladder Tissue Formation From Cultured Bladder Urothelium, 2006), Claassen et al., (ROCK Inhibition Enhances the Recovery and Growth of Cryopreserved Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells, 2009) and Li et al. (Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors, 2006) ) as evidenced by Corning Life Sciences Hepatocyte Culture Media Kit (item 355056)
Regarding Claim 23: Shen et al. teaches a method of culturing bladder cell lines or organoids from bladder tissue. (0007) Shen discloses collecting tumors from patients (0541) and normal bladder tissue (0352) and dissociating the tissue into a single-cell suspension. (0349) This reads on the claimed method of an organoid culture of human bladder tissue, either normal or cancerous bladder tissue being used. Example 1 teaches a method for isolation of human bladder tissue in a medium consisting of a basal media (Hepatocyte culture kit, Corning), FBS, Glutamax, an antimicrobial, and ROCK inhibitor. (Example 1, 0370-0376) Notably, Shen does not make use of a supernatant of primary fibroblasts in the media composition. Shen discloses a method of isolating bladder tumor samples and enzymatically digesting them in order to obtain a cell suspension of bladder epithelial cells, which encompass urothelial cells as urothelial cells are a type of bladder epithelial cell. (0349, 0370-0376) This reads on the claimed method of a suspension of urothelial cells and epithelial adult stem cells originating from the human bladder in a suspension medium comprising a basal medium, ROCK inhibitor, and being devoid of a supernatant of primary fibroblasts.
Shen teaches a method of producing bladder organoids which have been embedded in Matrigel. (0540-0546) This involves mincing the bladder tissue with scissors followed by enzymatic dissociation, mixing the resulting single cell solution in Matrigel, and culturing the resulting organoids in complete hepatocyte medium. (0540-0546) Shen discloses the number of passages the patient-derived organoids underwent in Table 4 (pg 41) as anywhere from 2-20 passages. This reads on the claimed method of culturing the urothelial cells in an extracellular matrix and expansion medium suitable for differentiation of the cells as well as obtaining a 3D tissue and cultured between 2-5 or 10-15 passages. Shen also discloses use of fetal bovine serum (FBS) in the media preparation (Example 1, 0370-0376) which reads on the claimed method of use of a receptor tyrosine kinase ligand in the cell culture expansion medium.
Shen fails to disclose the absence of ROCK inhibitor in media changes following initial plating of the cell suspension. Claassen teaches that use of ROCK inhibitor during the thawing and recovery period of cryopreserved human stem cells acted to “kick-start” slow growing cells. (Pg 1, Abstract) In addition to this, Claassen teaches that use of ROCK inhibitor in long term culture may provide benefits in the form of continued increased cell colony size when exposed to ROCK inhibitor for up to 96 hours. (Pg 3, Results) This would give a person skilled in the art motivation to use ROCK inhibitor, but fails to teach motive for the cessation of use after plating.
Kümper teaches that use of a ROCK inhibitor leads to a decrease in cell proliferation through G1 arrest and a block in cytokinesis. (pg 15, first two paragraphs) As Applicant correctly states in the Remarks dated 02/06/2026, Kümper states that even after 48h of culture, cells exposed to ROCK inhibitor had reduced proliferation. (Pg 3, Results) As such, the teachings of Claassen and Kümper , when taken together, strongly suggest a benefit to using ROCK inhibitor during plating as it aids in colony size and proliferation (“kick-starting”), but a detriment to prolonged use as Kümper demonstrates loss of proliferation at 48h. Taken together, a person skilled in the art would interpret these teachings to suggest use of ROCK inhibitor during plating is beneficial, but prolonged use is detrimental to culture.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Shen (using ROCK during initial cell suspension plating) with the teaching of Kümper and Claassen to only use ROCK during initial cell suspension plating. One would have had motivation to do so based on the teaching of Kümper regarding the detrimental effects of long-term use of ROCK inhibitor, and Claassen, who demonstrate increased cell colony size and proliferation in the recovery of human stem cells post cryopreservation.
Shen fails to disclose use of primary fibroblast supernatant in their taught expansion media. Saito teaches the harvesting of primary esophageal fibroblasts and use of the supernatant from said culture to influence the proliferation of various esophageal cancer cell lines. (Pg 1, Abstract) Saito further teaches that fibroblasts secrete matrix metalloproteinases (pg 10, Discussion) and secrete extracellular matrix components (pg 2, Background). Saito utilized both primary and immortalized esophageal fibroblasts (HEF75 denotes primary, HEF75-hTERT denotes immortalized) in culture to harvest their respective supernatant, and found that both primary and immortalized fibroblasts resulted in a higher rate of proliferation of nearly every cell line tested (pg 2-3, Methods), as seen in the chart below (edited by Examiner solely to distinguish the abbreviations used for cell lines):
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Shen, Saito, Li, and XXX fail to disclose use of bovine pituitary extract (hereafter BPE). Oottamasathien et al. teaches a culture method to obtain bladder tissue sheets from primary cultured bladder urothelium with use of collagen ECM. (Pg 1, Abstract) During the culture method, bovine pituitary extract was included in the culture method (pg 5, Experimental Procedures) due to its known property of promoting cell growth. (Pg 4, Discussion) Due to this teaching, a person of ordinary skill in the art would be motivated before the effective filing date of the claimed invention to incorporate BPE in the culture method of Shen. One would have had motivation and expectation of success due to the teachings of Oottamasathien, who teach that BPE promotes cell growth.
Regarding Claim 24: Shen discloses a method of preparing a suspension of bladder epithelial tissue from a patient biopsy which involves both mechanical tissue processing (0540-0542) and enzymatic tissue processing (0391-0395) to achieve a cell suspension. (0396-0400) This reads on the claimed method of taking a sample of bladder tumor tissue from a patient, dissecting it to recover urothelium (a type of epithelial cell), and recovering a suspension of dissociated cells. Shen further teaches centrifugating the dissociated cells to form a pellet and then recovering said pellet. (0399-0400) This reads on the claimed method of centrifugating the suspension of urothelial cells and recovering the cell pellet.
Regarding Claim 25: Shen discloses a method of mincing the resected tissue with scissors prior to enzymatic digestion (0541-0543) and also use of bladder tumors (0541) for the culture protocol. This reads on the claimed method of dissecting the bladder tumor into pieces and reads on the claimed range of said tissue pieces being about 3mm-5mm in size. Shen also teaches use of centrifugating the dissociated cells to form a pellet and then recovering said pellet. (0399-0400) This reads on the claimed method of centrifugating the suspension of urothelial cells and recovering the cell pellet.
Regarding Claim 26: Shen teaches a method of processing bladder tissue from a human subject (0006) to recover bladder epithelial cells and recovering a suspension of bladder epithelial cells as a supernatant after enzymatic dissociation. (0391-0400) This reads on providing bladder tissue of a human subject, dissecting the tissue to recover urothelium cells, and recovering a suspension of urothelial cells as a supernatant after dissociation. Lastly, Shen teaches centrifugating the dissociated cells to form a pellet and then recovering said pellet. (0399-0400) This reads on the claimed method of centrifugating the suspension of urothelial cells and recovering the cell pellet.
Regarding Claim 27: Shen and Saito both fail to disclose a ratio used in their respective media recipes or culture protocols that read on the claimed range of 80%-40% basal medium to 20%-60% primary fibroblast supernatant. However, Saito teaches that use of primary fibroblast supernatant led to increased proliferation rates in multiple cell lines. Due to the teachings of Saito regarding the benefits of using primary fibroblast supernatant, one would have been motivated to include it in a basal medium to be used for bladder urothelial culture. Given absence of criticality otherwise, the specific ratio of basal medium to primary fibroblast supernatant would have been routinely optimized as part of experimental design as referenced in MPEP 2144.05.
Regarding Claim 28: Shen teaches a Matrigel embedding protocol where bladder tumors taken from human patients were manually dissociated with scissors, followed by enzymatic dissociation, and then mixed with a Matrigel: hepatocyte media mixture and allowed to set for 30 minutes prior to plating. (0540-0546) Shen discloses that the reaction Matrigel undergoes is polymerization at the correct temperature. (0424) Shen also discloses a method of collagen embedding where the collagen solution is not mixed with complete hepatocyte media prior to plating. (0552-0556) This reads on the claimed method of the extracellular matrix being provided to the cell suspension prior to the expansion medium and the extracellular matrix is polymerized to allow the cells to be embedded therein.
Regarding Claim 29: In Example 2, Materials and Methods for Establishing Bladder Organoid, Shen teaches that it takes about 3-5 weeks in culture for the organoids to reach a size large enough to warrant being re-plated. (0501) Table 4 also discloses the number of passages of several patient-derived organoids, ranging from 2-20 passages. (pg 41, Table 4) see below:
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Shen also discloses an embodiment where bladder organoids can be grown anywhere from 3 weeks to at least 8 months. (0298) This reads on the claimed method of keeping organoids in culture that show viability for at least 10 days while being maintained in the extracellular matrix and culture medium.
Regarding claim 33: Shen states that the invention discloses a methodology for the culture of bladder cell lines and organoids which can be from both non-cancerous and cancerous tissue. (57)
Regarding claim 34: Shen teaches an embodiment of the invention in which the cells are maintained in culture for “at least 10 passages” and “at least 15 passages”. (0191)
Regarding claim 35: Li teaches use of either KSFM or William’s medium in a comparison study using liver hepatocytes to see which medium caused the hepatocytes to maintain their differentiated properties in culture. (Pg 541, Abstract) It was found that after a culture period of 3-4 weeks, the hepatocytes maintained in KSFM supplemented with dexamethasone, epidermal growth factor, and pituitary gland extract maintained their transcriptional phenotype in culture for the duration of the culture time. Comparatively, cells cultured in William’s medium lost their expression profile after about 3 days. (Pg 541, Abstract) This shows that KSFM medium is better suited for the culture of epithelial cells (including blader cells, which are a type of epithelial cell) as it helps the cells maintain their phenotype in culture for longer periods of time. For that reason, one skilled in the art would have had motivation and a reasonable expectation of success at combining the teachings of Shen, Kümper, and Saito to create a bladder organoid culture system which uses KSFM in order to ensure retention of the transcriptional profiles of the bladder organoids. It would have been obvious to one skilled in the art to combine the teachings due to the motivation provided by Li, who demonstrates that use of KSFM results in retained transcriptional profiles in long-term culture of epithelial cells.
Regarding claim 36: Shen teaches an embodiment of the invention in which the media comprises 5% Matrigel. (0193) Shen further teaches a method of the invention involving contacting the dissociated bladder tissue with a Matrigel solution and then placing the construct in a cell culture support system in which the Matrigel solution comprises hepatocyte medium. (0010) This reads on the method of claim 36 of keeping the organoids both in Matrigel and in the culture medium.
Regarding claims 37 and 38: Shen teaches embodiments of the invention which use EGF in the culture medium (0008)
Response to Arguments
Applicant's arguments filed 02/06/2026 have been fully considered but they are not persuasive.
Applicant argues that Examiner has used “hindsight reasoning” to “overhaul Shen” by picking specific features of Kümper and Khan. Examiner clarifies that, when combining references, it is appropriate to include the teachings of outside methods to incorporate into a 35 U.S.C. 103 rejection based on the “teaching, suggestion, or motivation” rationale. To further clarify why a person skilled in the art would have been motivated to only incorporate ROCK inhibitor to the initial plating phase of culture, the teachings of Claassen promoting the benefits of ROCK inhibitor as a “kick starter” for stem cell culture (as discussed above) have been incorporated into the rejection. As Claassen teaches that ROCK inhibitor demonstrated increased proliferation and colony size during recovery after cryopreservation, a person skilled in the art would be motivated to incorporate it in culture. This teaching, taken along with Kümper’s teachings that, as pointed out by the Applicant, use of ROCK inhibitor in culture media even after only 48 hours led to cell senescence, would clearly motivate a person skilled in the art to remove ROCK inhibitor from the culture media during subsequent media changes after initial plating. Based on the combined teachings of Kümper and Claassen taken in the context of the protocol taught by Shen, strongly suggests that the only logical timeframe to incorporate ROCK inhibitor is during plating as that will allow for the “kick starter” effect but avoid risk of senescence.
In addition to this, a person skilled in the art is also a person of ordinary creativity, as referenced in MPEP 2141.03.I: “A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.”
Lastly, in response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Applicant further argues that the unexpected benefit of ROCK inhibitor as recited in the Specification was interpreted incorrectly under MPEP 716.02(a). Following the discussion above, the 35 U.S.C. 103 rejection has been updated to incorporate the teachings of Claassen, who state that use of ROCK inhibitor leads to an increase of proliferation and larger colony sizes upon thawing. Examiner would like to restate that although the claims are examined in light of the specification, the features recited in the specification pertaining to “undifferentiated basal cells and differentiated cells with hierarchical organization with preserved response capability to drugs” (Arguments, Pg 11) is not recited in the claims themselves. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant further argues that Khan et al. is non-analogous art and fails to teach the actual use of PFS in culture, instead teaching well-known properties of fibroblasts and that, as ECM is now amended into the claims as a requirement, Khan fails to read on the claimed method. This is persuasive. However, as necessitated by amendment and for further clarification, the teachings of Saito have been incorporated into the 35 U.S.C. 103 rejection. As discussed above, Saito teaches both that fibroblasts secrete extracellular matrix components and that use of said supernatant (both of primary and immortalized fibroblast cell lines) directly benefits cells in culture. This satisfies both the use of PFS and the newly required use of ECM components. Applicant is again reminded that though the specification demonstrates that “PFS in the expansion medium as currently claimed is critical to robust urothelial organoid formation and growth”, the claims are again examined in light of the specification. Furthermore, incorporation of the teachings of Saito negate this argument as Saito fully demonstrates the benefits of use of PFS in culture.
Lastly, as necessitated by amendment, the use and benefit of BPE has been accounted for via incorporation of the teachings of Oottamasathien, who state that use of BPE promotes the growth of urothelial cells, making it directly relevant to the claimed method of the culture of bladder tissue.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to HANNA M THUESON whose telephone number is (571) 272-3680. The examiner can normally be reached M-F 7:30-5 EST. 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.
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/HANNA MARIE THUESON/Examiner, Art Unit 1638
/Tracy Vivlemore/ Supervisory Primary Examiner, Art Unit 1638
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