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 .
Applicant’s amendment filed on 12/31/2025 has been entered.
Claims 1-11, 13 and 16-18 are pending in the present application.
Claims 1-10 were withdrawn previously from further considerations because they are directed to a non-elected invention.
Accordingly, claims 11, 13 and 16-18 are examined on the merits herein.
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 11 and 17-18 are still rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al (World J. Gastroenterol. 11:2906-2911, 2005) in view of Cao et al (Cell Prolif. 44:19-32, 2011; IDS), Zanini et al (PLoS ONE 6(12):e28175.doi:10.1371/journal.pone.0028175, 13 pages; 2011); Liu et al (In Vitro Cell. Dev. Biol. 50:464-474, 2014) and Rouy et al (US 2010/0278783) for the same reasons already set forth in the Non-Final Office action dated 07/03/2025 (pages 3-7). The same rejection is restated below.
The instant claims are directed to a method for preparing stem cells, comprising: a) collecting a pancreatic islet from a neonatal pig; b) culturing the pancreatic islet in suspension culture to obtain cells, cell fraction or cell cluster, wherein said cells, cell fraction, or cell cluster contain stem cells; c) freezing the collected cells, cell fraction, or cell cluster, wherein the collected cells, cell fraction, or cell cluster are not cultured in an adherent culture prior to freezing; d) thawing the frozen cells, cell fraction, or cell cluster to obtain thawed cells, cell fraction, or cell cluster; e) inoculating and culturing said thawed cells, cell fraction, or cell cluster; f) isolating adherent cells, cell fraction or cell cluster from a culture of thawed cells, cell fraction, or cell cluster, wherein said adherent cells, cell fraction, or cell cluster comprise primary cultured stem cells; and g) sub-culturing (preferably sub-culturing comprising 1 to 20 subcultures, dependent claim 17) said isolated adherent cells, cell fraction or cell cluster comprising the primary cultured stem cells after 3 to 12 days from the inoculation; and the same method further comprising freezing the isolated primary sub-cultured stem cells (dependent claim 18). It is noted that the term “neonatal pig” is defined by the present application to refer to a fetal pig or a pig less than one month after birth (paragraph [0013]).
Zhang et al already disclosed a method for isolating nestin-positive progenitor cells from human fetal pancreas that have phenotypic markers identical to mesenchymal stem cells, the method comprises: (a) enzymatically digesting human fetal pancreata; (b) incubating the washed digested tissue containing islet-like cell clusters (ICCs) in RPMI 1640 media supplemented with 10% fetal bovine serum, 10mmol/L HEPES, 1mmol/L sodium pyruvate and 71.5 umol/L β-mercaptoethanol for 96 hrs; (c) pipetting out the floating ICCs and culturing them in new dishes with fresh media supplemented with 20 ng/mL bFGF and 20ng/mL EGF, wherein the ICCs attached to the bottom and a monolayer of epithelium-like cells started the outgrowth from the ICCs within 24 hrs; and (d) after confluence, the cells were repeated passaged and the cells were able to passaged more than 16 times in vitro (see at least Abstract; particularly section titled “Isolation, culture and induction of pancreatic progenitor cells” on page 2907; section titled “Nestin-positive progenitor cells isolated from human fetal pancreas” on page 2907-2908; and Figure 1). Zhang et al also disclosed that FACS analysis revealed the nestin-positive cells are positive for CD44, CD90 and CD147 while negative for CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR, that suggests that the phenotype of these cells is identical to that of mesenchymal stem cells derived from bone marrow (section titled “Surface markers of the nestin-positive pancreatic progenitor cells” on page 2908).
Zhang et al did not teach specifically a method for preparing mesenchymal stem cells derived from pancreatic islets of a neonatal/fetal pig comprising at least the steps of freezing the collected cells, cell fraction, or cell cluster that have not been cultured in an adherent culture (step c); then thawing the frozen cells, cell fraction, or cell cluster to obtain thawed cells, cell fraction, or cell cluster (step d) prior to inoculating and culturing the thawed cells, cell fraction, or cell cluster; followed by isolating adherent cells, cell fractions, or cell cluster comprising primary cultured stem cells; and sub-culturing the isolated adherent cells, cell fraction or cell cluster after 3 to 12 days from the inoculation.
Before the effective filing date of the present application (09/08/2017), Cao et al already demonstrated that pancreatic mesenchymal stem cells (MSCs) are present in fetal porcine pancreas, and they can be immortalized by transfection with hTERT to establish immortalized pancreatic mesenchymal stem cells (iPMSCs) which may provide an unlimited source of such cells for regenerative medicine, tissue engineering and basic research (see at least Abstract; and left column, first full paragraph at page 31). Cao et al also taught that fetal porcine pancreas were removed, minced, and digested with collagenase; the digested tissue was filtered, centrifuged, rinsed and suspended in a supplemented RPMI medium; the resultant suspensions were seeded in culture dishes and after 96 h incubation, suspended islets were physically manually picked out and transferred to six-well plates in a subculture medium; and specimens were sub-cultured at 70-80% confluency after dissociation with 0.25% trypsin with the culture medium was replenished every 2-3 days (section titled “Isolation, culture and transfection of foetal porcine pancreatic cells” on page 20).
Additionally, Zanini et al also disclosed a method for isolating human pancreatic islet-derived mesenchymal stem cells (HI-MSC), the method comprises the step of transferring digested islets to fibronectin-coated flasks in the presence of a supplemental Minimum essential medium/endothelial cell basal medium-1, and within 4 days an islet outgrowth of adherent, monomorphic and spindle-shaped cells was observed; and confluence was achieved by day 7-8 when the cell monolayer was detached by trypsin-EDTA treatment (Abstract; and section titled “Isolation and characterization of human Bone Marrow and Islet-derived Mesenchymal Stromal Ceslls (BM-MSCs and HI-MSCs)” on page 11). Zanini et al also taught that bone marrow (BM-) and HI-MSCs were plastic-adherent cells when maintained in expansion conditions; BM- and HI-MSC preparations expressed CD105, CD73, CD90 and CD29 but they did not express hematopoietic markers, like CD45, CD14 and CD34 (section titled “Characterization of BM- and HI-MSCs” on page 2-3). Zanini et al further demonstrated that both BM-MSCs and HI-MSCs are capable of differentiating into Islet-like cells (ILCs) (Abstract and Figure 4).
Moreover, Liu et al already disclosed an optimal method of isolating and purifying Mongolian sheep bone marrow-derived mesenchymal stem cells (BMSCs), said method comprises: harvesting bone marrow aspirates from 1-year-old Mongolian sheep with DMEM-F12 medium supplemented with 10% FBS; BMSCs were isolated from marrow cells by density-gradient centrifugation using Percoll; mononuclear cells were collected and cultured with DMEM-F12 supplemented with 10% FBS at 370C in a 5% CO2 humidified incubator; the nonadherent cells were removed after 72-96h while the adherent cells were harvested using trypsinization when reaching 90% confluence; and then replated (see at least Abstract; particular sections titled “Bone Marrow (BM) collection” and “Cell Isolation and culture” on page 465). Liu et al also taught that after three passages, eight separate BMSC cell lines were trypsinized, and each divided into two aliquots; with half of the samples were prepared for cryopreservation using 10% DMSO as a cryoprotectant (section titled “Cryopreservation and reconstitution” on page 465). Liu et al further taught that the cells were stored at 40C for 1 h, at -200C for 2 h, at -800C for 10.5 h, and, finally, in liquid nitrogen (-1960C); and the cells were recovered after freezing for 3 min by shaking the cryovial in a 370C water bath for 1 min, and the contents in the vials were transferred into a 15-ml centrifuge tube containing a complete growth medium and centrifuged at 1,000 rpm for 10 min to remove the DMSO. The recovered cells in the pellet were resuspended in fresh media and seeded into flasks, followed by culturing at 370C with saturated humidity and 5% CO2. Liu et al demonstrated that the passage 3 (P3) BMSCs are capable of differentiating into adipocytes, osteoblasts, chondrocytes, neural cells and cardiomyocytes under appropriate in vitro differentiating conditions (pages 468-469).
Furthermore, before the effective filing date of the present application Rouy et al disclosed a method of preparing mesenchymal cells derived from autologous bone marrow in autologous culture medium which can be used in a clinical setting, said method comprises the preparation step of autologous bone marrow mononuclear cells isolated by density gradient on Ficoll-Plaque to be frozen as backup for future use if the number of cultured hMSCs do not reach the acceptable 20 million level (Abstract; Summary; and particularly paragraphs [0011], [0020]-[0024]; and Figure 1). Rouy et al stated explicitly “A second fraction will be frozen for future use if the number of cultured hMSCs do not reach the acceptable 20 million level. This second fraction may be used in an attempt to delivery of MNCs as shown in FIG.1 or saved to attempt an expansion of the frozen cells at a later date” (paragraph [0023]).
Accordingly, it would have been obvious for an ordinary skilled artisan to modify the teachings of Zhang et al by also preparing mesenchymal stem cells derived from pancreatic islets of a neonatal/fetal pig in a method that includes using back-up frozen/cryopreserved floating pancreatic islets that have not been subjected to an adherent culture, in light of the teachings of Cao et al, Zanini et al, Liu et al and Rouy et al as set forth above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because: (i) Cao et al already demonstrated that pancreatic mesenchymal stem cells (MSCs) are present in fetal porcine pancreas and at least immortalized pancreatic mesenchymal stem cells (iPMSCs) may provide unlimited source of such cells for regenerative medicine, tissue engineering and basic research; (ii) Zanini et al taught that adherent primary cultured pancreatic islet-derived reached confluence by day 7-8 after plating suspended islets to fibronectin-coated flasks when the monolayer was detached by trypsin-EDTA treatment; (iii) Liu et al already taught successfully using frozen/cryopreserved samples of sub-cultured BMSCs to reconstitute/recover BMSCs after thawing, as well as using the fraction of mononuclear cells obtained via Ficoll-Plaque density-gradient centrifugation to prepare adherent primary BMSCs; and (iv) Rouy et al also taught autologous bone marrow mononuclear cells isolated by density gradient on Ficoll-Plaque to be frozen as backup for future use, including for the expansion of the frozen cells at a later date. Thus, it would have been readily apparent and advantageous for an ordinary skill in the art to use the back-up frozen/cryopreserved floating neonatal/fetal pancreatic islets that have not been subjected to an adherent culture for preparation and expansion of neonata/fetal pig pancreatic MSCs at a future or later date without the need to start from scratch for various applications.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Zhang et al, Cao et al, Zanini et al, Liu et al and Rouy et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Zhang et al, Cao et al, Zanini et al, Liu et al and Rouy et al as set forth above is indistinguishable and encompassed by the presently claimed method.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 13 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al (World J. Gastroenterol. 11:2906-2911, 2005) in view of Cao et al (Cell Prolif. 44:19-32, 2011; IDS), Zanini et al (PLoS ONE 6(12):e28175.doi:10.1371/journal.pone.0028175, 13 pages; 2011); Liu et al (In Vitro Cell. Dev. Biol. 50:464-474, 2014) and Rouy et al (US 2010/0278783) as applied to claims 11 and 17-18 above, and further in view of Hu et al (J. Lab. Clin. Med. 141: 342-349, 2003) and She et al (WO 2018/064976) for the same reasons already set forth in the Non-Final Office action dated 07/03/2025 (pages 8-10). The same rejection is restated below.
The combined teachings of Zhang et al, Cao et al, Zanini et al, Liu et al and Rouy et al were presented above. However, none of the cited references teach explicitly that the pancreatic islet collected from the neonatal pig contains cells in a mononuclear cell fraction (claim 13), and the (c) freezing of the cells in the mononuclear cell fraction comprises freezing at -200C for 1 hour, followed by freezing at -800C for 24 hours.
Before the effective filing date of the present application (09/08/2017), Hu et al already demonstrated that pancreas-derived mesenchymal stem cells (PMSCs) can be isolated from human fetal pancreas by means of their adherent ability and that they are capable of self-renewal, propagation, and multipotent differentiation; and that PMSCs were isolated from pancreatic mononuclear cells obtained by means of Ficoll-Paque density-gradient centrifugation and depleted of CD45- and GlyA-positive cells by means of micromagnetic-bead sorting (see at least Abstract; and section titled “Cell separation” on page 343).
Additionally, She et al also disclosed a method for the cryopreservation of biological materials such as cells (e.g., mammalian cells that include porcine and human stem cells) and tissues using a composition comprising a permeating cryoprotectant, a saccharide and a macromolecule that has a low toxicity to biological materials and promotes survival and retention of viability of the biological materials (Abstract; Summary; and page 5, lines 11-16). She et al also taught specifically a stepped method of cryopreserving cells that comprises cooling the cells to a first temperature, holding at that first temperature for a first period of time, then cooling the cells to a second temperature for storing the cells; for example the freezing container is transferred to 40C for about 1 hour, and then placed in a chamber at about -800C for at least 12 hours; in some embodiments, the biological is subjected to at least one intermediate storing temperature for a desired period of time with the intermediate storing temperature may range from about 00C to about -1000C before transfer to longer term storage; and the cells may be stored for any desired period such as about 1-5 hours, about 5-12 hours, about 12-24 hours, about 24-48 hours, about 48 hours, about 1 week, about 2 weeks or longer (page 23, lines 28-31; page 25, line 16 continues to line 25 on page 26).
Accordingly, it would have been obvious for an ordinary skilled artisan to further modify the combined teachings of Zhang et al, Cao et al, Zanini et al, Liu et al and Rouy et al by also at least collecting pancreatic islet mononuclear cells derived from the neonatal pig for stem cell preparation, as well as freezing the cells in the mononuclear cell fraction at -200C for 1 hour, followed by freezing at -800C for 24 hours prior to finally, in liquid nitrogen (-1960C) in step c) with a reasonable expectation of success, in light of the teachings of Hu et al and She et al as set forth above.
An ordinary skilled artisan would have been motivated to further carry out the above modifications because: (i) Hu et al already taught that human fetal pancreas-derived mesenchymal stem cells (PMSCs) were isolated from pancreatic mononuclear cells obtained by means of Ficoll-Paque density-gradient centrifugation; and (ii) She et al also disclosed a stepped method of cryopreserving cells (e.g., mammalian cells that include porcine and human stem cells) that comprises cooling the cells to a first temperature, holding at that first temperature for a first period of time, then cooling the cells to a second temperature for storing the cells; for example the freezing container is transferred to 40C for about 1 hour, and then placed in a chamber at about -800C for at least 12 hours; and in some embodiments, the biological is subjected to at least one intermediate storing temperature for a desired period of time with the intermediate storing temperature may range from about 00C to about -1000C before transfer to longer term storage; and the cells may be stored for any desired period such as about 1-5 hours, about 5-12 hours, about 12-24 hours, about 24-48 hours, about 48 hours, about 1 week, about 2 weeks or longer; and particularly Liu et al already taught that passaged Mongolian sheep bone marrow-derived mesenchymal stem cells (BMSCs) were stored at 40C for 1 h, at -200C for 2 h, at -800C for 10.5 h, and, finally, in liquid nitrogen (-1960C). Moreover, both Liu et al and Rouy et al also taught that BMSCs were isolated from collected mononuclear cells by density-gradient centrifugation.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Zhang et al, Cao et al, Zanini et al, Liu et al, Rouy et al, Hu et al and She et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Zhang et al, Cao et al, Zanini et al, Liu et al, Rouy et al, Hu et al and She et al as set forth above is indistinguishable and encompassed by the presently claimed method.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Arguments
Applicant’s arguments related to the above 103 rejections in the Amendment filed on 12/31/2025 (pages 2-3) have been fully considered but they are respectfully not found persuasive for the reason discussed below.
Applicant argued basically that in order to show obviousness, it must be shown that one or ordinary skill in the art would have had a motivation to combine or modify the teachings of the cited references with a reasonable expectation of success to achieve the claimed method. Specifically, Applicant argued that none of the cited references teach or suggest that pancreatic islet cells can be frozen and maintain viability without being cultured in an adherent culture prior to freezing. Applicant argued that Rouy discloses freezing bone marrow cells which are a different type of cells than the pancreatic islet cells, and the reference does not disclose culturing or freezing pancreatic islet cells. Applicant also argued that the paragraphs [0011], [0020]-[0024] of Rouy that are cited by the examiner refer to bone marrow aspirated cells, and mesenchymal stromal stem cells from the bone marrow as depicted in figures 2 and 3 of Rouy showed adherent cells. Applicant also argued that none of Rouy and other cited references provide any suggestion that pancreatic islet cells can be frozen in a manner taught by Rouy. Applicant further argued that since Cao and Zanini teach culturing pancreatic stem cells in adherent culture, one of ordinary skill in the art would have had no motivation to omit this step prior to freezing the cells.
First, since the above rejections were made under 35 U.S.C. 103 none of the cited references have to teach every limitation of the instant claims. For example, none of Zhang, Cao, Zanini and Liu have to teach freezing collected cells which are not cultured in an adherent culture prior to freezing. Nor does Rouy have to disclose culturing and/or freezing pancreatic cells. It is also apparent that Applicant considered each of the cited references in total isolation one from the others, particularly focusing solely on Rouy, without taking into account of the specific combination of Zhang et al, Cao et al, Zanini et al, Liu et al and Rouy et al.
Second, an ordinary skilled artisan would have been motivated to modify the teachings of Zhang et al by also preparing at least mesenchymal stem cells derived from pancreatic islets of a neonatal/fetal pig in a method that includes using back-up frozen/cryopreserved floating pancreatic islets that have not been subjected to an adherent culture because: (i) Cao et al already demonstrated that pancreatic mesenchymal stem cells (MSCs) are present in fetal porcine pancreas and at least immortalized pancreatic mesenchymal stem cells (iPMSCs) may provide unlimited source of such cells for regenerative medicine, tissue engineering and basic research; (ii) Zanini et al taught that adherent primary cultured pancreatic islet-derived reached confluence by day 7-8 after plating suspended islets to fibronectin-coated flasks when the monolayer was detached by trypsin-EDTA treatment; (iii) Liu et al already taught successfully using frozen/cryopreserved samples of sub-cultured BMSCs to reconstitute/recover BMSCs after thawing, as well as using the fraction of mononuclear cells obtained via Ficoll-Plaque density-gradient centrifugation to prepare adherent primary BMSCs; and (iv) Rouy et al also taught autologous bone marrow mononuclear cells isolated by density gradient on Ficoll-Plaque to be frozen as backup for future use, including for the expansion of the frozen cells at a later date. Thus, it would have been readily apparent and advantageous for an ordinary skill in the art to use the back-up frozen/cryopreserved floating neonatal/fetal pancreatic islets that have not been subjected to an adherent culture for preparation and expansion of neonata/fetal pig pancreatic MSCs at a future or later date without the need to start from scratch for various applications.
Third, since Cao already disclosed that pancreatic mesenchymal stem cells are present in fetal porcine pancreas and Zanini taught that human pancreatic mesenchymal stem cells are similar to human bone marrow-derived mesenchymal stem cells, coupled with the teachings of Liu showing that cryopreserved sheep bone marrow-derived mesenchymal stem cells can be thawed, cultured and successfully differentiated into adipocytes, osteoblasts, chondrocytes, neural cells and cardiomyocytes under appropriate in vitro differentiating conditions; an ordinary skill in the art would readily recognize that pancreatic islet cells such as fetal porcine pancreatic mesenchymal stem cells can be successfully frozen and are viable, including fetal porcine pancreatic mesenchymal stem cells that are not cultured in an adherent culture prior to freezing or cryopreserving. Moreover, She (WO 2018/064976) already taught successfully a method for the cryopreservation of biological material such as cells (e.g., mammalian cells that include porcine and human stem cells) and tissues using a composition comprising a permeating cryoprotectant, a saccharide and a macromolecule that has a low toxicity to biological materials and promoters survival and retention of viability of the biological materials.
Fourth, Rouy taught clearly to use 50 ml of bone marrow aspiration to prepare mononuclear cells (MNCs) containing mesenchymal stem cells that are isolated by density gradient on Ficoll-Plaque to be frozen as backup for future use (see at least Fig. 1 and paragraphs [0020]-[0023]), and not adherent mesenchymal stem cells in Figs. 2-3. The MNCs were not subjected to adherent culture prior to freezing.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. 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 Quang Nguyen, Ph.D., at (571) 272-0776.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s SPE, James Douglas (Doug) Schultz, Ph.D., may be reached at (571) 272-0763.
To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Group Art Unit 1631; Central Fax No. (571) 273-8300.
Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to (571) 272-0547.
Patent applicants with problems or questions regarding electronic images that can be viewed in the Patent Application Information Retrieval system (PAIR) can now contact the USPTO’s Patent Electronic Business Center (Patent EBC) for assistance. Representatives are available to answer your questions daily from 6 am to midnight (EST). The toll-free number is (866) 217-9197. When calling please have your application serial or patent number, the type of document you are having an image problem with, the number of pages and the specific nature of the problem. The Patent Electronic Business Center will notify applicants of the resolution of the problem within 5-7 business days. Applicants can also check PAIR to confirm that the problem has been corrected. The USPTO’s Patent Electronic Business Center is a complete service center supporting all patent business on the Internet. The USPTO’s PAIR system provides Internet-based access to patent application status and history information. It also enables applicants to view the scanned images of their own application file folder(s) as well as general patent information available to the public.
/QUANG NGUYEN/Primary Examiner, Art Unit 1631