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 .
Priority
The instant application is a national stage entry of PCT application PCT/IL2023/050306, filed 09/23/2024 under 35 USC 371. Acknowledgement is made of the applicant’s claim for benefit to prior-filed U.S. provisional patent applications 63/323,093, which was filed 03/24/2022.
Election/Restrictions
Applicant’s election without traverse of group I, claims 1, 4, 6, 8, 12, 14, 16, 19, 21-22, 24-26, 29-30, 38, 41 and 43, drawn to a method of producing mesoderm-committed nonhuman-animal cells, in the reply filed on 09/11/2026 is acknowledged. Accordingly, claims 1, 4, 6, 8, 12, 14, 16, 19, 21-22, 24-26, 29-30, 38, 41 and 43 have been considered on the merits. Claims 31 and 46 are withdrawn from consideration pursuant 37 CFR 1.142(b).
Claim Objections
Claims 1 and 38 are objected to because of the following informalities: Claims 1 and 38 use multiple periods. MPEP 608.01(m) states “[E]ach claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations.” Herein the steps “a.”, “b.”, etc. should be amended to (a), (b), etc.. Appropriate correction is required.
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.
Claims 1, 4, 6, 8, 12, 14, 16, 19, 21-22, 24-26, 29-30, 38, 41 and 43 are 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.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. The omitted steps are: the preamble requires the process is conducted “under variable gravity conditions”. However, there is no such condition limited in any of the steps (a) to (d).
Claim 1 recites “at least one type of polysaccharide” renders instant claim indefinite. The addition of the word "type" to an otherwise definite expression extends the scope of the expression so as to render it indefinite. Ex parte Copenhaver, 109 USPQ 118 (Bd. Pat. App. & Inter. 1955). See MPEP 2173.05(b).
Claim 1 recites “inducing conditions enabling the transition of the suspension to a solid or semisolid state” renders instant claim indefinite. The phrase “inducing conditions enabling the transition” is not defined in the claim, the specification does not provide a definition or clarification, therefore an ordinary in the art would not be reasonably apprised of the meaning and/or step of “inducing condition enabling the transition”. The scope of the claim is not clear.
Claims 4, 6, 8, 12, 14, 16, 19, 21-22, 24-26 and 29-30 depend from, at least, claim 1, and thus inherit the deficiency and are rejected on the same basis.
Claim 30, which depends upon claim 1, recites the limitation "the micro- or zero-gravity conditions” in lines 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 38 recites the limitation "steps (a) to (e)" in lines 22 and 24. There is insufficient antecedent basis for this limitation in the claim.
Claims 41 and 43 depend from claims 38, and thus inherit the deficiency and are rejected on the same basis.
Claim Interpretation
As stated above, claims 1, 30 and 38 are indefinite. In the interest of compacted prosecution, claim 1 is interpreted that all the steps (a) to (e) are conducted under same gravity condition (i.e., one of the gravity conditions as recited in claim 8). Claim 30 is interpreted as depending upon claim 8, which limits the types of gravities. In claim 38, the "steps (a) to (e)" is interpreted as "steps (e) to (i)".
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.
Claims 1, 4, 6, 8, 14, 16, 22, 25, 26 and 29-30 are rejected under 35 U.S.C. 103 as being unpatentable over Lavon et al. (WO 2020/230138 A1, cited in IDS) in view of Eleftheriadou et al. (RSC Adv. 2022 Feb 1;12(7):4005-4015) and Cao (thesis, 2011), evidenced by MSD manual (2012).
Lavon et al. teach compositions and methods for mass production of pluripotent stem cells derived from non-human-animal (Abstract).
Regarding claim 1, Lavon et al. teach a method comprising the steps of: (a) seeding at least one non-human-animal derived pluripotent stem cells (PSC, same as NHAPSCs as recited in instant claim) in an expansion medium to form a seeding suspension culture; (b) growing the suspension culture under conditions enabling aggregate formation and aggregate expansion, thereby forming homogenous aggregates of said PSCs (see p56, claim 1). Lavon et al. teach according to certain embodiments, step (a) of seeding the at least one PSC further comprises encapsulating at least one of said PSCs within a particle and seeding a plurality of the particles within the expansion medium (p13, L1-3). Lavon et al. teach according to certain embodiments, the particle comprises an inner core comprising the at least one PSC surrounded by at least one outer shell layer (p13, L4-5). According to certain embodiments, the outer shell layer is of a fluid-permeable food grade material (p13, L5-6). According to certain embodiments, the outer shell layer comprises at least one food grade pre-polymer and/or polymer and/or copolymer (p13, L10-11). According to certain embodiments, the food grade polymer is selected from the group consisting of alginate, Gellan-Gum, agar, agarose, chitosan, hyaluronic acid, curdlan, carrageenan, pectin, modified starch and combinations thereof (p13, L14-16). Herein non-human-animal derived pluripotent stem cells are the NHAPSCs, the polymer such as alginate is a polysaccharide, and the particle comprises an inner core comprising the at least one PSC surrounded by at least one outer shell layer indicates the particle is in solid or at least semisolid state encapsulating PSCs. Lavon et al. teach according to certain embodiments, the inner core provides a microenvironment supporting proliferation of the at least one PCS (p13, L18-19). Lavon et al. also teach according to these embodiments, the PSCs are further differentiated to at least one of adipocyte cells, muscle cells, stromal cell, and endothelial lineages (p16, L17-19), i.e., the differentiation of bESC (see i.e., p43, Example 3), which use the differentiation medium for differentiating PSCs. Moreover, Lavon et al. teach the intended use of the PSC aggregates produced according to the teachings is in the industry of cultured meat products. Cultured meat preferably comprises the various cells comprising the muscle tissue, including myoblasts, adipocytes, stromal and endothelial cells, all being derivatives of the mesoderm lineage (p43, L4-7). All the teachings above teach the method for producing mesoderm-committed non-human-animal cells (MCNHACs) comprising steps (a) seeding the cells in culture medium, and the culture medium comprises at least one type of polysaccharide; (c) (obtain and) maintain a solid or semisolid hydrogel comprising the NHAPSCs under proliferation condition; and (d) use / replace to differentiation medium to put the cells under differentiation condition for differentiation the cells to producing MCNHACs.
Instant claim differs from Lavon et al. that: instant claim disclose the step (b) inducing conditions enabling the transition of the suspension to a solid or semisolid state, thereby forming a solid or semisolid hydrogel comprising the NHAPSCs, while Lavon et al. teach the particles formed in the culture medium, but do not expressively disclose how to form the particle or how the suspension comprising the NHAPSCs become solid or semisolid state. However, such was disclosed by Eleftheriadou et al. and Cao at the time of instant invention.
Eleftheriadou et al. teach the development of a novel approach for protecting cells after implantation into the central nervous system (CNS), by developing dual-layer alginate beads that encapsulate therapeutic cells and release an immunomodulatory compound in a sustained manner (Abstract).
Cao teaches study on the dispensing-based biofabrication of 3D cell encapsulated alginate hydrogel scaffolds (pii, parag 1).
Regarding step (b) inducing conditions enabling the transition of the suspension to a solid or semisolid state, thereby forming a solid or semisolid hydrogel comprising the cells, Eleftheriadou et al. teach sodium alginate was dissolved in Dulbecco's Modified Eagle's medium (DMEM). For cell encapsulation, cells at the desired density were dispersed in the alginate solution (see p4007, left column), extruded into CaCl2 solution to induce gelation (see p4007, left column). Cao teaches in methodology of hydrogel formation, both the method of adding alginate solution to CaCl2 solution and the method of adding CaCl2 solution to alginate solution works: most of the methods are based on the strategies of internal gelling and diffusion gelling. In the first strategy, i.e., internal gelling, the calcium chloride solution is introduced into the reservoir of alginate and the gelation starts at the interface of two reagents, then gelation proceeds into the alginate solution as calcium ions diffuse into the alginate body. In the second strategy, an alginate solution is typically added to a reservoir of divalent ions such as a calcium chloride solution and then the gelation process starts from the outermost layer of alginate as the calcium ions diffuse into the core of the material (p9, parag 2).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Lavon et al.’s method of culturing non-human-animal derived pluripotent stem cells (PSC), which forming particles comprising an inner core comprising the at least one PSC surrounded by at least one outer polysaccharide shell layer (i.e., alginate) in the culture medium, and induce conditions enabling the transition of the suspension to a solid or semisolid state by adding CaCl2 solution to alginate solution as taught by Eleftheriadou et al. and Cao. The only difference between instant claim and Lavon et al.’s method of culturing non-human-animal derived PSCs is instant claim teaches a step of inducing the transition of the suspension to a solid or semisolid state. Given that Eleftheriadou et al. and Cao teach mixing alginate solution with CaCl2 solution (by adding alginate solution to CaCl2 solution, or adding CaCl2 solution to alginate solution) leads to gelation, one of ordinary skill in the art would have substituted Lavon et al.’s method of forming particle, and adding CaCl2 solution to the culture medium comprising cells and alginate for gelation/ form solid or semisolid state. This simple substitution of one known element (mixing the culture medium comprising cells and alginate with CaCl2 solution to form a solid or semisolid state) for another known element (forming alginate particles comprising cells in the culture medium) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claims 4, 6 and 29, Lavon et al. teach the entire production of aggregates is performed in a closed system. The use of a closed system is of significant advantage in the mass
production of PSC aggregates at a large-scale allowing to keep a sterile environment and
automated process (p7, L4-5).
Regarding claim 8, Lavon et al. teach compositions and methods for mass production of pluripotent stem cells derived from non-human-animal (Abstract). This methos is conducted in earth gravity since Lavon et al. did not point out that the method needs to be conducted in a special gravity condition (i.e., particle gravity condition).
Regarding claims 14 and 16, following the discussion above, Eleftheriadou et al. teach using CaCl2 for crosslinking, which is chemical crosslinking comprises using Ca+2 (see p4007, left column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Lavon et al.’s method of culturing non-human-animal derived pluripotent stem cells (PSC), which forming particles comprising an inner core comprising the at least one PSC surrounded by at least one outer polysaccharide shell layer (i.e., alginate) in the culture medium, and induce conditions enabling the transition of the suspension to a solid or semisolid state by chemical crosslinking CaCl2 (Ca+2) with alginate as taught by Eleftheriadou et al. and Cao. The only difference between instant claim and Lavon et al.’s method of culturing non-human-animal derived PSCs is instant claim comprises a step of inducing the transition of the suspension to a solid or semisolid state. Given that Eleftheriadou et al. and Cao teach crosslinking alginate with CaCl2 leads to gelation, one of ordinary skill in the art would have substituted Lavon et al.’s method of forming particle, and chemical crosslinking CaCl2 (Ca+2) with alginate for gelation/ form solid or semisolid state. This simple substitution of one known element (chemical crosslinking CaCl2 (Ca+2) with alginate to form a solid or semisolid state) for another known element (forming alginate particles comprising cells in the culture medium) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claim 22, following the discussion above, Lavon et al. teach the use of a closed system is of significant advantage in the mass production of PSC aggregates at a large-scale allowing to keep a sterile environment and automated process (p7, L4-5). Lavon et al. teach the use of a closed system in which steps of cell aggregation and disaggregation take place and an incubation temperature compatible with non-human-animal body temperature (p12, L1-2). This teaching indicates that the temperature is pre-set temperature which compatible with non-human-animal body temperature (i.e., 36.7-39.1 °C in cow, as evidenced by MSD manual), which is in the range of about 35°C to about 39°C.
Regarding claim 25, following the discussion above, Lavon et al. teach the intended use of the PSC aggregates produced according to the teachings is in the industry of cultured meat products. Cultured meat preferably comprises the various cells comprising the muscle tissue, including myoblasts, adipocytes, stromal and endothelial cells, all being derivatives of the mesoderm lineage (p43, L4-7). Lavon et al. also teach cell differentiation to adipocytes (see i.e., p51, Example 10), which use different growth medium, reads on the step (e) replacing the differentiation conditions to maturation condition and further differentiation cells to adipocytes as recited in instant claim.
Regarding claim 26, following the discussion above, Lavon et al. teach the intended use of the PS Cs aggregates produced is in the industry of cultured meat products (p43, L4-5).
Regarding claim 30, the micro- or zero-gravity conditions are outer space conditions are inherent features since earth is known to have gravity.
Claims 1, 4, 6, 8, 12, 14, 16, 22, 25, 26 and 29-30 are rejected under 35 U.S.C. 103 as being unpatentable over Lavon et al. (WO 2020/230138 A1, cited in IDS) in view of Eleftheriadou et al. (RSC Adv. 2022 Feb 1;12(7):4005-4015) and Cao (thesis, 2011), evidenced by MSD manual (2012), as applied to claims 1, 4, 6, 8, 14, 16, 22, 25, 26 and 29-30, further in view of Kapr et al. (Adv Healthc Mater. 2021 Aug;10(16):e2100131, cited in IDS).
The teaching of Lavon et al., Eleftheriadou et al. and Cao is set forth above.
Regarding claim 12, Eleftheriadou et al. teach concentrations of alginate is 1.5% or 2% (w/v). Lavon et al. in view of Eleftheriadou et al. and Cao do not teach the concentration of the at least one type of polysaccharide (i.e., herein the alginate) is from about 0.3 to about 0.8% w/v of the suspension. However, this was disclosed by Kapr et al. at the time of instant application.
Kapr et al. teach two alginate/gellan gum/laminin (ALG/GG/LAM) hydrogel blends are presented for the fabrication of human induced pluripotent stem cell (hiPSC)-based 3D neural models (Abstract).
Regarding claim 12, Kapr et al. teach GG and the 0.3% ALG/0.8% GG blend retained their structural integrity (p2, right column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Lavon et al.’s method of culturing non-human-animal derived pluripotent stem cells (PSC), which forming particles comprising an inner core comprising the at least one PSC surrounded by at least one outer polysaccharide shell layer (i.e., alginate) in the culture medium, and induce conditions enabling the transition of the suspension to a solid or semisolid state by adding CaCl2 in alginate solution as taught by Eleftheriadou et al. and Cao, and have the concentration of alginate at a concentration of 0.3% w/v as taught by Kapr et al.. The skilled artisan would have been motivated to have the concentration of alginate at 0.3% w/v since Kapr et al. teach the 0.3% ALG blend retained the structural integrity (p2, right column). There would be a reasonable expectation of success of having the concentration of alginate at 0.3% w/v since it is daily operation to prepare a solution having 0.3% w/v alginate in the art.
Claims 1, 4, 6, 8, 14, 16, 19, 21-22, 24-26 and 29-30 are rejected under 35 U.S.C. 103 as being unpatentable over Lavon et al. (WO 2020/230138 A1, cited in IDS) in view of Eleftheriadou et al. (RSC Adv. 2022 Feb 1;12(7):4005-4015) and Cao (thesis, 2011), evidenced by MSD manual (2012), as applied to claims 1, 4, 6, 8, 14, 16, 22, 25, 26 and 29-30 , further in view of Lim et al. (Biochemical Engineering Journal 150 (2019) 107258).
The teaching of Lavon et al., Eleftheriadou et al. and Cao is set forth above.
Regarding claim 19, Lavon et al. teach the culture method is in a closed system with automation, but do not teach the closed system is configured to automatically inject (i)proliferation medium through the solid or semisolid hydrogel comprising the NHAPSCs at a pre-determined injection interval, volume and/or flow rate and/or (ii) differentiation medium through the solid or semisolid hydrogel comprising the NHAPSCs and/or MCNHACs at a pre-determined interval, injection volume and/or flow rate. However, this was disclosed by Lim et al. at the time of instant invention.
Lim et al. teach a fully automated bioreactor system (fABS) for precisely controlling the stem cell fate (Abstract).
Regarding claim 19, Lim et al. developed a water-jacketed culture chamber (WJCC) to provide a stable growth environment and physical stimulation to stem cells (p2, right column). Lim et al. teach culture medium was pumped into the WJCC through a recirculating process using variable speed peristaltic pumps. The flow rate could be controlled (p3, left column). Since Lavon et al. teach the outer shell layer is of a fluid-permeable food grade material (p13, L5-6), the pumped medium can be automatically injected through the solid or semisolid hydrogel comprising the NHAPSCs at a pre-determined injection flow rate.
Regarding claim 21, following the discussion above, Lim et al. teach fully automated bioreactor system (see p2, figure 1), in addition to the compartment comprising cells, there is at least on additional compartment such as the media feeding system.
Regarding claim 24, following the discussion above, Lim et al. teach fully automated bioreactor system (see p2, figure 1), the system enables the monitoring of cell movement using an inverted microscope (p7, right column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Lavon et al.’s method of culturing non-human-animal derived pluripotent stem cells (PSC), which forming particles comprising an inner core comprising the at least one PSC surrounded by at least one outer polysaccharide shell layer (i.e., alginate) in the culture medium, and induce conditions enabling the transition of the suspension to a solid or semisolid state by adding CaCl2 in alginate solution as taught by Eleftheriadou et al. and Cao, and conduct this method in a fully automated bioreactor system which configured to automatically inject medium, have additional support system for cell culture (i.e., media feeding system), as well as obtain microscopic images as taught by Lim et al.. The skilled artisan would have been motivated to have a fully automated bioreactor system which configured to automatically inject medium, have additional support system for cell culture (i.e., media feeding system), as well as obtain microscopic images since Lim et al. teach this fully automated bioreactor system (fABS) can stringently regulate environmental cues as well as induce biophysical and biochemical cues (p9, right column), and can precisely controlling the stem cell fate (Abstract). There would be a reasonable expectation of success of having the automated system which configured to automatically inject medium, having additional support system for cell culture (i.e., media feeding system), as well as the ability for obtaining microscopic images since Lim et al. teach the block diagram of a fully automated bioreactor system (see figure 1).
Claims 38, 41 and 43 are rejected under 35 U.S.C. 103 as being unpatentable over Lavon et al. (WO 2020/230138 A1, cited in IDS) in view of Eleftheriadou et al. (RSC Adv. 2022 Feb 1;12(7):4005-4015), Cao (thesis, 2011), Wang et al. (FASEB J. 2019 May;33(5):5903-5913.) and Sønderholm et al. (Appl Environ Microbiol. 2017 Apr 17;83(9):e00113-17), evidenced by Biocompare (published in 2007).
Regarding claim 38, it is noted that the preamble “for obtaining RNA and/or RNA-marker profile of cells differentiating under variable gravity condition” is an intended use. The purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention’s limitations, then the preamble is not considered a limitation and is of no significance to claim construction. See MPEP 2111.02. Lavon et al. teach a method comprising the steps of: (a) seeding at least one non-human-animal derived pluripotent stem cells (PSC, same as NHAPSCs as recited in instant claim) in an expansion medium to form a seeding suspension culture; (b) growing the suspension culture under conditions enabling aggregate formation and aggregate expansion, thereby forming homogenous aggregates of said PSCs (see p56, claim 1). Lavon et al. teach according to certain embodiments, step (a) of seeding the at least one PSC further comprises encapsulating at least one of said PSCs within a particle and seeding a plurality of the particles within the expansion medium (p13, L1-3). Lavon et al. teach according to certain embodiments, the particle comprises an inner core comprising the at least one PSC surrounded by at least one outer shell layer (p13, L4-5). According to certain embodiments, the outer shell layer is of a fluid-permeable food grade material (p13, L5-6). According to certain embodiments, the outer shell layer comprises at least one food grade pre-polymer and/or polymer and/or copolymer (p13, L10-11). According to certain embodiments, the food grade polymer is selected from the group consisting of alginate, Gellan-Gum, agar, agarose, chitosan, hyaluronic acid, curdlan, carrageenan, pectin, modified starch and combinations thereof (p13, L14-16). Herein non-human-animal derived pluripotent stem cells are the NHAPSCs, the polymer such as alginate is a polysaccharide, and the particle comprises an inner core comprising the at least one PSC surrounded by at least one outer shell layer indicates the particle is in solid or at least semisolid state encapsulating PSCs. Lavon et al. teach according to certain embodiments, the inner core provides a microenvironment supporting proliferation of the at least one PCS (p13, L18-19). Lavon et al. also teach according to these embodiments, the PSCs are further differentiated to at least one of adipocyte cells, muscle cells, stromal cell, and endothelial lineages (p16, L17-19), i.e., the differentiation of bESC (see i.e., p43, Example 3), which use the differentiation medium for differentiating PSCs. Moreover, Lavon et al. teach the intended use of the PSC aggregates produced according to the teachings is in the industry of cultured meat products. Cultured meat preferably comprises the various cells comprising the muscle tissue, including myoblasts, adipocytes, stromal and endothelial cells, all being derivatives of the mesoderm lineage (p43, L4-7). All the teachings above teach the method for producing mesoderm-committed non-human-animal cells (MCNHACs) comprising steps (e) seeding the cells in culture medium, and the culture medium comprises at least one type of polysaccharide; (g) (obtain and) maintain a solid or semisolid hydrogel comprising the NHAPSCs under proliferation condition; and (h) use / replace to differentiation medium to put the cells under differentiation condition for differentiation the cells to producing MCNHACs.
Instant claim differs from Lavon et al. that: (1) instant claim disclose the step (f) inducing conditions enabling the transition of the suspension to a solid or semisolid state, thereby forming a solid or semisolid hydrogel comprising the NHAPSCs, while Lavon et al. teach the particles in the culture medium, but do not expressively disclose how to form the particle or how the suspension comprising the NHAPSCs become solid or semisolid state; (2) step (i) stabilizing cellular RNA in situ within said solid or semisolid hydrogel under unfrozen conditions; and (3) the method is conducted in first system and second system, and in the first system at least part of steps (e) to (i) are performed under gravity conditions selected from the group consisting of partial, micro- or zero- gravity conditions and in the second system the entire steps (e) to (i) are performed under earth gravity conditions. However, such was disclosed by Eleftheriadou et al., Cao, Wang et al. and Sønderholm et al. at the time of instant invention.
Eleftheriadou et al. teach the development of a novel approach for protecting cells after implantation into the central nervous system (CNS), by developing dual-layer alginate beads that encapsulate therapeutic cells and release an immunomodulatory compound in a sustained manner (Abstract).
Cao teaches study on the dispensing-based biofabrication of 3D cellencapsulated alginate hydrogel scaffolds (pii, parag 1).
Wang et al. maintained mouse bone marrow HSPCs in the presence of stem cell factor for 12 d under spaceflight and simulated microgravity conditions, respectively, and analyzed cell proliferation and gene expression (Abstract).
Sønderholm et al. teach that alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h (Abstract).
Regarding step (f) inducing conditions enabling the transition of the suspension to a solid or semisolid state, thereby forming a solid or semisolid hydrogel comprising the cells, Eleftheriadou et al. teach preparing the alginate beads: sodium alginate was dissolved in Dulbecco's Modified Eagle's medium (DMEM). For cell encapsulation, cells at the desired density were dispersed in the alginate solution (see p4007, left column), mix with CaCl2 induce gelation (see p4007, left column). Cao teaches in methodology of hydrogel formation, both the method of adding alginate solution to CaCl2 solution and the method of adding CaCl2 solution to alginate solution works: most of the methods are based on the strategies of internal gelling and diffusion gelling. In the first strategy, i.e., internal gelling, the calcium chloride solution is introduced into the reservoir of alginate and the gelation starts at the interface of two reagents, then gelation proceeds into the alginate solution as calcium ions diffuse into the alginate body. In the second strategy, an alginate solution is typically added to a reservoir of divalent ions such as a calcium chloride solution and then the gelation process starts from the outermost layer of alginate as the calcium ions diffuse into the core of the material (p9, parag 2).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Lavon et al.’s method of culturing non-human-animal derived pluripotent stem cells (PSC), which forming particles comprising an inner core comprising the at least one PSC surrounded by at least one outer polysaccharide shell layer (i.e., alginate) in the culture medium, and induce conditions enabling the transition of the suspension to a solid or semisolid state by adding CaCl2 in alginate solution as taught by Eleftheriadou et al. and Cao. The only difference between instant claim and Lavon et al.’s method of culturing non-human-animal derived PSCs is instant claim comprises a step of inducing the transition of the suspension to a solid or semisolid state. Given that Eleftheriadou et al. and Cao teach mixing alginate solution with CaCl2 solution (by adding alginate solution to CaCl2 solution, or adding CaCl2 solution to alginate solution) leads to gelation, one of ordinary skill in the art would have substituted Lavon et al.’s method of forming particle, and adding CaCl2 solution to the culture medium comprising cells and alginate for gelation/ form a solid or semisolid state. This simple substitution of one known element (mixing the culture medium comprising cells and alginate with CaCl2 solution to form a solid or semisolid state) for another known element (forming alginate particles comprising cells in the culture medium) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding (2) stabilizing cellular RNA insitu within said solid or semisolid hydrogel under unfrozen condition, and (3) conduct steps (e) to (i) in under gravity conditions and earth gravity conditions, Wang et al. teach HSPCs display reduced proliferation in a microgravity environment. Cell cycle analysis demonstrated that simulated microgravity inhibited the G1/S transition. RNA-sequencing (RNA-Seq) and bioinformatics analysis clarified clearer gene signatures and deeper molecular mechanisms for microgravity inhibiting HSPC proliferation (p5904, left column). Sønderholm et al. teach isolation of RNA: for alginate bead cultures, the beads were harvested and rinsed three times in sterile, prewarmed 0.9% NaCl to remove planktonic bacteria before mixing with two volumes of RNAlater. The samples were stored ON at 4°C before freezing at -80°C until further use (see p12-13, Methods, Microarray analysis). RNAlater is an RNA stabilizer, as evidence by Biocompare.
Regarding claims 41 and 43, following the discussion above, Wang et al. teach HSPCs display reduced proliferation in a microgravity environment. Cell cycle analysis demonstrated that simulated microgravity inhibited the G1/S transition. RNA-sequencing (RNA-Seq) and bioinformatics analysis clarified clearer gene signatures and deeper molecular mechanisms for microgravity inhibiting HSPC proliferation (p5904, left column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Lavon et al.’s method of culturing non-human-animal derived pluripotent stem cells (PSC), which forming particles comprising an inner core comprising the at least one PSC surrounded by at least one outer polysaccharide shell layer (i.e., alginate) in the culture medium, and induce conditions enabling the transition of the suspension to a solid or semisolid state by adding CaCl2 solution to alginate solution as taught by Eleftheriadou et al. and Cao, and conduct the method in two systems with different gravity conditions, then stabilize RNA for RNA sequence and compare RNA sequence under different system/gravity condition as taught by Wang et al. and Sønderholm et al.. The skilled artisan would have been motivated to conduct the method in two systems with different gravity conditions, then stabilize RNA for RNA sequence and compare RNA sequence under different gravity condition since Wang et al. teach stem cells (i.e., HSPCs) display reduced proliferation in a microgravity environment, and using RNA-sequencing (RNA-Seq) and bioinformatics analysis can clarify clearer gene signatures and deeper molecular mechanisms. There would be a reasonable expectation of success of conducting the method in two systems with different gravity conditions, and stabilizing RNA for RNA sequence and comparing RNA sequence under different gravity condition since Sønderholm et al. teach using RNAlater for RNA stabilization, and Lavon et al. teach the culture system.
Conclusion
No claims are allowed.
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/Q.G./Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699
