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
Acknowledgement is made of Applicants’ claim for priority Chinese patent application CN202310978630.5 (filed 08/04/2023). A copy of the foreign priority document is present in the application file.
Claim Objections
Claims 1 and 4 are rejected for minor informalities:
In claim 1, line 5, the colon following the phrase “gelatin solution” should be replaced with a semicolon.
Claim 4 recites “wherein comprising cultivating”. The word “wherein” should be removed.
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 1-7 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.
Regarding claims 1-3: In claim 1(1), the method of synthesizing GelMA is understood to require the following steps:
Dissolving type A gelatin in PBS at a mass/volume ratio of 1:(10-20) g/mL to obtain a gelatin solution;
Adding MAA dropwise to the gelatin solution at a mass ratio of 1:(0.6-0.8) (gelatin : MAA);
stirring the resulting mixture at 40° C – 60°C to allow a reaction for 1-3 hrs;
Subjecting the resulting reaction to dialysis with deionized water at 40° C for 3-7 days to remove unreacted MAA and by-products;
Aseptically filtering a purified GelMA monomer solution through a microporous filter membrane
Lyophilizing a resulting filtrate to obtain the GelMA
It is however unclear that step iv of the GelMA synthesis method produces “a purified GelMA monomer”. Therefore, it is unclear whether the “purified GelMA monomer” in step v is required to be produced by the method of steps i-iv or whether any purified GelMA monomer can be used. Claims 2 and 3 depend from claim 1 and thus inherit the deficiencies of claim 1
Further regarding claims 1-3: The term “slowly” in claim 1 is a relative term which renders the claim indefinite. The term “slowly” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear whether adding methacrylic anhydride to the gelatin solution of claim 1(1) dropwise is considered “slowly” or if the term “slowly” requires the drops to be added to the gelatin solution at a specific rate. For the purpose of compact prosecution, adding “dropwise” will be considered as adding “slowly”. Claims 2 and 3 depend from claim 1 and thus inherit the deficiencies of claim 1.
Claim 1 contains the trademark/trade name Glutamax. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe an L-alanyl-Lglutamine dipeptide and, accordingly, the identification/description is indefinite. Claims 2 and 3 depend from claim 1 and thus inherit the deficiencies of claim 1.
Regarding claims 4-7: Claim 4 recites the limitation “using hydrogel material according to claim 1”. Claim 1 recites the following hydrogels: gelatin, GelMA, and ns-GelMA-PEO. Thus, the metes and bounds of the “hydrogel material according to claim 1” are unclear. Claims 5-7 depend from claim 4 and thus inherit the deficiencies of claim 4.
The term “gently” in claim 5 is a relative term which renders the claim indefinite. The term “gently” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear whether the of “gently pipetting… up and down to obtain single cells” in claim 5(2) requires pipetting to be performed at a specific speed/force. Given that pipetting “gently” results in a single cell suspension, the term is being interpreted as pipetting with sufficient force to produce a single cell suspension. Claims 6 and 7 depend from claim 5 and thus inherits the deficiencies of claim 5.
Claim 5 contains the trademark/trade name Matrigel. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a basement membrane extract and, accordingly, the identification/description is indefinite. Claims 6 and 7 depend from claim 5 and thus inherits the deficiencies of claim 5.
Claims 5 and 7 contain the trademark/trade name StemPro. Claim 5 further contains the trademarks/ trade names mTeSR1 and B27. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademarks/trade names are used to identify/describe cell culture mediums and, accordingly, the identification/description is indefinite. Claim 6 depends from claim 5 and thus inherits the deficiencies of claim 5.
Claim 6 contains the trademark/trade name KnockOut. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a cell culture medium and, accordingly, the identification/description is indefinite.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim 4 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Chen et al (Adv Funct Mater, 2012).
Chen et al discloses a method for culturing 3D vascular networks in a GelMA hydrogel (See abstract).
Regarding claim 4: Chen et al discloses culturing 3D vascular networks (reads on BVO) in a GelMA hydrogel. The “hydrogel material according to claim 1” reads on any of the following hydrogels: gelatin, GelMA, ns-GelMA-PEO (See 112(b) rejection above). Furthermore, the GelMA synthesis method of claim 1(1) is undefined (i.e. step v can include any GelMA (See 112(b) rejection above). Therefore, the GelMA of claim 1 can include any GelMA. Thus, the method of culturing 3D vascular networks in GelMA of Chen et al reads on the instant claim.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4 are rejected under 35 U.S.C. 103 as being unpatentable over Ying et al (Advanced Materials, 2018) in view of Loessner et al (Nature Protocols, 2016), STEMCELL (Generation and Maturation of Blood Vessel Organoids Using STEMdiff Blood Vessel Organoid Kit, May, 2023), and Chen et al (Adv Func Mater, 2012).
The teachings of Chen et al are set forth above.
Chen anticipates claim 4.
Ying et al disclose a method of synthesizing a gelatin-methacryloyl (GelMA) bioink for bioprinting of cell-laden porous hydrogel constructs. The bioink preparation method of Loessner comprises mixing 5 or 10% GelMA with a LAP solution comprising 0.5% LAP, 0.5%, 1%, or 1.6% PEO and PBS then crosslinking under UV light (0.5 W cm−2 for 15 s) to obtain a final GelMA concentration at 10 w/v%. For cell encapsulation, cells can be mixed with the GelMA pre-gel solution (See supporting information pgs. 2-3, Sec. 2. Emulsion preparation and characterization). Additionally, Ying et al teaches phase separation is affected by the molecular weight of PEO.
Regarding claims 1 and 2: Ying et al discloses a method of synthesizing a GelMA bioink by mixing a GelMA pre-gel foam comprising 5% or 10% GelMA with a LAP solution comprising 0.5% LAP and 0.5%, 1% or 1.6% PEO with reads on obtaining an ns-GelMA-PEO comprising 5-10% GelMA, 0.1% to 0.5% LAP and 0.5 to 1.6% PEO in mass percentages.
Ying et al does not disclose a method of synthesizing the GelMA pre-gel.
Loessner discloses a method of preparing a GelMA based hydrogel (See abstract). The method of Loessner et al comprises the following steps (See pgs. 19-21; Procedure):
Soak gelatin (type A from porcine skin – See pg. 11; Reagents) to a final concentration of 10% (wt/vol) in demineralized or ultrapure water at RT in a round bottom flask with a magnetic stir bar. Stir the resulting mixture for 10–60 min to facilitate gelatin dissolution. Alternatively, PBS can be used as a solvent for gelatin.
While moderately stirring, heat the mixture to (and keep at) 50 °C in a water bath until the gelatin is fully dissolved and the solution becomes clear.
While stirring vigorously, slowly add 0.6 g of methacrylic anhydride (MAA) per 1 g of dissolved gelatin for a high degree of methacryloyl-functionalization using a glass pipette as organic solvents 19 may dissolve plastic pipette tips (adding slowly with a glass pipette reads on dropwise). Continue stirring vigorously for 60 min. If mixing is sufficient, the solution will turn homogeneously opaque due to dispersion of methacrylic anhydride. Alternatively, this reaction can be run for up to 3 h; however, the reaction time and temperature, as well as the mass ratio of methacrylic anhydride to gelatin are used determine the degree of GelMA functionalization.
After the reaction period, transfer the solution into 50-ml tubes and remove unreacted methacrylic anhydride by centrifugation at 3,500 g for 3 min at RT. Decant the GelMA-containing supernatant and discard the unreacted methacrylic anhydride deposited at the bottom of the 50-ml tubes (opaque and viscous ‘pellet’).
Dilute the supernatant solution with two volumes of pre-heated (40 °C), demineralized or ultrapure water.
Transfer the solution to a dialysis membrane with a 12 kDa molecular weight cut-off and dialyze at 40 °C against a large volume of demineralized (reads on deionized) or ultrapure water for 5–7 days in a chemical safety fume hood.
Adjust the pH of the GelMA solution to 7.4 using 1 M NaHCO3.
In a class II biological safety cabinet, sterile-filter the GelMA solution using 0.2-μm syringe filter units or disposable vacuum filtration units with a PES membrane.
Aliquot the GelMA solution into 50-ml tubes and snap-freeze them in liquid nitrogen.
Transfer all aliquots to the freeze-dryer without allowing the solutions to thaw, and lyophilize until GelMA is fully dehydrated (typically 4–7 days). To maintain a sterile barrier during lyophilization, the 50-ml tubes need to be sealed with vented screw-top caps or press-fitted with 0.2-μm syringe filter units prior to lyophilization.
Step 1 of Loessner et al reads on dissolving type A gelatin in phosphate buffered saline according to a mass/volume ration of 1:(10-20) g/mL to obtain a gelatin solution. Steps 2-3 of Loessner et al read on slowly adding MAA dropwise to the gelatin solution and stirring a resulting mixture at 40° C to 60°C to allow a reaction for 1h to 3h wherein a mass ratio of type A gelatin to MAA is 1:(0.6-0.8). Steps 5-6 of Loessner et al read on subjecting a resulting reaction system to dialysis with deionized water at a 40°C for 3d to 7d to remove unreacted MAA and by-products. Steps 8 of Loessner et al reads on aseptically filtering a purified GelMA monomer solution through a microporous filter membrane and Steps 9-10 of Loessner et al read on lyophilizing a resulting filtrate to obtain the GelMA with a microporous filter membrane that is 0.22µm filter membrane.
Given than Ying et al teaches a method of synthesizing a GelMA bioink comprising a step of mixing GelMA foam and Loessner et al discloses a method of synthesizing a GelMA foam (See pg. 24, step 18, critical step), it would have been prima facie obvious to substitute the GelMA foam of Ying et al with the GelMA foam produced by the method of Loessner et al et al. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
Additionally, Ying et al does not teach the LAP and PEO are mixed in a solvent prepared from 82% PBS, 6% 10x DMEM, 1.2% HEPES, 0.6? GlutaMAX, 9.2% HAM’s F12 medium, and 1% NAOH.
STEMCELL discloses a Collagen medium solution used for culturing of blood vessel organoids (See pg. 11, Sec. 3.6.1) To prepare 4997.7 µL of the collagen medium solution, 626 µl of 5X DMEM (reads on 12% which is equivalent to 6% 10X DMEM), 32 µL GlutaMAX (reads on 0.6%), 795 µL Ham’s F12 (reads on 15.9%), 63µL (reads on 1.2%) HEPES, 49µL sodium bicarbonate, 61 µL sterile-filtered water, and 41.7 µL 1 N sodium hydroxide solution (reads on approximately 1%) are combined with a collagen Solution See Table 2).
Chen et al teaches GelMA hydrogels are advantageous over Collagen hydrogels for culturing vascular networks because collagen-based gels have poor mechanical stability and suboptimal durability compared to GelMA (See pg. 3, first paragraph and figure 7).
Given that STEMCELL teaches a collagen medium solution for culturing blood vessel organoids and Chen et al teaches GelMA hydrogels are advantageous over collagen hydrogels for culturing vascular networks (reads on blood vessels) and Ying and Loessner teach a method of producing a GelMA hydrogel, it would have been prima facie obvious to modify the culture medium of STEMCELL by replacing the collagen with GelMA prepared by the method of Ying et al and Loessner et al. One would have been motivated to replace collagen with GelMA in the culture medium of STEMCELL because Chen teaches GelMA has better mechanical stability and durability than collagen. There is a reasonable expectation of success because Chen teaches GelMA can be used for growing vasculature and blood vessel organoids comprise vasculature.
Additionally, it would have been prima facie obvious to a person of ordinary skill in the art to optimize the amount of PBS and Ham’s F12 in the medium composition in order to optimize cell growth conditions. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Regarding claim 3: Ying et al, Loessner et al, STEMCELL, and Chen et al disclose a method of preparing a GelMA hydrogel according the method of claim 1. Ying further teaches the molecular weight of PEO affects phase separation of the hydrogel.
Ying et al, Loessner et al, STEMCELL, and Chen et al do not teach using PEO with a molecular weight of 7000 kDa to 8000 kDa.
Given that Ying et al teaches phase separation of the hydrogel is affected by the molecular weight of PEO, it would have been prima facie obvious to a person or ordinary skill in the art to optimize the molecular weight of the PEO used to prepare the GelMA-PEO hydrogel in order to optimize the amount of phase separation in the hydrogel and arrive at the claimed range through routine experimentation. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Regarding claim 4: STEMCELL discloses a method of generating blood vessel organoids comprising a step of culturing in a Collagen/Matrigel matrix comprising a collagen medium solution and Matrigel which reads on a method for cultivating BVOs in vitro. To prepare 4997.7 µL of the collagen medium solution, 626 µl of 5X DMEM (reads on 12% which is equivalent to 6% 10X DMEM), 32 µL GlutaMAX (reads on 0.6%), 795 µL Ham’s F12 (reads on 15.9%), 63µL (reads on 1.2%) HEPES, 49µL sodium bicarbonate, 61 µL sterile-filtered water, and 41.7 µL 1 N sodium hydroxide solution (reads on approximately 1%) are combined with a collagen Solution See Table 2).
Chen et al teaches GelMA hydrogels are advantageous over Collagen hydrogels for culturing vascular networks because collagen-based gels have poor mechanical stability and suboptimal durability compared to GelMA (See pg. 3, first paragraph and figure 7).
Loessner discloses a method of preparing a GelMA based hydrogel (See abstract). The method of Loessner et al comprises the following steps (See pgs. 19-21; Procedure):
Soak gelatin (type A from porcine skin – See pg. 11; Reagents) to a final concentration of 10% (wt/vol) in demineralized or ultrapure water at RT in a round bottom flask with a magnetic stir bar. Stir the resulting mixture for 10–60 min to facilitate gelatin dissolution. Alternatively, PBS can be used as a solvent for gelatin.
While moderately stirring, heat the mixture to (and keep at) 50 °C in a water bath until the gelatin is fully dissolved and the solution becomes clear.
While stirring vigorously, slowly add 0.6 g of methacrylic anhydride (MAA) per 1 g of dissolved gelatin for a high degree of methacryloyl-functionalization using a glass pipette as organic solvents 19 may dissolve plastic pipette tips (adding slowly with a glass pipette reads on dropwise). Continue stirring vigorously for 60 min. If mixing is sufficient, the solution will turn homogeneously opaque due to dispersion of methacrylic anhydride. Alternatively, this reaction can be run for up to 3 h; however, the reaction time and temperature, as well as the mass ratio of methacrylic anhydride to gelatin are used determine the degree of GelMA functionalization.
After the reaction period, transfer the solution into 50-ml tubes and remove unreacted methacrylic anhydride by centrifugation at 3,500 g for 3 min at RT. Decant the GelMA-containing supernatant and discard the unreacted methacrylic anhydride deposited at the bottom of the 50-ml tubes (opaque and viscous ‘pellet’).
Dilute the supernatant solution with two volumes of pre-heated (40 °C), demineralized or ultrapure water.
Transfer the solution to a dialysis membrane with a 12 kDa molecular weight cut-off and dialyze at 40 °C against a large volume of demineralized (reads on deionized) or ultrapure water for 5–7 days in a chemical safety fume hood.
Adjust the pH of the GelMA solution to 7.4 using 1 M NaHCO3.
In a class II biological safety cabinet, sterile-filter the GelMA solution using 0.2-μm syringe filter units or disposable vacuum filtration units with a PES membrane.
Aliquot the GelMA solution into 50-ml tubes and snap-freeze them in liquid nitrogen.
Transfer all aliquots to the freeze-dryer without allowing the solutions to thaw, and lyophilize until GelMA is fully dehydrated (typically 4–7 days). To maintain a sterile barrier during lyophilization, the 50-ml tubes need to be sealed with vented screw-top caps or press-fitted with 0.2-μm syringe filter units prior to lyophilization.
Ying et al discloses a method of synthesizing a GelMA bioink by mixing a GelMA pre-gel foam comprising 5% or 10% GelMA with a LAP solution comprising 0.5% LAP and 0.5%, 1% or 1.6% PEO with reads on obtaining an ns-GelMA-PEO comprising 5-10% GelMA, 0.1% to 0.5% LAP and 0.5 to 1.6% PEO in mass percentages.
Given that STEMCELL teaches a collagen medium solution for culturing blood vessel organoids and Chen et al teaches GelMA hydrogels are advantageous over collagen hydrogels for culturing vascular networks (reads on blood vessels) and Ying and Loessner teach a method of producing a GelMA hydrogel, it would have been prima facie obvious to modify the culture medium of STEMCELL by replacing the collagen with GelMA prepared by the method of Ying et al and Loessner et al. One would have been motivated to replace collagen with GelMA in the BVO culture method of STEMCELL because Chen teaches GelMA has better mechanical stability and durability than collagen. There is a reasonable expectation of success because Chen teaches GelMA can be used for growing vasculature and blood vessel organoids comprise vasculature.
Claims 1-5 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Ying et al (Advanced Materials, 2018) in view of Loessner et al (Nature Protocols, 2016), STEMCELL (Generation and Maturation of Blood Vessel Organoids Using STEMdiff Blood Vessel Organoid Kit, May, 2023), Chen et al (Adv Func Mater, 2012), STEMCELL (Coating Plates with Matrigel for Pluripotent Stem Cell culture, 2020), Matsumoto et al (Bioengineering, 2022), Salmon et al (Lab Chip, 2022) and Salewskij et al (Circulation Research, Feb. 2023).
The teachings of Ying et al, Loessner et al, STEMCELL, and Chen et al are set forth above.
Chen anticipates claim 4.
Ying et al, Loessner et al, STEMCELL, and Chen et al render claims 1-4 obvious.
Regarding claims 5 and 7: Following the discussion of claim 4 above, STEMCELL teaches a method of forming aggregates from iPS cells. STEMCELL teaches iPS cells are maintained in mTeSR medium at 37° C, on 6 wells of Matrigel coated dishes (See Sec. 3.3). STEMCELL further teaches the iPS cell cultures should be passaged when they are no more than 70-80% confluent (See Sec. 3.3). The cell aggregate formation method comprises the following steps:
Use a microscope to visually identify regions of differentiation in the ES/iPS culture. Remove regions of differentiation by scraping with a pipette tip or by aspiration.
Aspirate medium from ES/iPS culture.
Add 0.6 mL/well of warm 0.5 mM EDTA (prepared in step 1). Incubate at 37°C and 5% CO2 for 5 minutes.
Carefully remove the plate from the incubator.
Aspirate EDTA. Add 0.6 mL/well of room temperature ACCUTASE™.
Incubate at 37°C and 5% CO2 for 5 minutes.
Monitor cells under the microscope; cells should be round and completely detached from the plate.
Using a 1 mL pipettor, gently resuspend cells by pipetting up and down slowly 3 - 5 times. Transfer the cell suspension from each well to a sterile 50 mL conical tube.
Rinse each well with 1 mL of Aggregate Formation Medium and add this rinse to the tube containing cells.
Count cells using Trypan Blue and a hemocytometer.
Calculate the volume of this cell suspension required to seed 3 wells per density as follows:
2 x 105 and 4 x 105 cells/well in TeSR™-E8™ or mTeSR™1, or
1 x 105 and 2 x 105 cells/well in mTeSR™ Plus
Transfer the calculated volumes to 2 x 15 mL conical tubes. Note: It is recommended to test both cell densities to determine the optimal density for each cell line.
Centrifuge the tubes at 300 x g for 5 minutes.
Remove and discard the supernatant. Add 9 mL of Aggregate Seeding Medium to each tube to resuspend the cells.
For each cell suspension, add 3 mL/well to 3 wells of a 6-well ultra-low adherent plate.
Shake the plate to distribute cells evenly, then incubate at 37°C and 5% CO2. Do not disturb the plate for at least 24 hours.
Observe aggregates under a microscope. Aggregates should reach a diameter of < 50 μM and exhibit round and smooth edges (see Figure 1). Note: If not many aggregates are observed, incubate for one additional day at 37°C and 5% CO2. Do not disturb the plate.
Steps 4-19 occur on Day 0 and Step 20 occurs on Day 1.
Step (1) of claim 5 requires allowing iPSCs to grow adherently on a 1% Matrigel coated 6-well plate until a cell density reaches about 80%. While STEMCELL teaches iPSCs should be grown on 6-wells of a Matrigel coated plate and that the iPSCs should be passaged when they are no more than 80% confluent, STEMCELL does not teach coating 1% Matrigel.
STEMCELL (Coating Plates with Matrigel) teaches Matrigel should be diluted based on protein concentration which should be calculated for each lot.
Therefore, a person of ordinary skill in the art would have found it prima facie obvious to optimize the concentration of Matrigel used to coat plates in the aggregate formation method of STEMCELL and arrive at the claimed concentration of 1% based on the protein concentration of the specific lot used. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II)
Step (1) further requires adding 0.5 mM EDTA-containing digestion solution, incubating the plate in 37° C incubator for 5-8 min, observing cells under an inverted microscope for 95% or more cells becoming rounded, removing the digestion solution and resuspending the cells with a mTeSR1 medium comprising Y-27632 at a ratio of 1:4000 to obtain a cell suspension.
Steps 6-15 of STEMCELL’s aggregate formation method disclose a procedure for dissociating cells in 0.5mM EDTA comprising incubating the cells at 37° for 5 min, visualizing the cells under a microscope until they become round and completely detached (reads on 95% or more) from the plate, aspirating the EDTA and resuspending the cells in a culture TeSR-E8, mTeSR1, or mTeSR Plus.
STEMCELL does not teach the mTeSR1 medium comprises Y-27632 at a ratio of 1:4000
Matsumoto teaches long-term exposure to ROCK inhibitor Y-27632 enhances growth abilities of hiPSCs (See abstract).
Given that STEMCELL teaches a method of growing iPSC aggregates and Matsumoto teaches exposure to Y-27632 enhances growth if iPSCs it would have been prima facie obvious to modify the mTeSR medium of STEMCELL by adding Y-27632 in the method of STEMCELL. One would have been motivated to add Y-27632 to the mTeSR medium of STEMCELL because STEMCELL teaches a method for forming iPSC aggregates and Matsumoto teaches Y-27632 enhances growth of iPSCs. There is a reasonable expectation of success because Matsumoto teaches Y-27632 enhances growth of iPSCs and STEMCELL is culturing iPSCs.
Additionally, it would have been prima facie obvious to optimize the ratio of Y-27632 in the culture medium in order to improve growth of the hiPSCs and arrive at the claimed ratio of 1:4000 through routine experimentation. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Step (1) of claim 5 further requires inoculating the cell suspension into a Matrigel coated 6-well plate and cultivating the cells in a 37° C and 5% CO2 incubator, during which the original medium is replaced with a mTeSR1 complete medium on day 2 to remove unadherent cells.
Steps 14-20 of STEMCELL disclose steps comprising suspending cells in mTeSR and adding the cell suspension to 3 wells of a 6-well plate then incubating the cells at 37°C and 5% CO2. STEMCELL further teaches iPSCs should be cultured on Matrigel coated culture ware. Thus STEMCELL teaches replacing the original medium with mTeSR1 and inoculating the cell suspension into a 6-well plate and cultivating the cells in a 37° C and 5% CO2 incubator.
STEMCELL does not disclose the culture medium is changed on day 2. However, given that STEMCELL teaches steps of monitoring cells and changing culture medium and further teaches cells should be passaged at 70-80% confluency it would have been prima facie obvious to optimize the time period between culture medium changes to arrive at day 2 through routine experimentation based on cell confluency and monitoring of growth conditions. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Step (2) of claim 5 requires steps of adding 0.5 mM EDTA at 0.6mL/well to 1 mL/well and removing EDTA step 6 of the aggregate formation method of STEMCELL reads on this step. Step (2) further requires adding a marine-origin enzyme for cell dissociation at 0.6 mL/well to 1 mL/well and incubating the plate in a 37° C incubator for 3 to 5 min. ¶0019 of the specification of the instant application discloses Accutase as the marine origin enzyme used in this step. Therefore, steps 8 and 9 of the STEMCELL aggregate formation protocol read on this step. Next, Step (2) requires pipetting cells up and down when they become rounded and brightened to obtain single cells then resuspending the single cells with a cell aggregation medium. Seps 10-12 of STEMCELL read on this portion of step (2). Step (2) further requires inoculating the single cells into an ultra-low attachment 6-well plate at a cell density of 2 x105 to 5 x 105/well and cultivating the single cells in a 37°C and 5% CO2 incubator for 1-3 days to obtain spherical iPSC aggregates each with a smooth surface and a diameter of 50µm to 100µm. Steps 14-20 of STEMCELL read on these steps.
The BVO method of STEMCELL further comprises steps of Mesoderm Induction on days 1-4 followed by Vascular Induction on Days 4-6 (See Secs. 3.4 and 3.5).
STEMCELL does not disclose a mesoderm induction step comprising the limitations of Step (3) of claim 5.
Salmon et al discloses a method of differentiating hPSCs to Vascular cells comprising and intermediate step of mesoderm induction (See Sec. Vascular mix differentiation). The mesoderm induction step of Salmon et al comprises adding N2B27 medium supplemented with 12µm CHIR99021 and 30 ng/mL BMP4 and culturing for 3 days. The method further comprises changing the medium to vascular differentiation medium comprising 100 ng/mL VEGF-A and 2µM Forskolin on day 4. On day 6 the cells were collected and embedded in Matrigel (See Sec. Vascular mix differentiation). Salmon further teaches VEGF-A induces sprouting of vasculature (See Sec. Vascular mix differentiation).
Given that STEMCELL discloses a method of producing BVO from iPSCs with an intermediate steps of mesoderm induction and Salmon et al discloses mesoderm induction method, it would have been prima facie obvious to substitute the mesoderm induction steps of STEMCELL with the mesoderm induction method of Salmon et al in the BVO production method of STEMCELL. One would have expected the mesoderm induction method of Salmon et al to work equivocally with the mesoderm induction steps of STEMCELL in the method of STEMCELL because both methods/steps teach mesoderm induction from pluripotent stem cells. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
The method of Salmon et al differs from Step (3) of claim 5 in that Salmon uses 100 ng/mL of VEGF-A whereas step (3) of claim 5 uses 10 ng/mL of VEGF-A. However, given that Salmon et al discloses VEGF-A induces sprouting of vasculature, it would have been prima facie obvious to optimize the amount of VEGF-A in the method of Salmon et al to arrive at the claimed amount of 10 ng/mL through routine experimentation, in order to optimize the amount of vasculature sprouting. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Additionally, while Salmon discloses performing incubation steps at 37° C and STEMCELL discloses performing incubation steps at 37° C and 5% CO2. Therefore the culturing steps should be performed at 37° and 5% CO2.
Further regarding Step (3) of claim 5, it would have been prima facie obvious to use GelMA-PEO instead of collagen in the blood vessel network formation step (Step 3.6; Days 6-11) of STEMCELL and using the GelMA-PEO medium to embed cell aggregates.(See rejection of claim 4 above). The GelMA-PEO production method of Ying et al and Loessner et al comprises a step of UV crosslinking for 15s. However, it would have been prima facie obvious to optimize the length of UV exposure to arrive at the claimed time of 30 sec through routine experimentation in order to optimize the amount of polymerization in the hydrogel. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Further regarding step (3) of claim 5, STEMCELL discloses blood vessel network sprouting steps comprising steps of adding a maturation medium and incubating at 37°C and 5% CO2 for 2 days which reads on cultivating the iPSC aggregates in 37°C and 5% CO2 for 1-3 days to allow vascular budding to form a vascular network (See Sec. 3.6.3 step 2).
STEMCELL does not disclose the medium used in the blood vessel network sprouting steps is StemPro-34 SFM.
Salewskij et al teaches a medium composed of StemPro-34 SFM, 100 ng/mL VEGF-A, 100 ng/mL FGF2, and 15% FCS (reads on FBS) is a commonly used final medium used for culturing blood vessel organoids which reads on the limitations of claim 7 (See Table 1).
Therefore, it would have been prima facie obvious to substitute the maturation medium of STEMCELL with a medium composed of StemPro-34 SFM, 100 ng/mL VEGF-A, 100 ng/mL FGF2, and 15% FCS in the blood vessel network sprouting steps of STEMCELL. One would have expected the culture mediums to work equivocally in the method of STEMPRO because Salewskij et al teaches a medium composed of StemPro-34 SFM, 100 ng/mL VEGF-A, 100 ng/mL FGF2, and 15% FCS is commonly used for culturing blood vessel organoids. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
Claims 1-7 are rejected under 35 U.S.C. 103 as being unpatentable over Ying et al (Advanced Materials, 2018) in view of Loessner et al (Nature Protocols, 2016), STEMCELL (Generation and Maturation of Blood Vessel Organoids Using STEMdiff Blood Vessel Organoid Kit, May, 2023), Chen et al (Adv Func Mater, 2012), STEMCELL (Coating Plates with Matrigel for Pluripotent Stem Cell culture, 2020), Matsumoto et al (Bioengineering, 2022), Salmon et al (Lab Chip, 2022), Salewskij et al (Circulation Research, Feb. 2023), and Guo (WO2025006679A2, priority filing date: 06/2023).
The teachings of Ying et al, Loessner et al, STEMCELL, Chen et al, STEMCELL (Coating Plates with Matrigel), Matsumoto et al, Salmon et al, and Salewskij et al, are set forth above.
Chen anticipates claim 4.
Ying et al, Loessner et al, STEMCELL, and Chen et al render claims 1-4 obvious.
Ying et al, Loessner et al, STEMCELL, Chen et al, STEMCELL (Coating Plates with Matrigel), Matsumoto et al, Salmon et al, and Salewskij et al render claims 1-5 and 7 obvious.
Regarding claim 6: Following the discussion of claims 4 and 5 above, STEMCELL discloses a method of forming cell culture aggregates comprising adding cell aggregation medium.
STEMCELL does not disclose the cell aggregation medium is prepared from the following substance in volume percentages: 77% KnockOut DMEM/F12 medium 19% KnockOut serum replacement, 1% of a dipeptide additive of L-alanyl-L-glutamine, 1% NEAA additive, 1% 2-mercaptoethanol dilution with a dilution ratio of 1:100, and 1% penicillin-streptomycin.
Guo discloses a method of generating blood vessel organoids from iPSCs (See Example1, ¶0425-0435). The method of Guo comprises using an aggregation medium composed of 77% DMEM/F12, 99µM ß-mercaptoethanol (reads on 2-mercaptoethanol), 20% Knockout Serum Replacement, 1x GlutaMAX (reads on a dipeptide additive of L-alanyl-L-glutamine, 1x NEAA, and 1x penicillin-streptomycin (See ¶0429 and ¶0439).
Given that both Guo and STEMCELL disclose aggregation mediums used for differentiating iPSCs to BVOs it would have been prima facie obvious to substitute the aggregation medium of STEMCELL with the aggregation medium of Guo, in the method of STEMCELL. One would have expected the aggregation medium of Guo to work equivocally with the aggregation medium of STEMCELL in the method of STEMCELL because both mediums are aggregation mediums used in BVO production from iPSCs. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
Additionally, while the amounts of each component are not of the aggregation medium are not the same as the amounts required by claim 6, it would have been prima facie obvious to a person of ordinary skill in the art to optimize the concentrations of each of the components of the aggregation culture medium and arrive at the claimed amounts through routine experimentation to optimize viability and growth conditions. Where the general conditions of a claim are disclosed in the prior art it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Conclusion
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/MARISOL ANN O'NEILL/ Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633