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 .
Election/Restrictions
This action is in response to the papers filed on 08/07/2025. Claims 1-10 are currently pending as per claims filed on 08/07/2025. Claims 5 and 7-9 have been amended as per claims filed on 08/07/2025. Claims 1-4 have been withdrawn from consideration by Applicants’ amendment filed on 8/7/2025.
Applicant’s election without traverse of Group II, which include claims 5-10, drawn to a method of 3D culture in the reply filed on 08/07/2025 is acknowledged. Claims 1-4 have been withdrawn from further consideration by Applicants, pursuant to 37 CFR 1.142(b), as being drawn to non-elected inventions, there being no allowable generic or linking claim. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.03(a)). Claims that were drawn to a non-elected invention would have been withdrawn, as being directed to a non-elected invention. Reinstatement of claims drawn to non-elected inventions will be withdrawn during prosecution.
The requirement for restriction between Groups I-II is still deemed proper and is therefore made FINAL.
Therefore, claims 5-10 are subject to examination to which the following grounds of rejection are applicable.
Priority
The instant application is a 371 of PCT/CN2020/111261 filed on 08/26/2020. Thus, the earliest possible priority for the instant application is 08/26/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 07/20/2022 was filed before the mailing date of the non-final office action. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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 5-10 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 5, step (e) recites the phrase “arranging a Petri dish at a cryogenic component, adding the mixed solution into wells of the Petri dish, and allowing the mixed solution to stand for 1-7 minutes to form gel.”. Multiple “mixed solutions” are described in claim 5. Therefore, it is unclear which mixed solution is being referenced, which renders the claim indefinite. Applicant must amend the claim to specify which mixed solution is to be added in step (e).
Claims 6-10 inherit these deficiencies insofar as they depend from claim 5.
Claim 5 is indefinite in its recitation of “arranging a Petri dish at a cryogenic component”. The phrase “arranging a Petri dish at a cryogenic component” is not defined by the claim, the specification does not provide a standard for ascertaining how a Petri dish is arranged at a cryogenic component, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Appropriate action is required.
Claim 5 is indefinite in its recitation of “arranging the Petri dish at a temperature required until the cells form spheroids in the gel”. The term " required " in claim 5 is a relative term which renders the claim indefinite. What temperature conditions are considered "required" varies widely in the art depending on the individual situation as well as the person making the determination. Although it is acknowledged the specification some temperature conditions are considered by applicant to be "required” (¶ [0031], ¶ [0032] of the published application ), these are merely exemplary and non-limiting. The metes and bounds of the claims are unclear particularly since a temperature required to form spheroids would vary depending on cell type, growth medium, days of cell in culture, and conditions. As such, the metes and bounds of the claims cannot be determined.
Claim 8 is indefinite in its recitation of “washing dissolved gel solution with the phosphate buffer solution”. The practitioner in the art would readily understand that a solution cannot be used to wash another solution but to dilute another solution. As such the metes and bounds of the claim are indefinite.
Claim 9 recites the phrase “…wherein the cryogenic component in the step (e) is obtained by a thermal conductive sheet placed and cooled on a cryogenic device; wherein temperature required is 30-37°C…”. Step (e) does not recite a required temperature. Therefore, the claim 9 is indefinite for lack of antecedent basis. See MPEP § 2173.05(e) Lack of Antecedent Basis [R-08.2017] “A claim is indefinite when it contains words or phrases whose meaning is unclear. In re Packard, 751 F.3d 1307, 1314 (Fed. Cir. 2014). The lack of clarity could arise where a claim refers to “said lever” or “the lever,” where the claim contains no earlier recitation or limitation of a lever … “.
Further, it is unclear what component of step 9 the recitation “wherein temperature required is 30-37°C…” is meant to encompass. Is this meant for the entire apparatus, a single component of the apparatus such as the “thermal conductive sheet”, cells recited within the instant claims? The range of temperature recited in the instant claim 9 includes room temperature and near room temperature values. The purpose of the thermal conductive sheet cooled on a cryogenic device would be to displace heat from the petri dish, therefore it would be expected the temperature of the sheet, device, and cells interacting with the device would be below room temperature. Therefore, the recitation of claim 9 is unclear as to what the intended function and relationship of the thermal conductive sheet cooled on a cryogenic device is to the petri dish. The specification describes “The temperature required in the step (e) means preferred temperature at which the cells are cultured.” (Pg. 10, Line 13-15 of the instant specification). However, the specification cannot breathe limitations into the claims. Therefore, the claim as written lacks clarity which renders the claim indefinite. It would be remedial for the applicant to clarify within the claim what exactly is meant to be at the “preferred temperature”.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claim(s) 5-6 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Colosi et al. (Colosi C, Adv Mater. 2016; hereafter “Colosi”) in view of Horiguchi et al. (Horiguchi I, J Vis Exp. 2015; hereafter “Horiguchi”) as evidenced by Sigma Aldrich (https://www.sigmaaldrich.com/US/en/product/sial/e7889, accessed 12/9/2025).
Regarding Claim 5 (a) – (b), Colosi teaches optimization and production of a bioink material that is a gel composition for the purpose of 3D cell culture (Pg. 4, final paragraph transitioning to Pg. 5) containing a first gel material that includes the following: a gelatin (GelMA, 4.5% w/v), alginate (from 1.0 to 4.0% w/v), HEPES, and water. Colosi et al. teaches a second mixture (crosslinking solution) containing a divalent cation (0.3M CaCl2) and HEPES prepared in water (Pg. 4, 2nd full paragraph). These mixtures read on the solutions recited in the instant claim step (a) and (b).
Regarding Claim 5 (c), Colosi teaches removal of Ca2+ to dissolve alginate, but does not specifically teach the making of a buffer solution D (EDTA + NaOH + HEPES + Pure Water).
Horiguchi teaches a 10mM EDTA solution prepared by mixing HEPES-buffered saline prepared in 1.1 (containing NAOH) and adjusting the pH to 7.0 at RT (Pg. 1, Preparing Materials section.) Disodium EDTA is the EDTA form recited in step (c). Disodium EDTA is a sodium salt form of EDTA prepared from EDTA as evidenced by Sigma-Aldrich (Description Section; Preparation note). As EDTA is insoluble in water, one of ordinary skill would understand a reference to EDTA as a chelating agent within a protocol step of dissolving a gelatin-alginate would require the salt form of EDTA.
Regarding Claim 5 (d), Colosi teaches cells grown and then mixed with the gel material “…in which the bioink mixture containing: alginate, GelMA, …and cells” (Pg. 4, 2nd Paragraph) and further teaches cells (HUVECs) grown in a growth medium and then integrated within the gel material described above in step (a) (Supplemental Information, Materials and Methods, Cell Culture section).
Colosi does not teach cells in a volume ratio of 1:1 in the gel of step (d).
One of ordinary skill in the art would understand that the cells would be suspended in the cell growth medium during a routine protocol of cell plating, growth, expansion, and collection. Moreover, a person of ordinary skill in the art would have been motivated to determine an appropriate concentration for the ratio of cell volume suspension to mix with a gel mixture through routine optimization within a 3d culture method for cells grown in a hydrogel to effectively achieve a desired population of viable cells.
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.") See MPEP 2144.05 II. A.
Regarding Claim 5 (e), Colosi teaches an understanding of thermal gelation control, “ The presence of alginate also prevents the spontaneous thermal gelation of GelMA, so that the bioink maintains its low viscosity for prolonged periods of time at room temperature.” (Pg. 5, 1st full sentence).
However, Colosi does not specifically teach arranging a Petri dish at a cryogenic component, adding the mixed solution into wells of the Petri dish, and allowing the mixed solution to stand for 1-7 minutes to form gel.
Horiguchi also teaches thermal gelation control within their method for stable alginate encapsulation that incorporates nitrogen gas flow for forming droplets, eliminating the need for additional reagents such as oil. (Pg. 1, Introduction, 3rd Paragraph).
Neither Colosi nor Horiguchi teach allowing the mixed solution to stand for a time frame of 1-7 minutes to form a gel. However, a person of ordinary skill in the art would have been motivated to determine an appropriate timeframe for the solution to form into a gel for use in a hydrogel culture protocol through routine optimization absent evidence to the contrary. See MPEP 2144.05 II. A.
Regarding Claim 5 (f), Colosi teaches a cross-linking solution which reads on the buffer solution C recited in the instant claim step (b). Colosi teaches “When the ionic crosslinking solution came in contact with the bioink mixture, the alginate was ionically crosslinked resulting in hydrogel microfibers (Figure 1c).” (Pg. 4, 2nd Paragraph). Colosi does not specifically describe the duration of the cross linking step to be 10-20 minutes.
However, Horiguchi teaches crosslinking solution to be provided to the gel solution for 10-20 minutes (Pg. 2, Section 2, step 4). Regarding Claim 5 (g), Colosi teaches removal (washing) of crosslinking solution (Figure 1a). Colosi teaches cells grown within 3d culture for at least 3 days (Figure 3a.) and 10 days (Figure 4a), but does not specifically teach a growth medium. However, one of ordinary skill in the art familiar with cell culture would understand the need for a growth medium to obtain a growing and viable cell culture.
Moreover, Horiguchi teaches that after washing cells encapsulated in hydrogel, DMEM with 10% FBS was added. This growth media further serves to remove electrical charge on the hydrogel surfaces (Pg. 2, Section 3, Step 2).
Regarding Claim 5 (h), step (h) recites, “arranging the Petri dish at a temperature required until the cells form spheroids in the gel while the growth medium in the Petri dish needs to be replaced once a day.”
Colosi teaches cells encapsulated in gel to form and retain their shape as spheroids,
“Interestingly, cells embedded in the printed constructs exposed to shorter UV crosslinking times (< 25sec) retained a spherical shape after 10 days of culture (Figure 3f, Figure S3).” (Pg. 6, final paragraph). Therefore, if the cells form spheroids in the gel, then the mixture being in the temperature required to form a spheroid was intrinsically present.
It would have been obvious to a person of ordinary skill in the art, at the time of the invention, to have modified the method, as previously disclosed by Colosi, to include the following teachings disclosed by Horiguchi: for step (c), a formulation shown to be effective at dissolving alginate based gels to isolate the cells within, the use of nitrogen gas as a cryogenic component for step (e), and to incubate gels with crosslinking solution 10-20 minutes in step (f).
One would have been motivated to combine these teachings as both Colosi and Horiguchi are focused on establishing a successful 3D culture of cells with a gelatin hydrogel. As the additionally described steps taken by Horiguchi were shown to successfully establish a stable gelatin encapsulation for cells, an individual in of ordinary skill in the art seeking to enhance the 3d culture method of Colosi would incorporate known elements in the art to improve the outcome of the protocol. There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results. The motivation to combine can arise from the expectation that the prior art elements will perform their expected functions to achieve their expected results when combined for their commonly known purpose. MPEP §2144.
Regarding Claim 6, Colosi and Horiguchi together render obvious the method of claim 5, as iterated above, the content of which is incorporated herein, in its entirety. Moreover, Colosi teaches a first gel material containing a gelatin (GelMA, 4.5% w/v), alginate (from 1.0 to 4.0% w/v), HEPES, and water. Colosi also teaches a second mixture (crosslinking solution) containing a divalent cation (0.3M CaCl2) and HEPES prepared in water (Pg. 4, 2nd full paragraph). Note that claim 6 requires the same percentages of for all the components of a gel material A and a buffer solution C as recited in claim 5 except the % of sodium alginate which is 0.5-2 wt% rather than 0.5-3 wt%.
However, Colosi does not specify the amount of HEPES used in the preparations.
A person of ordinary skill in the art would have been motivated to determine an appropriate concentration for HEPES within the solution through routine optimization to determine the amount required to maintain the pH necessary for 3D hydrogel culture protocols.
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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 MPEP 2144.05 II. A.
Regarding Claim 10, Colosi teaches a crosslinking solution containing a divalent cation (0.3M CaCl2) and HEPES prepared in water (Pg. 4, 2nd full paragraph).
Claim(s) 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over Colosi et al. (Colosi C, Adv Mater. 2016; hereafter “Colosi”) in view of Horiguchi et al. (Horiguchi I, J Vis Exp. 2015; hereafter “Horiguchi”) as applied to claims 5-6 above, in view of Chen et al. (Chen Q, Molecules. 2020; hereafter “Chen”).
Regarding Claim 7-8, Colosi and Horiguchi together render obvious the method of claim 5 and 6, as iterated above, the content of which is incorporated herein, in its entirety. Moreover, Horiguchi teaches the use of a buffer reading on solution D of the instant claims and teaches the cells in gelatin-alginate capsules are incubated in this solution for 5 minutes (Pg. 2, Section 4) as recited in instant claim 7. Horiguchi also teaches collection of cells after centrifugation (Pg. 2, Section 4). One of ordinary skill in the art would understand intrinsically that separation of sample (cells) for analysis means separation from the supernatant after centrifugation.
The washing steps of Colosi and Horiguchi use buffer solution other than PBS. However, the use of PBS as a wash buffer for sodium alginate 3d culture protocols was well known in the art at the time of the instant application.
Chen teaches preparation of alginate/gelatin hydrogels with improved
interpenetrating networks for in vitro 3D cell cultures and organ bioprinting (Abstract; Introduction, final paragraph). Chen teaches the use of DPBS, a species of PBS, used throughout their protocol as a wash buffer suitable for this methodology (Pg. 15, Section 4.7).
It would have been obvious to a person of ordinary skill in the art, at the time of the instant application, to have modified the combined teachings of Colosi and Horiguchi to optimize the protocol by determining whether a HEPES-buffered solution or a PBS buffered solution as taught by Chen would be preferable for a washing buffer in this culture methodology. As Chen specifically teaches an optimization strategy to improve the existing protocols of alginate-gelatin hydrogel culture, one would be motivated to include the teachings of Chen, such as use of a PBS buffered solution as a wash buffer.
There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results.
Claim 9 are rejected under 35 U.S.C. 103 as being unpatentable over Colosi et al. (Colosi C, Adv Mater. 2016; hereafter “Colosi”) in view of Horiguchi et al. (Horiguchi I, J Vis Exp. 2015; hereafter “Horiguchi”) as applied to claim 5-6 above, in view of Tan et al. (Tan Z, Sci Rep. 2017; hereafter “Tan”).
Regarding Claim 9, Colosi and Horiguchi together render obvious the method of claim 5, as iterated above, the content of which is incorporated herein, in its entirety. Horiguchi teaches cell density within the gel is desirable to be more than 106. Colosi and Horiguchi do not teach the cryogenic component in the step [e] is obtained by a thermal conductive sheet placed and cooled on a cryogenic device.
Tan teaches the use of a solid carbon dioxide (dry ice) and an isopropanol thermal conductive bath that was used to achieve the cryogenic stage, which is a safer alternative to liquid nitrogen (Introduction, 5th Paragraph). This provides a time-reducing method, owing to the instantaneous freezing step (Pg. 7, 7th full Paragraph). The setup includes a stainless-steel bed atop of a thermal conductive bath on which the gel material is placed (Figure 4.). The instant claims do not specify what constitutes a thermal conductive sheet, however the instant specification provides a non-limiting example of the thermal conductive sheet to be a metal sheet cooled on a cryogenic device (Pg. 9, Lines 15-20 of instant specification). It would have been obvious to a person of ordinary skill in the art, at the time of the instant application, to have modified the combined teachings of Colosi and Horiguchi to optimize the protocol from the use of nitrogen gas as taught by Colosi to instead use the thermal conductive bath as taught by Tan et al. One would be motivated to make use of the Tan’s method as Tan teaches it to be a safer alternative to other methods and provides a time-reducing method.
There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results.
Conclusion
Claims 5-10 are rejected. No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KODYE LEE ABBOTT whose telephone number is (703)756-1111. The examiner can normally be reached M-F 8-5.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Maria G. Leavitt can be reached at (571) 272-1085. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KODYE LEE ABBOTT/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634