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
Applicant’s election without traverse of Group I, drawn to a method of recovering cells cultured on a substrate in the reply filed on October 17, 2025 is acknowledged.
Claims 12-13 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-11 are examined on the merits.
Priority
Applicant claims foreign priority to Japanese patent applications JP2022-040723 filed on March 15, 2022 and JP2022-197317 filed on December 9, 2022. Receipt is acknowledged of certified copies of papers received in the original language required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on February 24, 2023 is in compliance with the provisions of 37 CFR 1.97 and 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 1-11 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 recites the limitation "the cells singulated…" in line 7. There is insufficient antecedent basis for this limitation in the claim as there is no previous mention a population of singulated cells. Appropriate correction is required. It is recommended that Applicant amend with language such as “obtaining singulated cells”.
Claims 2-11 which depend from claim 1 are similarly rejected for incorporating the limitation of a rejected claim while failing to correct the deficiency.
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.
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.
Claims 1 and 8-11 are rejected under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. (JP 4775218B2, found in IDS dated 2/24/2023, Espacenet translation attached) and Kurashina et al. (2019). Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves. Communications biology, 2(1), 393, found in IDS dated 2/24/2023, hereafter “Kurashina”).
With regard to claim 1, Chiyoda Corp. teaches a closed system cell culture device capable of subculturing adhesion-depended cells and a method for detaching and dispersing cells (Para. [0001]), the method comprising supplying a detachment solution that reduces cell adhesiveness of cultured cells (Para. [0019], lines 2-3) wherein the detachment solution is a chelating solution for a divalent metal (Para. [0020]), which is considered to reasonably read on a detachment solution containing a metal ion chelating agent. Chiyoda Corp. additionally teaches that the method comprises a detachment step of vibrating the culture vessel horizontally (Para. [0022]) and subsequently where the cells “in which the adhesion between cells…has been released” are released into a cell collection containers (Para. [0026], lines 4-5), which is considered to reasonably read on obtaining separated cells from the detached cells.
While Chiyoda Corp. does teach use of vibration to detach cells via shear stress (Para. [0040], lines 2-3), Chiyoda Corp. does not specifically teach ultrasonic vibration.
Kurashina teaches use of ultrasonic waves in a method of detaching cells from a culture substrate without use of trypsin (Abstract). Kurashina teaches that the ultrasonic vibration generates forces, specifically the combination of acoustic pressure and sloshing, which are responsible for increased cell detachment (Pg. 8, left col., 2nd para.) over horizontal fluid motion alone (Pg 7, right col., last para., lines 6-8). Kurashina further teaches that use of ultrasonic vibration for detachment instead of trypsin detaches a comparable number of cells but reduces cell damage and improves cell survival (Abstract).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to apply ultrasonic vibration as taught by Kurashina as a detachment mechanism to the method of recovering cells from cell culture comprising use of a chelating agent and horizontal vibration as taught by Chiyoda Corp. with a reasonable expectation of success. A skilled artisan would have been motivated to make this combination in order to increase cell detachment thereby increasing yield from subsequent culturing as Kurashina teaches that use of ultrasonic vibration results in superior cellular detachment.
With regard to claim 8, Chiyoda Corp. teaches wherein the chelating agent in the detachment solution is 4mM EDTA (Para. [0079]).
With regard to claim 9, Chiyoda Corp. teaches that using proteases can cause negative effects on cells including degradation/destruction of cell surface markers, channels, and receptors (Para. [0003]) and that use of proteases should be avoided in culturing cells for medical purposes as proteases can be contaminated with prions/viruses due to their isolation from living organisms. Thus, a skilled artisan would be motivated to use a protease-free detachment solution in order to avoid harmful effects on cells and potential contaminants which may limit the future applications of the cultured cells.
With regard to claim 10, as detailed above, the combined teachings of Chiyoda Corp. and Kurashina teach a method of cellular detachment from a substrate comprising detaching cells using application of a chelating agent and ultrasonic vibration before collection of cells which have been separated. Chiyoda teaches exemplary embodiments of the method using HepG2 cells and HIT-T15 cells.
Chiyoda Corp. does not teach wherein the cells are stem cells.
Kurashina teaches wherein the method of cellular detachment via ultrasonic vibration in a trypsin-free system was performed using human mesenchymal stem cells (Pg. 10, Culturing of multiple cell types).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention to choose mesenchymal stem cells as taught by Kurashina for use in the method of recovery of cultured cells using detachment via use of a metal chelating agent and ultrasonic vibration as taught by the combined teachings of Chiyoda Corp. and Kurashina with a reasonable expectation of success. A skilled artisan would have been motivated to choose mesenchymal stem cells for use in the method of cellular detachment which reduces cell damage resulting in order to generate larger numbers of healthy stem cells for use in tissue engineering or other clinical applications (p. 2, 1st para. of Kurashina).
With regard to claim 11, Chiyoda Corp. teaches wherein cell detachment is performed at 37°C (Example 2, see Para. [0085]).
Claims 2-3 are rejected under 35 U.S.C. 103 as being unpatentable over over Chiyoda Corp. and Kurashina as applied to claim 1 above, and further in view of Qiu et al. (2018, Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells. Lab on a Chip, 18(18), 2776-2786, hereafter “Qiu”).
With regard to claims 2 and 3, as detailed above an incorporated herein, the combined teachings of Chiyoda Corp. and Kurashina teach a method of detaching cells from a culture substrate comprising application of a chelating agent and ultrasonic vibration followed by collection of the separated cells.
Chiyoda Corp. does not teach a step of passing dissociated cells through a mesh filter having a size of 20 µm or more to 40 µm or less.
Qui teaches a method of dissociating cellular aggregates into single cells via use of mesh filters in a microfluidic device (Abstract) which can be used to remove cellular aggregates produced by standard enzymatic digestion procedures (Pg. 2777, right col, last para.). Qui teaches investigation of single cell recovery and viability for several filter sizes including 25 µm (Pg. 2779, left col, last para. and Table 1) showing that filer size of 25 µm was effective as dissociating cells without affecting cell viability (Fig. 2).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention to combine and choose the 25 µm filter size used to dissociate cells as taught by Qui in the method of recovering cultured cells as taught by the combination of Chiyoda Corp. and Kurashina with a reasonable expectation of success. A skilled artisan would have been motivated to make this combination in order to choose a mesh filter size which is most effective for dissociating cellular aggregates into single cells while still maintaining high cell viability which is important for downstream applications of dissociated cell cultures.
Claims 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. and Kurashina as applied to claim 1 above, and further in view of Hutcheson et al. (2010, Saving cells from ultrasound-induced apoptosis: quantification of cell death and uptake following sonication and effects of targeted calcium chelation. Ultrasound in Med. & Bio., 36(6), 1008-1021, hereafter “Hutcheson”).
With regard to claim 4, as detailed above and incorporated herein, combined teachings of Chiyoda Corp. and Kurashina teach a method of detaching cells from a culture substrate comprising application of a chelating agent and ultrasonic vibration followed by collection of the separated cells. Chiyoda Corp. teaches wherein the detached cells are passed through a thin tube which breaks adhesion between cells to allow recovery of separated cells (Para. [0032], lines 2-4) via use of shear stress (Para. [0033], line 5).
Although Chiyoda Corp. teaches a chelating agent concentration of 4mM EDTA (Para. [0079]), Chiyoda Corp. does not teach adjustment of the chelating agent concentration after application of ultrasonic vibration.
Hutcheson teaches that application of ultrasound to cells can cause apoptotic cell death hours after sonication and that this apoptosis can occur due to cellular membrane damage which allows intracellular influx of Ca2+ (Pg. 1009, left col., 1st full para.). Hutcheson teaches that application of the Ca2+ chelator, which is considered to reasonably read on a metal ion chelating agent, following ultrasound application reduces apoptosis and results in increased cellular viability (Abstract).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to apply thel chelating agent after ultrasonic vibration as taught by Hutcheson to the method of recovering cultured cells comprising detachment using the chelating agent and ultrasonic vibration as taught by the combination of Chiyoda Corp and Kurashina with a reasonable expectation of success. Since Hutcheson teaches that post-ultrasound application of a metal ion chelating agent reduces ultrasound-induced apoptosis, a skilled artisan would have been motivated to make this combination in order to increase cell viability after ultrasound application. As Chiyoda Corp. already teaches a concentration of 4mM EDTA, and EDTA was a known calcium chelator, a skilled artisan would easily be able to arrive at a metal ion chelating agent concentration which is 1.0mM or greater.
With regard to claim 5, Chiyoda Corp. teaches that the shear stress is due to passage of the cells through syringe connected to a capillary tube (Para. [0033]).
Claim 6 are rejected under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp., Kurashina and Hutcheson, as applied to claims 1, 4, and 5 above, and further in view of Laermer et al. (US 2021/0114024)
With regard to claim 6, as detailed above and incorporated herein, combined teachings of Chiyoda Corp. and Kurashina and Hutcheson teach a method of detaching cells from a culture substrate comprising application of a chelating agent and ultrasonic vibration followed by collection of the separated cells. Chiyoda Corp. teaches wherein the detached cells are passed through a thin tube which breaks adhesion between cells to allow recovery of separated cells (Para. [0032], lines 2-4) via use of shear stress (Para. [0033], line 5).
Although Chiyoda Corp. teaches a syringe to apply the shear force, Chiyoda Corp. does not teach a pipette and pipette tip to apply the shear force.
Laermer teaches that application of tools of similar function such as syringes or pipettes in a microfluidic device for handling cells [0008].
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the syringe taught by Chiyoda with the pipette taught by Laermer with a reasonable expectation of success. Since Laermer teaches syringes and pipettes serve similar function, one of ordinary skill in the art could have substituted one known element for another, and the results of the substitution would have been predictable.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. and Kurashina as applied to claim 1 above, and further in view of Gigout et al. (2005, Low calcium levels in serum-free media maintain chondrocyte phenotype in monolayer culture and reduce chondrocyte aggregation in suspension culture. Osteoarthritis and cartilage, 13(11), 1012-1024, hereafter “Gigout”).
With regard to claim 7, as detailed above and incorporated herein, the combined teachings of Chiyoda Corp. and Kurashina teach a method of detaching cells from a culture substrate comprising application of a chelating agent and ultrasonic vibration followed by collection of the separated cells. Chiyoda Corp. teaches an additional step of application of a calcium-containing solution (Para. [0038], [0049], Para. [0066]) such as culture medium in order to replenish calcium lost by the stripping solution and to strengthen and protect cellular membranes which had been damaged by application of the detachment solution (Paras. [0038], [0049], [0069]).
Chiyoda Corp. does not teach addition of serum to the cell suspension comprising detached cells.
Gigout teaches use of serum in cell culture (Abstract) and that serum contains 4mM of calcium (Pg. 1013, left col., 1st para.).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date, to choose serum as taught by Gigout in the calcium-containing solution to be applied to repair cellular damage caused by detachment with a chelating agent as taught by Chiyoda Corp. with a reasonable expectation of success. A skilled artisan would have been motivated to make this combination as serum is a normal component of cell culture medium and is widely used and commonly available to those skilled in the art. Thus, one having ordinary skill in the art of cell culture is likely to already have serum either alone or in prepared cell culture media.
In regard to the order of the steps, although Chiyoda Corp. details a method wherein the calcium containing solution is applied after application of the detachment solution and prior to vibration (Paras. [0066], [0070]), they disclose the steps individually, and their combination into the claimed order would have been obvious and would have been recognized to achieve a similar results. See Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.). Additionally, as Chiyoda Corp. teaches that application of calcium-containing solution will cause increase cell adhesiveness and reattachment to the culture vessel (Para. [0070], lines 2-4), a skilled artisan would be motivated to rearrange the order of steps to apply the calcium-containing solution which provides the benefit of protecting the cells after the detachment steps comprising application of a chelating agent and ultrasonic vibration in order to minimize the chances of reattachment to the culture substrate.
Conclusion
No claims are allowed.
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/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631