DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s submission filed on April 7, 2026 has been entered and considered. Rejections and/or objections not reiterated from the previous action mailed January 7, 2026 are hereby withdrawn. The following rejections and/or objections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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Claims 12-13 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Status of the Claims
Claim 1 has been amended to more clearly define the invention and to improve form. Claims 2, and 5-6 have been canceled. Claims 3-4, 7-8, 11 and withdrawn claim 12 have been amended to adjust dependencies and improve form.
Claims 1, 3-4, and 7-11 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 has been considered by the examiner.
Withdrawn Claim Rejections - 35 USC § 112
Claims 1-11 were 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. Claims 2 and 5-6 have been canceled rendering the rejection of those claims moot. In light of Applicant’s amendment to claim 1 to more clearly define the instant invention, the rejection of claims 1, 3-4, and 7-11 under 35 U.S.C. 112 has been withdrawn.
Withdrawn Claim Rejections - 35 USC § 103
Claims 1 and 8-11 were 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”). In light of Applicant’s amendment to claim 1 to incorporate the mesh filter step of claim 2, the rejection of claims 1 and 8-11 under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. and Kurashina et al. has been withdrawn.
Claims 2-3 were 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 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”). Claim 2 has been canceled, rendering the rejection of claim 2 moot.
Claims 4-5 were rejected under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. and Kurashina as applied to claim 1, 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”). Claim 5 has been canceled, rendering the rejection of claim 5 moot. In light of Applicant’s amendment to claim 1, the rejection of claim 4 under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. and Kurashina et al. and further in view of Hutcheson et al. has been withdrawn.
Claim 6 was 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). Applicant has canceled claim 6, rendering the rejection of claim 6 moot.
Claim 7 was rejected under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. and Kurashina as applied to claim 1, 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”). In light of Applicant’s amendment to claim 1, the rejection of claim 7 under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp. and Kurashina et al. and further in view of Gigout et al. has been withdrawn.
New Claim Rejections - 35 USC § 103
This is a new rejection necessitated by Applicant’s amendment. However, this rejection shares substantial similarity to the rejection as previously set forth in the office action dated January 7, 2026. Any aspect of Applicant’s traversal that pertains to the rejection as newly set forth will be provided following the new statement of rejection.
Claims 1, 3-4, 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) in view of 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”) and 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 1 and 3, 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.
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 mesh filter sizes including 25 µm (Pg. 2779, left col, last para. and Table 1) showing that the mesh filter size of 25 µm was effective at 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 mesh 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.
With regard to claim 4, Chiyoda Corp. teaches use of 4mM EDTA as the detachment solution, which is considered to reasonably read on a concentration of metal ion chelating agent which is 1.0mM of more. Claim 4 recites a wherein clause comprising adjusting the concentration of the metal ion chelating agent which is interpreted as not required to be performed based on the concentration of metal ion chelating agent taught by Chiyoda Corp. Further, Qiu teaches use of a mesh filter applied to detached cell aggregates which is considered to reasonably read on application of a shear force to the cells.
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., Kurashina, and Qiu teach a method of cellular detachment from a substrate comprising detaching cells using application of a chelating agent and ultrasonic vibration before passage of detached cells via a mesh filter in order to dissociate cells. 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 followed by use of a mesh filter as taught by the combined teachings of Chiyoda Corp., Kurashina, and Qiu 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]).
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Chiyoda Corp., Kurashina, and Qui 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., Kurashina, and Qiu teach a method of detaching cells from a culture substrate comprising application of a chelating agent, ultrasonic vibration, followed by passaged of detached cells through a mesh filter in order to dissociate cell aggregates. 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.
Response to Arguments
Applicant's arguments filed April 7, 2026 have been fully considered but they are not persuasive.
Applicant’s newly amended claim 1 is drawn towards recovery of cells cultured on a substrate via a method comprising addition of a cell detachment solution comprising a metal ion chelating agent, application of ultrasonic vibration to detach cells, and passage of a cell suspension containing detached cells through a mesh filter in order to dissociate cells (Pg. 6). First, Applicant traverses on Pg. 7 the Office’s assertion of obviousness for the instantly claimed invention based on the combination of Chiyoda, which teaches use of a metal ion chelating agent for cell detachment; Kurashina, which teaches ultrasonic vibration as a method of cell detachment; and Qiu, which teaches use of a mesh filter to dissociate cell aggregates into single cells. Specifically, Applicant traverses that Chiyoda indicates that use of a filter causes a problem of cell recovery reduction and specifically designs a recovery method to avoid use of a filter. Applicant asserts that Chiyoda “actively discourages passing a solution with detached cells through a filter” and teaches that “use of a filter should be avoided” and, therefore, there is no motivation to modify the recovery method of Chiyoda to use a mesh filter.
Applicant’s arguments have been fully considered but are not persuasive.
With regard to Applicant’s arguments of teaching away and lack of motivation to modify the method of Chiyoda, the teachings of Chiyoda indicate that the prior art of JP 2005198626A uses a filter which prevents the passage of cultured cells Para. [0005] but allows for exchange of detachment solution and/or media Para. [0006]. Chiyoda teaches that the filter of JP 2005198626A causes cells to be trapped and that Chiyoda’s system eliminates the need to temporarily accumulate a cultured cell population on a filter thereby preventing entrapment of cells. (Para. [0034]). However, the filter as taught in JP 2005198626 and as referenced by Chiyoda is not the same structure as the instantly claimed mesh filter or the mesh filter as taught by Qiu. JP 2005198626A teaches a cell culture vessel that comprises a “filter” which allows liquids (i.e., culture medium, washing solution, and detachment solution) to pass through but “prevents the passage of cells” (Para. [0005]). Thus, as “filter” is a broad term, the filter as taught by JP 2005198626A is interpreted to be a semi-permeable membrane which is used in order to allow the passage of liquids but prevent the passage of cells. The semi-permeable membrane of JP 2005198626A is not considered to be the same as the instantly claimed mesh or the mesh filter as taught by Qiu, particularly given the instantly claimed pore size of the mesh filter of 20 – 40 µm. Since the generally accepted size of an animal cell is well known to be 10 – 20 µm (See Alberts et al., Molecular Biology of the Cell), based on the instantly claimed pore size, a skilled artisan would recognize that the instantly claimed mesh filter is not intended to allow for the passage of liquids while retaining cells.
Second, Applicant traverses on Pg 7, 3rd para. that the instant specification indicates that the cells detached by the ultrasonic method of Kurashina were associated with each other, resulting in cell masses and that neither Kurashina nor Qiu provides teaching or suggestion that cell masses formed after ultrasonic detachment can be effectively dissociated by a mesh filter without compromising cell viability. Applicant asserts that Kurashina does not teach passage of a cell suspension through a mesh filter after ultrasonic detachment and that Qiu discloses a mesh filter which is applied to cell aggregates obtained from enzymatic digestion or tissue dissociation (on Pg. 7, last para and Pg. 8, 1st para.). Applicant asserts “there is a fundamental difference between such enzymatically digested aggregates and those resulting from ultrasonic detachment with respect to how separation is to be carried out.”
Applicant’s arguments have been fully considered but are not persuasive.
Applicant is reminded that arguments presented by applicant cannot take the place of factually supported objective evidence. See, e.g., In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965); In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). (See MPEP 2145). It is noted that Applicant has not provided any objective evidence that application of a mesh filter to cells which have been detached ultrasonically would alter cell viability nor that the methods of cell separation must be altered for cells dissociated via ultrasound compared to enzymatic digestion.
As detailed in the rejections above, Qui teaches that in cell aggregates detached from a substrate enzymatically (Pg. 2784, right col, 2nd full para.) mesh pore sizes of 25 µm were able to increase the number of dissociated cells without compromising cell viability (See Fig. 2A and 2C). Kurashina teaches that enzymatic detachment compared to ultrasonic detachment results in cell aggregates which are comparable in number (Fig. 2c) and size (Fig. 2e and g) and, further, that use of ultrasound reduces cell damage and improves cell survival (Abstract). Therefore, a skilled artisan would have every reason to believe that the mesh filter as taught by Qui could be applied to similarly sized cell aggregates formed by ultrasonic detachment in order to dissociate cells and that use of a mesh filter would not negatively affect cell viability as ultrasonically detached cells are less damaged than those detached enzymatically.
Third, Applicant asserts on Pg. 8 that the teachings of Hutcheson, Laermer, and Gigout do not cure the deficiencies of Kurashina and Qiu and therefore do not provide the motivation to modify Chiyoda.
Applicant’s arguments have been fully considered but are not persuasive.
The motivation to modify Chiyoda in view of Kurashina and Qiu has been detailed above. Applicant traverses that Hutcheson demonstrates that ultrasonic treatment can induce delayed cell death and therefore, a skilled artisan would have not been motivated to use a mesh filter to dissociate tissue. It is noted that in the new grounds of rejection necessitated by Applicant’s amendment does not rely on Hutcheson, thus Applicant’s arguments as to the teachings of Hutcheson are not considered persuasive. Applicant additionally traverses on Pg. 8 that the teachings of Laermer do not cure the deficiencies of Kurashina, Qiu, and Hutcheson as to the modification of Chiyoda. As Applicant has canceled claim 6, the new grounds of rejection does not rely on the teachings of Laermer and thus Applicant’s arguments as to the teachings of Laermer are also not considered persuasive.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERIN V PAULUS whose telephone number is (571)272-6301. The examiner can normally be reached Mon-Fri 8 AM-5 PM.
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/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631
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