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 .
Claims 2-6, of record 1/2/2026, are pending and subject to prosecution. Claims 2-6 are amended. Claims 1 and 7-10 are cancelled.
Status of Prior Rejections
RE: Objection to the specification:
The amendment to the specification is effective to obviate the objection. The objection is withdrawn.
RE: Objection to claims 1-3:
The cancellation of claim 1 renders the objection thereto moot.
The amendment to claims 2-3 is effective to obviate the objection. The objection is withdrawn.
RE: Rejection of claims 1-10 are rejected under 35 U.S.C. 112(b):
The cancellation of claims 1 and 7-10 renders the rejection thereto moot.
The amendment to claim 6 is effective to obviate the rejection. The rejection is withdrawn.
RE: Rejection of claims 6-10 are rejected under 35 U.S.C. 101:
The cancellation of claims 7-10 renders the rejection thereto moot.
The amendment to claim 6 is effective to obviate the rejection. The rejection is withdrawn.
RE: Rejection of claims 1 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021), evidenced by Marshall (Cell Migration, 2011), in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), Liu et al. (eFood, 2021), and Lin (US 20210077526 A1):
RE: Rejection of claims 1-2 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021) in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), Liu et al. (eFood, 2021), and Lin (US 20210077526 A1), further in view of Tao et al. (Frontiers in Pharmacology, 2022) and Kitamoto et al. (Virology, 1991):
RE: Rejection of claims 1 and 3-4 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021) in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), Liu et al. (eFood, 2021), and Lin (US 20210077526 A1), further in view of Sun et al. (PLoS One, 2010):
RE: Rejection of claims 1 and 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021) in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), Liu et al. (eFood, 2021), and Lin (US 20210077526 A1), further in view of Tao et al. (Frontiers in Pharmacology, 2022) and Wu et al. (Pharmazie, 2013):
The cancellation of claim 1 renders the rejection thereto moot.
The amendments to claims 2-5, requiring the claims to depend upon amended claim 6, are effective to obviate the rejections. The rejections are withdrawn.
New Rejections
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.
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 4 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021), of record, evidenced by Marshall (Cell Migration, 2011), of record, in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), of record, Michl et al. (Communications Biology, 2019), Masters et al. (Nature Protocols, 2007), Liu et al. (eFood, 2021), of record, Lin (US 20210077526 A1), of record, and Liang et al. (Molecules, 2017),.
Regarding claim 6: Kim et al. teach a Caco-2 cell/RAW 264.7 cell co-culture model of intestinal inflammation for assessing antioxidant activity of acai berry extract (See Abstract). Caco-2 cells were cultured in DMEM comprising FBS, 1% non-essential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin (See page 3, col. 1, full ¶4). RAW 264.7 cells were cultured in DMEM comprising 10% FBS, 1% HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin (See page 3, col. 1, full ¶4 and col. 2, ¶1). Both cell types were cultured in a humidified incubator (which reads on “constant humidity”) at 37°C with 5% CO2 (which reads on “95% air”) (See page 3, col. 1, ¶1). The Caco-2 cells were seeded at a density of 1 × 105 cells/well on the apical surface in a six-well Transwell plate and cultured for 21 days (See page 2, col. 1, full ¶2 and page 3, col. 2, full ¶1-2). RAW 264.7 cells were seeded at a density of 1 × 10 cells/well on the basolateral surface (See page 3, col. 2, full ¶1). Assembly of the Transwell apparatus necessarily reads on “transferring the upper chamber of the Transwell plate loaded with the Caco-2 cells… to the… plate loaded with the RAW 264.7 cells”. LPS was added to the basolateral side, and secretion of IL-6, IL-8, PGE2, and TNF-α (which read on “inflammatory factor” was measured in Caco-2 cell supernatants (which reads on “AP side”) by ELISA kits (See page 3, col. 2, full ¶1).
Kim et al. do not expressly teach a Transwell membrane of 4 µm, however Marshall teaches that Transwell pore sizes include 3 and 5 µm pore diameters (See page 105, full ¶4). Where claimed amounts do not overlap with the prior art but are merely close, a prima facie case of obviousness exists. See MPEP 2144.05(I).
Kim et al. do not expressly teach culturing at pH 7.4 or for 21 days with media changes every 1-2 days prior to Transwell seeding and every day after seeding. Kim et al. do not teach the culture of Caco-2 cells in MEM, the use of a 24-well Transwell plate, incubation of the RAW 264.7 cells for one day after seeding in the Transwell plate, or preparation of a stock solution of LPS and the use of LPS in the assay at 1 µg/ml for 3 h. Kim et al. also do not teach the use of cyanidin-3-glucoside nanoliposomes in the Transwell assay.
Michl et al. teach that optimal growth for Caco-2 cells occurs in pH 7.4 medium (See fig. 2E).
Masters et al. provide an overview of cell culture protocols and teach that how frequently the culture medium needs to be changed depends on the cell line and type of medium and that medium changes must be related to cell health and any experiments (See page 2278, col. 2, full ¶3-4).
Chen et al. teach that Caco-2 cells can be cultured in either DMEM or MEM (See page 335, col. 1, full ¶3). Chan et al. also teach seeding of Caco-2 cells at 1 × 105 cells on 24-well inserts (See page 335, col. 1, full ¶4 and col. 2, ¶1).
Liu et al. teach the addition of 1 µg/ml LPS to the basolateral compartment for 3 h in a Caco-2 cell/RAW 264.7 cell Transwell co-culture for stimulating inflammatory factors (See page 93, col. 2, full ¶4-5). The Transwell assay is used 24 h after RAW 264.7 cell seeding (See page 93, col. 2, ¶1).
Lin teaches the preparation of LPS for in vivo administration (See ¶0059). LPS was dissolved in PBS at a concentration of 2 µg/ml and filtered through a 0.22 µm pore size membrane (which reads on “0.22 µm pinhole filter”) (See ¶0059).
Liang et al. teach the optimization of cyanidin-3-O-glucoside uptake by Caco-2 cells (See Abstract). The anthocyanin cyanidin-3-O-glucoside was formulated as nanoliposomes, and its effects on Caco-2 cancer cell morphology and survival were measured (See fig. 7-8).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Kim et al. to substitute MEM for DMEM and six-well plates for 24-well plates, as taught by Chen et al. Substitution of a known element for another known element is considered prima facie obvious, absent a showing that the substitution yields more than predictable results. See MPEP 2144.06(II).
The use of MEM at pH. 7.4 would have been obvious, as well, because Michl et al. teach this pH as optimal for Caco-2 cell growth (See fig. 2E). Such a modification could be readily performed. Increasing the frequency of media changes to every 1-2 days would have obvious, as Masters et al. indicate that the timing of media changes is a result-effective variable (See page 2278, col. 2, full ¶3-4), and therefore ripe for optimization. See MPEP 2144.05((II)(B).
It also would have been obvious to modify the Transwell assay of Kim et al. to comprise its use 24 h after seeding the RAW 264.7 cells and incubation with 1 µg/ml LPS for 3 h, as taught by Liu et al. One would be motivated to make these modifications because Liu et al. show that Transwell assays comprising co-culture of Caco-2 cell/RAW 264.7 for studying inflammation can successfully begin 24 h after seeding RAW 264.7 cells and disclose that incubation with 1 µg/ml LPS for 3 h is sufficient to stimulate secretion of inflammatory factors from RAW 264.7 cells. A skilled artisan would have had a reasonable expectation of success in practicing the claimed invention as successful culture times after seeding RAW 264.7 cells in co-cultures Caco-2 cell/RAW 264.7 used in inflammation assays, and LPS concentrations successfully capable of stimulating secretion of inflammatory factors from RAW 264.7 cells were known in the art at the time of the invention .
It would have been further obvious to modify the method of Kim et al. to comprise the LPS solution preparation method taught by Lin. One would be motivated to make this modification for sterilization of the LPS (See ¶0059). There would be a reasonable expectation of success in doing so because the LPS in the method of Kim et al. could be readily prepared in such a manner. While Lin does not expressly teach a concentration of 50 µg/ml, one of ordinary skill would recognize that solution concentrations can be increased during optimization for an intended use.
Finally, it would have been obvious to modify the method of Kim et al. to comprise testing cyanidin-3-O-glucoside nanoliposomes, as taught by Liang et al., in place of acai berry extract. One would be motivated to make this modification because the results of Kim et al. suggest that the assay is appropriate for examining the effects of anthocyanins on Caco-2 cells in a Transwell co-culture model (See fig. 1-2). There would be a reasonable expectation of success in doing so because the liposomes could be readily incorporated.
Regarding claim 4: Following the discussion of claim 6, Kim et al., evidenced by Marshall, and modified by Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al., render obvious a Caco-2 cell/RAW 264.7 cell Transwell assay but do not teach TEER measurement every three days.
However, Liu et al. teach TEER measurement approximately every 2-3 days (which reads on “once every three days on average”) (See fig. S1A). Liu et al. also teach that values of approximately 800 Ω·cm2 suggest formation of a tight junction structure (which reads on “transepithelial resistance of greater than 300 Ω·cm2 is regarded that a monolayer is dense and intact”) (See page 94, col. 2, ¶2).
It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to further modify the method of Kim et al., modified by Chen et al., Michl et al., Masters et al., Liu et al., and Lin, to comprise the TEER measurement taught by Liu et al. One would be motivated to make this modification in order to verify the Caco-2 monolayer prior to conducting subsequent experiments (See 94, col. 2, ¶2), and such a modification could be readily done during the 21 days after seeding.
Claims 2, 4, and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021), of record, evidenced by Marshall (Cell Migration, 2011), of record, in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), of record, Michl et al. (Communications Biology, 2019), Masters et al. (Nature Protocols, 2007), Liu et al. (eFood, 2021), of record, Lin (US 20210077526 A1), of record, and Liang et al. (Molecules, 2017), further in view of Tao et al. (Frontiers in Pharmacology, 2022), of record, and Kitamoto et al. (Virology, 1991), of record.
The teachings of Kim et al., Marshall, Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al. are set forth in the rejection above and are incorporated herein in their entirety.
Regarding claim 2: Following the discussion of claims 4 and 6, Kim et al., evidenced by Marshall, and modified by Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al., render obvious a Caco-2 cell/RAW 264.7 cell Transwell assay but do not teach the Caco-2 medium as comprising 20% FBS, 1% HEPES, 1% L-glutamine, and 1% sodium pyruvate.
Tao et al. teach the development of a Caco-2 Transwell model for studying drug transport (See Abstract). Caco-2 cells were maintained in MEM comprising 20% FBS, 1% glutamine, and 1% sodium pyruvate (See page 2, col. 2, full ¶2).
Kitamoto et al. teach Caco-2 cell culture in MEM comprising 10 mM HEPES (See page 730, col. 2, ¶1).
It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Kim et al., evidenced by Marshall, and modified by Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al., to comprise the addition of 20% FBS, 1% glutamine, 1% sodium pyruvate, and 10 mM HEPES to the Caco-2 cell culture medium. One would be motivated to make this modification because the inclusion of these components by Tao et al. and Kitamoto et al. suggest that they are appropriate for Caco-2 cell culture, and these components could be readily added. While Kitamoto et al. teach the use of 10 mM HEPES (approximately 2.38 g/L) rather than 1% HEPES (10 g/L), it would have been obvious to one of ordinary skill that the amount of HEPES could be increased through routine experimentation for maintaining a particular pH.
Claims 3-4 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021), of record, evidenced by Marshall (Cell Migration, 2011), of record, in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), of record, Michl et al. (Communications Biology, 2019), Masters et al. (Nature Protocols, 2007), Liu et al. (eFood, 2021), of record, Lin (US 20210077526 A1), of record, and Liang et al. (Molecules, 2017), further in view of Sun et al. (PLoS One, 2010), of record.
The teachings of Kim et al., Marshall, Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al. are set forth in the rejection above and are incorporated herein in their entirety.
Regarding claim 3: Following the discussion of claims 1 and 4, Kim et al., evidenced by Marshall, and modified by Chen et al., Liu et al., Lin, and Liang et al., render obvious a Caco-2 cell/RAW 264.7 cell Transwell assay but do not teach the RAW 264.7 cell medium as comprising 1% non-essential amino acids and 1% L-glutamine.
Sun et al. teach the culture of RAW 264.7 cells in DMEM comprising 1% L-glutamine and 1% non-essential amino acids (See page 10, col. 2, full ¶2).
It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Kim et al., evidenced by Marshall, and modified by Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al., to comprise the addition of 1% L-glutamine and 1% non-essential amino acids to the RAW 264.7 cell culture medium. One would be motivated to make this modification because their inclusion by Sun et al. suggest that they are appropriate components for RAW 264.7 cell culture, and such a modification could be readily performed.
Claims 4-6 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Journal of Food Biochemistry, 2021), of record, evidenced by Marshall (Cell Migration, 2011), of record, in view of Chen et al. (Journal of Pharmacological and Toxicological Methods, 2010), of record, Michl et al. (Communications Biology, 2019), Masters et al. (Nature Protocols, 2007), Liu et al. (eFood, 2021), of record, Lin (US 20210077526 A1), of record, and Liang et al. (Molecules, 2017), further in view of Tao et al. (Frontiers in Pharmacology, 2022), of record, and Wu et al. (Pharmazie, 2013), of record.
The teachings of Kim et al., Marshall, Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al. are set forth in the rejection above and are incorporated herein in their entirety.
Regarding claim 5: Following the discussion of claims 1 and 4, Kim et al., evidenced by Marshall, and modified by Chen et al., Liu et al., Lin, and Liang et al., render obvious a Caco-2 cell/RAW 264.7 cell Transwell assay but do not teach detection of alkaline phosphatase activity.
Tao et al. teach detection of alkaline phosphatase activity on the apical and basolateral sides of a Caco-2 cell monolayer in a Transwell plate (See page 3, col. 2, full ¶1). The activity on the apical side was higher, indicating cell polarization (which reads on “a greater ratio of the AP side of the upper chamber to the BL side of the lower chamber indicates a higher degree of cell polarization”) (See page 4, col. 2, full ¶4).
Wu et al. teach the establishment of a Caco-2 cell Trans well model (See Abstract). Alkaline phosphatase activity was measured on days 8, 15, and 23 (which reads on “once every 7 days on average” (See page 806, col. 2, full ¶1 and fig. 4).
It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Kim et al., evidenced by Marshall, and modified by Chen et al., Michl et al., Masters et al., Liu et al., Lin, and Liang et al., to comprise analysis of alkaline phosphatase activity. One would be motivated to make this modification in order to assess the degree of polarization in the Caco-2 cell monolayer (See Tao et al., page 4, col. 2, full ¶4). There would be a reasonable expectation of success because such testing could be readily performed. It also would have been obvious to perform alkaline phosphatase testing at weekly intervals, as taught by Wu et al., in order to track polarization of the developing monolayer at different time points (See page 806, col. 2, full ¶1 and fig. 4), which could be readily done.
Conclusion
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 JENNIFER S SPENCE, whose telephone number is 571-272-8590. The examiner can normally be reached M-F 8:30-5:30.
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/J.S.S./Examiner, Art Unit 1633
/CHRISTOPHER M BABIC/Supervisory Patent Examiner, Art Unit 1633