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 .
DETAILED ACTION
Claims 4, 6 and 10-12 are cancelled. Claims 1-3, 5, 7-9, and 13-23 are pending.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03/02/2026 has been entered.
Priority
This application is a 371 of PCT/EP2020/076559 filed on 9/23/2020, which claims benefit of EUROPEAN PATENT OFFICE (EPO) 19199296.5 filed on 9/24/2019. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
The effective filing date of the current application is September 24, 2019.
Election/Restrictions
Newly submitted claim 23 is directed to an in vitro drug testing kit, which is a non-elected invention. Applicant traversed the restriction requirement in the reply filed on 03/12/2025. Claims 13-21 and 23 are withdrawn.
Claims 1-3, 5, 7-9 and 22 are under examination.
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 1-3, 5, 7-9 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (US 20190055510A1, published on February 21, 2019; previously cited) in view of Lu et al. (“Designing compartmentalized hydrogel microparticles for cell encapsulation and scalable 3D cell culture”, Journal of Materials Chemistry B, 2015, Vol. 3, pp.353-360; previously cited) and Akasov et al. (“3D in vitro co-culture models based on normal cells and tumor spheroids formed by cyclic RGD-peptide induced cell self-assembly”, Biotechnology Letters, 2017, Vol. 39, pp.45-53).
Regarding claim 1, Lee teaches an in vitro multiwell plate-based perfusion culture device (title). Lee teaches a co-culture of three or more cell types [patient lymphocytes, patient dendritic cells, patient epithelial cells] (See Figure 11). Lee teaches testing therapeutic efficacy of a therapeutic agent on the viable MMCs (description p.12, [0123]). Lee teaches the ex vivo response of the patient’s MM cells (PMMCs) to the therapeutic agent is cell viability, which can be measured by enzyme activity, cell membrane permeability, ATP production, co-enzyme production, nucleotide uptake activity, apoptosis, and the like (description p.27, [0360]).
Lee teaches a panel of cell death analysis methods; for example, annexin V/PI staining using flow cytometry and in situ detection of cleaved caspase-3 using immunofluorescence can be used to determine cell death; with the percentage of dead cells calculated as [% of Annexin V+/PI+ cells in co-cultures - % of Annexin V+/PI+ cells in IEC alone cultures] (relevant to claim 1a) (description p.30, [0422]).
Lee teaches the cells to be cultured can be encapsulated in suitable hydrogels, matrices and/or scaffolds (relevant to claim 1b) (description p.29, [0417]).
Lee teaches bringing the BMMCs in contact with the endosteal microenvironment perfused by nutrients and gas molecules (relevant to claim 1c) (p.44, claim 17(c)). Lee teaches testing therapeutic efficacy of a therapeutic agent on the viable MMCs maintained by the endosteal microenvironment (relevant to claim 1d) (description p.44, claim 17(d)). Lee further teaches PMMCs cultured and treated in the tissue models, after 1-day post-treatment, will be stained for surface markers and cell viability for high-content screening analyses (relevant to claim 1e) (description p.35, [0475]). Lee teaches the following stains will be further tested and optimized to follow multiple PMMC populations: CD38 VioBright FITC (496/522), CD138 Alexa Fluor 546 (556/573), and CD56 APC-eFluor® 780 (633/780). MM stem cell-like cells will be assessed with ALDEFLUOR™ Kit for aldehyde dehydrogenase (ALDHl, 490/525). Cell viability will be assessed using 7-AAD (546/647). BMMCs will be pre-labeled with CellTrace violet (405/450) in order to easily differentiate OSB from BMMCs. IC50 values will be obtained as a function of dormancy/proliferation (i.e., CFSEhigh vs. CFSElow or CellTracehigh vs. CellTracelow), cell cycle and sternness (Table 3) (p.35-36, [0475]).
Lee does not teach the capsules are made of alginate. Lee does not teach three or more cell types organized in 3D multicellular structures inside alginate capsules.
However, Lu teaches double-layer hydrogel microparticles made of fluorescently labeled alginate (p.354, Fig. 1). Lu teaches that the compartmentalized hydrogel microparticles have a high surface area of mass transfer and also offer defined space and essential ECM support for various scalable efficient 3D cell culture, co-culture applications (abstract). Lu teaches that hydrogel microparticles have been used extensively for cell encapsulation, and protect cells from the environment while allowing facile mass transfer necessary for cell survival and function (p.353, 1st column 1st paragraph). Lu further teaches that alginate-based core-shell microparticles improve immunoprotection (p.353, 1st column 1st paragraph).
Akasov teaches 3D in vitro co-culture models based on normal cells and tumor spheroids (title). Akasov teaches mouse melanoma M-3 cells were stained with DiO; the obtained spheroids were mixed with previously DiI-stained L-929 cells; then the cell mixture was entrapped in alginate/chitosan microcapsules and cultivated for 14 days (p.47, 2nd column – Co-culture of tumor spheroids and single stromal cells in alginate-chitosan microcapsules). Akasov further teaches that the generation of co-culture spheroids within microcapsules was observed by confocal microscopy (p.47, 2nd column – Co-culture of tumor spheroids and single stromal cells in alginate-chitosan microcapsules). Akasov teaches 3D co-culture spheroids are proposed as a tool in tumor biology to study cell-cell interactions as well as test novel anticancer drugs and drug delivery vehicles (abstract). Akasov further teaches that the use of both normal and tumor tissue could allow the ranking of formulations according to their therapeutic safety ratio (p.46, 1st column 1st paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute alginate capsules taught by Lu and Akasov for the suitable hydrogel for encapsulation taught by Lee. Each of Lee, Lu and Akasov teach the encapsulation of cell types for in vitro applications. One of ordinary skill in the art would have reasonable expectation of success with this substitution because Lu teaches that hydrogel microparticles have been used extensively for cell encapsulation, and alginate-based microparticles improve immunoprotection.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute 3D co-culture spheroids taught by Akasov for the three or more cell types taught by Lee, because Akasov teaches that 3D co-culture spheroids would be a useful tool for testing novel anticancer drugs. One of ordinary skill in the art would have found it beneficial to utilize a 3D multicellular structure inside alginate capsules to rank cancer formulations according to their therapeutic safety ratio.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the method of flow cytometry taught by Lu with imaging the 3D co-culture spheroids inside the alginate capsules using confocal microscopy taught by Akasov, because Akasov teaches that the 3D co-cultures could be used as a tool to study cell-cell interactions and to test novel drugs. One of ordinary skill in the art would have found it beneficial to utilize confocal microscopy as an imaging method to allow observation of the intact spheroids within the alginate capsules.
Regarding claim 2, Lee teaches showing the effect of O2 tension on CD138+ PMMC viability and proliferation after 5-day culture in fibrin (description p.13, [0134] and Figure 6A/6B). Lee further teaches the described model is useful to study and identify new mechanisms associated with tumor progression (description p.11-12, [0121]).
Regarding claim 3, Lee further teaches a method for optimizing donor selection for allogeneic blood and marrow transplantation therapy comprising seeding a population of primary intestinal epithelial cells (IECs) in a first adjacent well; generating a population of conditional reprogrammed intestinal epithelial cells, and seeding and expanding in a second adjacent well the population of T lymphocytes derived from the potential donor (description p.28, [0389]-[0393]). Lee teaches that bone marrow niche refers to a well-organized architecture composed of osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells and adipocytes, which play an essential role in the survival, growth and differentiation of diverse lineages of blood cells (description p.1, [0004]).
Regarding claim 5, Lee teaches using a panel of cell death analysis methods to quantify cell death, for example annexin V/PI staining using flow cytometry and in situ detection of cleaved caspase-3 using immunofluorescence can be used to determine cell death (description p.30, [0422]).
Regarding claim 7, Lee teaches patient multiple myeloma cells, bone marrow mononuclear cells, and osteoblasts from a well culture device (description p.13-14, [0138]; FIG. 10).
Regarding claim 8, Lee does not teach wherein at least one of said three or more cell types are hepatocytes.
However, Lu teaches the utilization of microparticles in maintaining in vitro culture of hepatocytes (p.353, 2nd column last paragraph). Lu further teaches that in vitro culturing of primary hepatocytes, particularly in a scalable fashion, has important applications in drug screening and toxicity testing for pharmaceutical industries (p.356, 1st column last paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to select hepatocytes taught by Lu as one of the cell types in the capsules of Lu, because Lu teaches that in vitro culturing of primary hepatocytes in a scalable fashion is important for drug screening and toxicity testing. One of ordinary skill in the art would have found it beneficial to include hepatocytes as one of the cell types in an in vitro drug screening and toxicity application.
Regarding claim 9, Lee teaches patient’s multiple myeloma cells (description p.27, [0359]) and solid tumors that metastasize to bone including breast cancer and prostate cancer (description p.27, [0361]). Lee teaches a method for testing personalized therapeutics for MM patients and for evaluating new drugs without the need for costly animal models (description p.27, [0361]).
Regarding claim 22, Lee teaches a co-culture of three or more cell types [patient lymphocytes, patient dendritic cells, patient epithelial cells] (See Figure 11). Lee further teaches PMMCs cultured and treated in the tissue models, after 1-day post-treatment, will be stained for surface markers and cell viability for high-content screening analyses (description p.35, [0475]). Lee teaches the following stains will be further tested and optimized to follow multiple PMMC populations: CD38 VioBright FITC (496/522), CD138 Alexa Fluor 546 (556/573), and CD56 APC-eFluor® 780 (633/780).
Lee does not teach encapsulation in alginate capsules. Lee does not teach three or more cell types organized in 3D multicellular structures inside alginate capsules.
However, Lu teaches complex hydrogel microparticles with controlled ECM internal compartments for efficient and scalable 3D cell culture (p.353, 2nd column 2nd paragraph). Lu teaches using model cells (MDA-MB-231 expressing GFP; normal human lung fibroblasts (NHLFs) with RFP; and MCF-10A stained with Hoechst) to demonstrate that different types of cells could be encapsulated in distinct compartments within individual particles (p.353, 1st column 1st paragraph; Figure 1(k) and 1(l)).
Akasov teaches 3D in vitro co-culture models based on normal cells and tumor spheroids (title). Akasov teaches mouse melanoma M-3 cells were stained with DiO; the obtained spheroids were mixed with previously DiI-stained L-929 cells; then the cell mixture was entrapped in alginate/chitosan microcapsules and cultivated for 14 days (p.47, 2nd column – Co-culture of tumor spheroids and single stromal cells in alginate-chitosan microcapsules). Akasov further teaches that the generation of co-culture spheroids within microcapsules was observed by confocal microscopy (p.47, 2nd column – Co-culture of tumor spheroids and single stromal cells in alginate-chitosan microcapsules).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to analyze the fluorescence of the three or more cell types encapsulated in the alginate capsules using confocal microscopy taught by Akasov, because Lu teaches imaging three different cell types each with a different fluorescent marker encapsulated in alginate capsules, and Akasov teaches staining different cell types with different markers. One of ordinary skill in the art would have been motivated to use confocal microscopy taught by Akasov because this method allows visualization of the structure within the capsule without disrupting the cells. One of ordinary skill in the art would have found it beneficial to be able to visualize all three cell types at the same time to determine the location of the different cell types within the capsule.
Response to Arguments
Applicant argues that claim 1 recites “while the three or more cell types remain in their native 3D organization and encapsulated in their respective alginate capsules”, and Lee, Lu and Mawad do not teach or suggest at least this claim feature (See Remarks dated 3/2/2026, p.7, last full paragraph). Applicant argues that Lee fails to teach or suggest “3D multicellular structures inside alginate capsules” or analyzing cells in their “native 3D organization” (See Remarks dated 3/2/2026, p.7 last paragraph). Applicant argues that Lee’s cells are cultured in a perfused chamber but analysis requires removing them from this context; the pending application maintains the architectural integrity of the spheroid during measurement, a critical feature for physiological relevance that Lee cannot achieve (See Remarks dated 3/2/2026, p.7 last paragraph – p.8 top paragraph).
Applicant argues that the analysis by “high-content screening while the three or more ce3ll types remain in their 3D organization” is a direct consequence of combining capsule-specific fluorophores for cell-type ID and intracellular fluorescent reporter genes for process measurement, and neither Lee nor Lu teach this combination (See Remarks dated 3/2/2026, p.8 first paragraph).
Applicant argues that Lee teaches away from the claimed limitations, because Lee’s analysis method of flow cytometry requires destruction of the 3D culture (See Remarks dated 3/2/2026, p.8, 2nd paragraph). Applicant argues that a person having ordinary skill in the art (PHOSITA) would understand that the teachings of Lee require disruption of the culture, and Lee fails to provide a PHOSITA with motivation to develop an in situ high-content screening method, instead it teaches away from it (See Remarks dated 3/2/2026, p.8, 2nd paragraph).
Applicant argues that a PHOSITA would not have been motivated to modify the technique of Lee to use the encapsulated cell types of Lu with any reasonable expectation of success, because Lu is directed to a different problem (See Remarks dated 3/2/2026, p.8 last paragraph). Applicant argues that the techniques of Lee and Lu are fundamentally and technically incompatible, and it would have been illogical for a PHOSITA to take Lee’s complex perfusion system, replace its cellular components with Lu’s static alginate beads, and still use Lee’s destructive flow cytometry for analysis (See Remarks dated 3/2/2026, p.9 first paragraph).
Applicant argues that the pending application additionally teaches that the claimed method produces unexpected results, by successfully measuring two distinct biological processes: proliferation via FUCCI and apoptosis via zipGFP-Casp3 that occur independently in three different cell types within the same well (See Remarks dated 3/2/2026, p.9 2nd paragraph). Applicant further argues that this simultaneous, independent multiplexing in situ is not taught or suggested by Lee or Lu and the ability to deconvolute these signals in real-time, in 3D, based on capsule color is a wholly unexpected result that demonstrates a synergy that elevates the pending application beyond a simple aggregation of known elements (See Remarks dated 3/2/2026, p.9 2nd paragraph).
Applicant's arguments filed March 2, 2026 have been fully considered but they are not persuasive.
Claim 1 is directed towards an in vitro method, which requires active method steps of expressing in two or more of the three or more cell types at least one fluorescent reporter gene for each biological process; encapsulating each of the three or more cell types to be analyzed with a biopolymer of alginate; co-culturing all the encapsulated cell types to be analyzed; exposing said co-culture to one or more drugs; and measuring the activity of the two or more biological processes in each cell. Each of these limitations are taught in the prior art by Lee, Lu and Akasov, as discussed in the rejection above.
Lee teaches a method of expressing at least one fluorescent reporter gene for each biological process to be analyzed; encapsulating each of the three or more cell types to form capsules of the three or more cells; co-culturing all the encapsulated cell types; exposing said co-culture to one or more drugs; and measuring the activity of the two or more biological processes in each cell type by analyzing through high-content screening and in situ detection using immunofluorescence, the fluorescence of each fluorescent reporter gene. Lu is relied upon for teaching that the capsules are alginate capsules, which Lee does not teach. Akasov is relied upon for teaching encapsulation of 3D spheroids within alginate capsules, and using confocal microscopy to visualize each of the differently stained cell types within the capsules without disrupting the cells or capsules. Each of Lee, Lu and Akasov teach the encapsulation of cells in capsules for in vitro testing. One of ordinary skill in the art would be motivated to modify the method of Lee to use alginate microcapsules taught by Lu encapsulating 3D spheroids taught by Akasov and image the resulting structures using confocal microscopy taught by Akasov to create a system comprising cells encapsulated in alginate capsules that could be cultured, exposed to one or more drugs, and analyzed by measuring the activity of two more biological processes as required by the instant claims.
One of ordinary skill in the art would recognize that the teachings of Lee, Lu and Akasov provide guidance in encapsulating 3D multicellular spheroids comprising multiple cell types stained for identification that can be imaged and visualized using confocal microscopy without disturbing the capsule or cellular architecture within the capsule. Lee teaches using different dyes to identify metabolic processes including cell death, and co-culturing varied cell types of lymphocytes, dendritic cells and epithelial cells. Lee further teaches measuring cell viability using enzyme activity, cell membrane permeability, ATP production, co-enzyme production, nucleotide uptake activity, apoptosis, and the like. One of ordinary skill in the art would therefore by motivated to combine the teachings of Lee with the relevant teachings from Lu and Akasov to arrive at the claimed invention for the reasons set forth in the rejection above.
Regarding Applicant’s claim of unexpected results: one of ordinary skill in the art would reasonably expect that by combining stains that identify metabolic activity in a cell type would predictably result in detecting the desired metabolic activity in the cell. One of ordinary skill in the art would also reasonably expect that combining two or more types of cell types or encapsulated cell types together would predictably result in the detection of two or more processes within the cell types, because it would amount to a combination of known elements together in a predictable way. It was known in the art at the time of invention that different techniques and fluorescent imaging dyes exist to detect different metabolic processes and cell types. Thus, Applicant’s argument that ability to detect cellular proliferation and apoptosis using different dyes independently in three different cell types within the same well is not an unexpected result.
Conclusion
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/LOUISE W HUMPHREY/ Supervisory Patent Examiner, Art Unit 1657
/DEEPA MISHRA/Examiner, Art Unit 1657