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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 09/09/2025 has been entered.
Applicant' s amendment and response filed on 08/12/2025 has been received and entered into the case.
Amendments
In the reply filed 08/12/2025, Applicant has amended claims 73, 77 and 95.
Claim Status
Claims 73-81, 84-90 and 93-95 are pending and are considered on the merits.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 09/09/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner. The corresponding signed and initialed PTO form 1449 has been mailed with this action.
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 73-79, 84-89 and 93-95 are rejected under 35 U.S.C. 103 as being unpatentable over Lewis et al (Biomater. Sci. 2015; 3: 821-832. Cited in IDS 11/17/2021), in view of Sugaya et al (2012 International Symposium on Micro-NanoMechatronics and Human Science (MHS), Nagoya, Japan, 2012, pp. 435-438, doi: 10.1109/MHS.2012.6492486. Prior art of record), Souza et al. (Nature Nanotechnology. 2010;5:291-296 and Supplementary p. 1-17), Kolhatkar et al (ACS Omega. 2017, 2, 8010−8019. Prior art of record), Tong et al (Journal of Polymer Science: Part A: Polymer Chemistry. 2011; 49: 1513–1516. Cited in IDS 03/25/2022) and Nair et al (Polymer. 2012; 53: 2429-2434. Prior art of record).
It is noted that the prior rejection set forth in the Office action mailed on 06/12/2025 is maintained. The following rejection is edited to address the amendments.
With respect to the preamble of independent claim 73 and claim 95, Lewis teaches a method of culturing cells within a hydrogel to make an in vitro alveoli tissue model (title, abstract).
With respect to incubating cells seeded in a uniformly dispersed polymer microsphere composition in claims 73 and 95, and the polymer microspheres having a diameter of 116.1 μm to about 300 μm in claim 73, it is noted that the phrase “uniformly dispersed” is examined as being equivalent to the term “monodisperse”, which is defined as a particle-based composition comprising particles that are substantially uniform in size, shape and mass (specification, p. 16), and the specification and claim further exemplify the phrase as “having a diameter of 116.1 μm to about 300 μm”. Lewis teaches A549 cells or primary alveolar epithelial type II (ATII) cells are seeded with polymer microspheres and incubated for 1 day or 3 days “to enable full coating of the microspheres” to form pre-cysts (p. 824, left col, “Cell-microsphere seeding” para). Lewis teaches the cultured cysts have an average diameter of 180 ± 80 μm (i.e., about 100 μm to about 260 μm), “which is in the size range relevant to human alveoli (∼200 μm)” (p. 828, last para, see e.g. Fig 5A, C, D and Fig 6A, B, E), thus teaches a plurality of uniformly dispersed cell-coated polymer microspheres having a diameter of about 100 μm to about 260 μm, which overlaps with the claimed size range in claim 73.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have chosen a uniformly dispersed polymer microsphere composition having a diameter of 116.1 μm to about 300 μm as suggested by Lewis in the method of culturing alveolar epithelial cells in an in vitro alveoli model disclosed by Lewis with a reasonable expectation of success. Since Lewis suggests that an average diameter of 180 ± 80 μm (i.e., about 100 μm to about 260 μm) is in the size range relevant to human alveoli (∼200 μm) (p. 828, last para, see e.g. Fig 5-6), one of ordinary skill in the art would have had a reason to choose a polymer microsphere composition having a size range relevant to human alveoli to better mimic human alveoli in an in vitro alveoli model. Furthermore, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use, since it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. See M.P.E.P. §2144.05.
With respect to aggregating portions of the polymer microsphere composition to form alveoli-like clusters, Lewis teaches both A549 cells coated microspheres and ATII coated microspheres aggregate and form alveoli-like clusters (see Fig 5A attached below for A549 cells and see Fig 7C-7D for ATII).
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However, although Lewis clearly contemplates to make and use 3D alveoli-like clusters as an in vitro lung model by performing a 3D surface reconstruction of mouse lung tissue (shown in Fig 7E) and teaches “the epithelial cysts developed here capture many aspects of the structure and cellular arrangement found in alveoli in vivo” and are useful as in vitro 3D models of the distal airway epithelium (p. 830, right col, last sentence above “Conclusions” and 1st sentence in “Conclusion”), Lewis is silent on a step of aggregating the cell-coated microspheres to form 3D alveoli-like clusters having a diameter of 1200 μm to 2200 μm comprising magnetically levitating the microspheres to form aggregates, nor teach the polymer microspheres comprising magnetic particles having a diameter of about 100 nm to about 500 nm in claims 73 and 95.
With respect to the microspheres further comprising magnetic particles so that the microspheres can be moved by magnetic force, Sugaya teaches production of hydrogel beads (i.e., microspheres) containing magnetic nanoparticles and teaches the beads are incubated with cells to form cell-bead complexes or cell spheroid (i.e., cell-coated microspheres) (abstract, p. 435, right col, line 5 from bottom, see Fig 2). Sugaya teaches cell-microsphere complexes are “moved and collected by applying magnetic fields” and “these heterogeneous spheroids are also recovered by applying magnetic field” (Fig 2a, 2b and legend) to “transport, concentrate and recover the cell-bead complexes by applying magnetic force” (p. 436, left col, line 1, see Fig 2), thus teaches polymer microspheres comprise magnetic particles and the cell-coated microspheres or cell spheroid can be moved by magnetic force in the culture medium. Sugaya teaches the magnetic hydrogel microspheres “would be highly useful as new types of cell handling tools, cell cultivation matrices, and building blocks for tissue engineering” (abstract, conclusion).
With respect to forming 3D aggregates by magnetic levitation, Souza teaches a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved (abstract, see Fig 1 for an exemplary 1-mm spheroid, and Fig 2 diagram attached below). Souza teaches levitated three-dimensional culture with magnetized hydrogels more closely recapitulates in vivo protein expression and is more feasible for long-term multicellular studies (abstract).
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Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro tissue model comprising incubating cells seeded on microspheres and aggregating cell-coated microspheres to form alveoli-like clusters disclosed by Lewis, by combining magnetic particles to the microspheres as taught by Sugaya and combining magnetically levitating the magnetized hydrogels to form three-dimensional aggregates or clusters as taught by Souza with a reasonable expectation of success. Since Sugaya teaches the cell-coated magnetic hydrogel beads can be moved by applying magnetic force and are highly useful for tissue engineering (abstract, p. 436, left col, line 1, and Fig 2a), and since Souza teaches by spatially controlling the magnetic field, the geometry of the cell aggregates can be manipulated, and levitated three-dimensional culture with magnetized hydrogels more closely recapitulates in vivo protein expression and is more feasible for long-term multicellular studies (abstract), one of ordinary skill in the art would have had a reason to make this modification in order to form three-dimensional alveoli-like clusters to develop an in vitro 3D lung model that closely recapitulates in vivo protein expression and is more feasible for long-term multicellular studies (Souza, abstract).
Regarding the newly recited limitation of the diameter of the 3D alveoli-like clusters, as stated supra, Lewis teaches the epithelial cysts are useful as in vitro 3D models of the distal airway epithelium (p. 830, right col, last sentence above and 1st sentence in “Conclusion”), and demonstrates an exemplary mouse lung distal airway tissue section showing multiple cysts in which there are at least 6 alveoli along one axis within the optical field (see Fig 7E and legend). Lewis teaches human lung alveolar size in vivo is about 200 μm in diameter (p. 822, para 1). Thus, Lewis suggests that human lung distal airway tissue has at least a diameter of 1200 μm (i.e., 200 μm x 6).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of making an in vitro 3D model of human lung distal airway by magnetic levitation suggested by Lewis in view of Sugaya and Souza, by choosing the size of the 3D alveoli-like clusters being 1200 μm to 2200 μm as suggested by Lewis with a reasonable expectation of success. Since Lewis demonstrates the distal airway tissue comprises multiple cysts (at least 6 alveoli along one axis shown in Fig 7E) and human lung alveolar size is about 200 μm in diameter (p. 822, para 1), one of ordinary skill in the art would have had a reason to choose the 3D alveoli-like clusters having a diameter of 1200 μm to 2200 μm to mimic the human lung distal airway tissue. Furthermore, since Sugaya teaches cell-microsphere spheroids having a diameter of about 300 μm are effectively collected by magnetic force (e.g., p. 437, left col, last para and Fig 4d), and since Souza teaches long-term magnetically levitated culture results in large cell masses having a diameter of at least 1600 μm (see Supplementary Fig S8b in p. 13 of Supplementary Information, showing a cell mass having a diameter at least 8 times of the 200-μm scale bar), one of ordinary skill in the art would have had a reasonable expectation of success in incorporating magnetic particles in Lewis’ polymer microspheres having a diameter of 116.1 μm to about 300 μm, and magnetically levitating the microspheres to form three-dimensional alveoli-like clusters having a diameter of 1200 μm to 2200 μm.
With respect to the magnetic particle having a diameter of about 100 nm to about 500 nm; however, Sugaya teaches the paramagnetic particles have a diameter of 50 nm (p. 436, left col, para 3.3, line 2), and Souza teaches the magnetic iron oxide nanoparticles have a size of less than 50 nm (p. 295, right col, para “Hydrogel self-assembly”).
Kolhatkar teaches production of magnetic particles with a diameter of 100−325 nm (abstract, see Fig 2 and Table 1 for size range) and teaches “larger nanoparticles (>80 nm) exhibit higher magnetization and are thus preferred for signal amplification considerations” (p. 8011, right col, last para, line 9).
Therefore it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro tissue model comprising magnetically levitating the cell-coated microspheres comprising magnetic particles to form aggregates suggested by Lewis in view of Sugaya and Souza, by substituting with large magnetic particles with a diameter of 100-325 nm as taught by Kolhatkar with a reasonable expectation of success. Since Kolhatkar teaches larger nanoparticles exhibit higher magnetization and are preferred (p. 8011, right col, last para, line 9), yet would still be encompassed by the polymer microspheres of Lewis (having an average diameter of 180 ± 80 μm, p. 828, last para, see e.g. Fig 5-6), one of ordinary skill in the art would have had a reason to make this substitution in order to obtain higher magnetization to support three-dimensional clustering by magnetic levitation. Furthermore, it would have been obvious to choose the claimed diameter, since it has been held that where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. In re Aller, 105 USPQ 233.
With respect to encapsulating and incubating the alveoli-like clusters in an encapsulating matrix, Lewis teaches “cell-laden microspheres were encapsulated in thiol–ene gels and cultured for one day to enable formation of cellular attachments to the encapsulating gel” (p. 824, right col, line 1).
With respect to the polymer microspheres, Lewis teaches a multifunctional monomer PEG tetrathiol (4-arm PEG-SH) in the microspheres (p. 822, right col, last para, line 3, see Fig 2A) and teaches the 4-arm PEG4SH of about 5000 D, thus teaches m is 2 and the number of ethylene glycol units (n) is about 30 (5000g/mol/4arms divided by about 44g/mol for each ethylene glycol unit) in claim 75(a). Thus, Lewis teaches claims 73, 74(a), 75(a) and 95. Lewis teaches laminin, fibronectin, and CRGDS peptide in the microspheres (matrisome proteins. p. 823, left col, para 1, line 7, see Fig 2A), thus teaches one peptide segment. Lewis teaches a photodegradable crosslinker, PEG di-photodegradable acrylate (PEGdiPDA) in microspheres (p. 822, right col, last para, line 1, see Fig 2A), thus teaches one degradable crosslinker.
With respect to the encapsulating matrix material, Lewis teaches a multifunctional monomer, an 8-arm PEG-norbornene in the encapsulating matrix (note that the 8-arm PEG-norbornene can also be viewed as a non-degradable crosslinker) and it has molecular weight of ∼40 000 Da (p. 823, right col, “Thiol-ene hydrogel synthesis” para, see Fig 3A), thus teaches m is 6, and the number of ethylene glycol units (n) is about 114 (40000g/mol/8arms divided by about 44g/mol for each ethylene glycol unit) in claim 75(b). Lewis teaches a degradable crosslinker, an enzymatically-cleavable di-cysteine peptide in the encapsulating matrix (p. 823, right col, “Thiol-ene hydrogel synthesis” para, line 2, see Fig 3A), and a CRGDS peptide segment in the encapsulating matrix (p. 823, right col, “Thiol-ene hydrogel synthesis” para, line 9, see Fig 3A).
However, Lewis does not teach the multifunctional monomer of the encapsulating matrix material comprises alpha-methacrylate functionality in claims 73, 75(b) and 95, nor teach the monomer being PEG independently functionalized with at least two functional moieties in claim 74(b) or the non-degradable crosslinker comprises PEG functionalized with thiol in claim 78.
Tong teaches an end group structure of PEG for hydrolysis-resistant biomaterials (title, abstract). Tong teaches “the structure in which the PEG chain is linked to the α-carbon atom of a methacrylate (MA) group and forms an α-PEG-MA structure (Scheme 1)” (p. 1513, left col, para 3, line 1). Tong further teaches α-PEG-MA and a non-degradable crosslinker PEG-SH are mixed to generate a hydrogel, and the hydrogel has less swelling ratio (Fig 4) and is “much more resistant to hydrolysis” (p. 1516, left col, para 2, last sentence, Supplemental Fig S3) than the control PEG-MA because the control “hydrogel structure contains ester linkages at the crosslink points, and their hydrolysis (half time of the ester bonds in the water-rich environment is on the order of days at pH 7.4 and 37 °C) will result in disintegration of the gel” (p. 1513, left col, para 1, line 11). Tong teaches the hydrogel made from a monomer α-PEG-MA and a crosslinker PEG-SH is “hydrolysis-stable and, hence, suitable for long-term applications” (p. 1516, left col, “conclusion” para, line 6). Thus, Tong teaches a multifunctional monomer of the encapsulating matrix material comprises alpha-methacrylate functionality in claims 73, 75(b) and 95, a non-degradable crosslinker comprises PEG functionalized with a thiol in claim 78. Note that the non-degradable crosslinker PEG-SH can also be viewed as a multifunctional monomer, thus Tong also teaches a monomer being PEG independently functionalized with at least two functional moieties alpha-methacrylate and thiol in claim 74(b).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro 3D tissue model comprising a microsphere composition and an encapsulating matrix material comprising a multifunctional monomer PEG-norbornene suggested by Lewis in view of Sugaya, Souza and Kolhatkar, by combining an α-PEG-MA monomer and a non-degradable PEG-SH crosslinker taught by Tong with a reasonable expectation of success. One of ordinary skill in the art would have had a reason to make this modification for several reasons: (1) Lewis teaches the encapsulating matrix is important for stabilizing the alveoli model (p. 822, right col, para 2, lines 1&11), but the hydrogel of PEG-norbornene contains ester linkages at the crosslink points making it sensitive to hydrolysis (compare the similar ester structure in PEG-norbornene in Fig 3A (left) with Tong’s control PEG-A in Scheme 1 (right) below):
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(2) Tong teaches the hydrogel made from a monomer α-PEG-MA and a crosslinker PEG-SH is “hydrolysis-stable and, hence, suitable for long-term applications” (p. 1516, left col, “conclusion” para, line 6). Thus, combining an α-PEG-MA monomer and a non-degradable PEG-SH crosslinker as taught by Tong would improve the stability of the encapsulating matrix of Lewis to make a long-term alveoli model.
However, although Tong teaches a thiol/alpha-methacrylate polymer forming system in Scheme 1 (a monomer comprising an alpha-methacrylate functional group and a crosslinker comprising a thiol group, related to claim 89) that can be added to Lewis’ encapsulating matrix, Lewis, Sugaya, Souza, Kolhatkar and Tong do not teach a dual stage curing process to obtain a greater elastic modulus in claims 73 and 95, nor teach a Michael addition reaction in the first polymerization stage or an off-stoichiometric amount of the monomer and crosslinker in encapsulating matrix in claim 88.
Nair teaches a thiol/acrylate two-stage polymer network forming system (title, abstract). Nair teaches excess acrylate functional monomer is used (also see Table 2, related to being off-stoichiometric where the monomer is greater than the crosslinker in claim 88), and “a first stage polymer network is formed via a 1 to 1 stoichiometric thiol-acrylate Michael addition reaction (stage 1)” (related to claim 88) and “the excess acrylate functional groups are homopolymerized via a photoinitiated free radical polymerization to form a second stage polymer network (stage 2)” (abstract, see Fig 1, related to a dual stage curing process). Nair teaches the rubbery modulus (i.e., elastic modulus) is increased after stage 2 polymerization, ranging from 2.5 to 18 fold (p. 2432, right col, para 1, Fig 3b, related to greater elastic modulus after the second polymerization in claims 73 and 95).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro tissue model comprising a microsphere composition and an encapsulating matrix material comprising a multifunctional monomer PEG-alpha-methacrylate and a non-degradable PEG-SH crosslinker suggested by Lewis, in view of Sugaya, Souza, Kolhatkar and Tong, by adding a dual stage curing process as taught by Nair with a reasonable expectation of success. Since Lewis teaches “the elastic modulus of the encapsulating gel is also tunable by … changing the stoichiometric ratio of the thiol groups on the peptide crosslinker to the ene groups on the PEG” to result in the same range of the elastic modulus in healthy lung tissue (p. 827, left col, line 7) and this model can be used in “screening potential therapeutics for treating diseases, such as lung fibrosis” (p. 831, left col, line 2), which is known to have higher elastic modulus than healthy lung tissue, and since Nair teaches the dual stage curing process increases elastic modulus of the polymer (p. 2432, right col, para 1, Fig 3b), one of ordinary skill in the art would have been motivated to make this combination in order to adjust the elastic modulus of the encapsulating matrix to mimic both the healthy and pathological conditions of lungs for better modeling.
However, Lewis, Sugaya, Souza, Kolhatkar, Tong and Nair are silent on using human primary pulmonary cells in claim 73.
Nevertheless, Lewis uses human cells (A549 adenocarcinoma line) and primary ATII cells (from mouse) to make an in vitro alveoli model (p. 822, right col, para 2), and acknowledges adult human ATII cells have been used in forming cysts in culture (see Introduction section in p. 822, left col, para 1). Lewis teaches this model is a valuable tool for “screening potential therapeutics for treating diseases, such as lung fibrosis” (abstract and p. 831, left col, line 2).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing human cell line cells or mouse primary pulmonary cells in an in vitro alveoli model suggested by Lewis in view of Sugaya, Souza, Kolhatkar, Tong and Nair, by substituting with human primary pulmonary cells (e.g., human ATII cells) as suggested by Lewis with a reasonable expectation of success. Since Lewis acknowledges the human cell line cells have significant genetic modifications (p. 822, left col, para 1) and aims to develop a model relevant to human alveoli (p. 828, last para.) for screening potential therapeutics for treating diseases (abstract and p. 831, para 1), one of ordinary skill in the art would have had a reason to substitute with human primary pulmonary cells in order to develop a model relevant to human alveoli to screen therapeutics for treating human patients.
With respect to claim 76, directed to the peptide segment being a matrisome protein, Lewis teaches laminin, fibronectin, and CRGDS peptide in the microspheres (matrisome proteins. p. 823, left col, para 1, see Fig 2A) and CRGDS peptide in the encapsulating matrix (p. 823, right col, “Thiol-ene hydrogel synthesis” para, and Fig 3A).
With respect to claim 77 directed to further comprising additional cells of fibroblasts or endothelial cells, Lewis teaches “this model is particularly suited for co-culture experiments with epithelial cysts surrounded by a second cell type such as pulmonary fibroblasts or endothelial cells, both of which play key roles during lung development and disease progression” (p. 830, last para and abstract).
Accordingly, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing human primary pulmonary cells in an in vitro alveoli model suggested by Lewis in view of Sugaya, Souza, Kolhatkar, Tong and Nair, by combining additional cells of fibroblasts or endothelial cells as suggested by Lewis with a reasonable expectation of success. Since Lewis suggests co-culture with pulmonary fibroblasts or endothelial cells in the in vitro alveoli model because both play key roles during lung development and disease progression (p. 830, last para and abstract), one of ordinary skill in the art would have had a reason to combine the suggested cell types in order to use the in vitro alveoli model to study lung development and disease progression (Lewis, abstract).
With respect to claim 79 directed to the degradable crosslinker being an enzyme-degradable crosslinker or a photodegradable crosslinker, Lewis teaches a photodegradable crosslinker, PEG di-photodegradable acrylate (PEGdiPDA) in microspheres (p. 822, right col, last para, line 1, see Fig 2A) and an enzymatically-cleavable di-cysteine peptide in the encapsulating matrix (p. 823, right col, “Thiol-ene hydrogel synthesis” para, line 2, see Fig 3A).
With respect to claim 84 directed to the polymer microsphere being solid and cells being cultured on the surface of the microsphere, Lewis teaches the microspheres are “solid gels” (p. 825, right col, line 9 from bottom) and cells are cultured “to enable full coating of the microspheres” (p. 824, left col, “Cell-microsphere seeding” para) thus teaches cells being cultured on the surface of the microspheres.
With respect to claim 85 directed to the polymer microspheres being monodispersed, as stated supra, the term “monodisperse” is defined as a particle based composition comprising particles that are substantially uniform in size, shape and mass (specification, p. 16) and is equivalent to the phrase “uniformly dispersed” in claim 73, and the specification and claim further exemplify the phrase “uniformly dispersed” as “having a diameter of 116.1 μm to about 300 μm”. Thus, the polymer microspheres being monodispersed is being examined as the polymer microspheres “having a diameter of 116.1 μm to about 300 μm”. As discussed above, one of ordinary skill in the art would have had a reason to choose a polymer microsphere composition having a size range of 180 ± 80 μm that is relevant to human alveoli to better mimic human alveoli in an in vitro alveoli model. Furthermore, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. See M.P.E.P. §2144.05.
With respect to claim 86 directed to the degrading the polymer microsphere while leaving the encapsulating matrix intact through exposure to UV light, Lewis teaches after “the microsphere template was eroded with 365 nm light”, “the A549 cysts remained roughly spherical in culture” (p. 828, left col, last para, line 6 from bottom, Fig 5B-E), and using ATII cells, “the microsphere templates were eroded one day after encapsulation (Fig. 6B)”, “the primary cysts also remained spherical with empty central lumens” (p. 828, right col, para 2, lines 5&9), thus discloses incubating the cells in the encapsulating matrix with exposure to UV light degrades the degradable crosslinkers thereby degrades the microspheres while leaves the encapsulating matrix intact.
With respect to claim 87 directed to testing the cells for biological markers, Lewis teaches the primary cysts are stained for the cell junction proteins β-catenin (adherens junctions) and ZO-1 (tight junctions) (p. 829, right col, last sentence, see Fig 7A-7B).
With respect to claim 93 directed to the polymer microspheres having a diameter of 198.5 μm ± 82.4 μm, and claim 94 directed to the polymer microspheres having an average diameter of about 200 μm, as stated supra, it would have been obvious for one of ordinary skill in the art to have chosen a polymer microsphere composition having a size range of 180 ± 80 μm that is relevant to human alveoli (∼200 μm) (Lewis, p. 828, last para, see e.g. Fig 5-6), in order to better mimic human alveoli in an in vitro alveoli model. Furthermore, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. See M.P.E.P. §2144.05.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 08/12/2025 are acknowledged.
Applicant argues that the combination of references fails to teach or suggest the claimed “aggregating portions of the uniformly dispersed polymer microsphere composition to form three-dimensional alveoli-like clusters having a diameter of 1200 μm to 2200 μm” in claims 73 and 95. Additionally, the combination of cited art fails to teach or suggest “wherein the cells are human primary pulmonary cells and/or human iPSC-derived cells” required by claim 73 (Remarks, p. 15-17, originally underlined).
Applicant’s arguments have been fully considered but they are not persuasive. The prior art used in the prior rejection does suggest the size of the alveoli-like clusters and use of human primary pulmonary cells in the in vitro model as discussed above. Therefore, the prior rejection set forth in the Office action mailed on 06/12/2025 is maintained, and the prior art Lewis, Sugaya and Souza have been re-applied to make obvious the newly recited limitations of the alveoli-like clusters having a diameter of 1200 μm to 2200 μm (see p. 8-9 of instant Office action) and the cells being human primary pulmonary cells (see p. 15-16 of instant Office action).
Applicant further argues the combination of cited art would not have motivated one having ordinary skill in the art to modify the teachings in Lewis to result in the claimed method because Lewis would have to be modified in numerous ways (Remarks, 17-18).
Applicant’s arguments have been fully considered but they are not persuasive. As discussed above, primary reference Lewis teaches a method of developing an in vitro lung alveoli model for studying lung development and disease progression. Sugaya suggests incorporating magnetic particles in the hydrogel and Kolhatkar suggests the claimed magnetic particle diameter so as to enable clustering of cysts to form aggregates by magnetic levitation suggested by Souza to obtain aggregates having a particular size as suggested by Lewis and practiced by Souza. Tong suggests a modification with an alpha-methacrylate functionality to stabilize the hydrogel, and Nair suggests a dual stage curing process allowing adjustment of the elastic modulus of the encapsulating hydrogel to mimic the disease status. Finally, Lewis suggests using human primary pulmonary cells for studying lung development and disease progression and for screening therapeutics for human patients. Therefore, one of ordinary skill in the art would have had a reason to modify the teachings in Lewis by combining the teachings of Sugaya, Kolhatkar, Souza, Tong and Nair to arrive at the claimed method.
Claim 75 is rejected under 35 U.S.C. 103 as being unpatentable over Lewis et al (Biomater. Sci. 2015; 3: 821-832. Cited in IDS 11/17/2021), in view of Sugaya et al (2012 International Symposium on Micro-NanoMechatronics and Human Science (MHS), Nagoya, Japan, 2012, pp. 435-438, doi: 10.1109/MHS.2012.6492486. Prior art of record), Souza et al. (Nature Nanotechnology. 2010;5:291-296 and Supplementary p. 1-17), Kolhatkar et al (ACS Omega. 2017, 2, 8010−8019. Prior art of record), Tong et al (Journal of Polymer Science: Part A: Polymer Chemistry. 2011; 49: 1513–1516. Cited in IDS 03/25/2022) and Nair et al (Polymer. 2012; 53: 2429-2434. Prior art of record), as applied to claim 73 above, and further in view of Tan et al (J Biomed Mater Res A. 2010; 92(3): 979–987. Prior art of record).
Elected species of m=6 embodiments
Claim 75 is directed to the multifunctional monomer of the polymer microspheres being a PEG-SH with PEG arms from 2-12 (m=0-10) and the number of ethylene glycol units (n) being 1-500; and the multifunctional monomer of the encapsulating matrix material being a PEG-alpha-methacrylate with 8 PEG arms (m=6) and the number of ethylene glycol units (n) being about 30.
As stated supra, Lewis teaches a PEG tetrathiol (4-arm PEG-SH) in the microspheres (p. 822, right col, last para, line 3, see Fig 2A) and an 8-arm PEG-norbornene in the encapsulating matrix (p. 823, right col, “Thiol-ene hydrogel synthesis” para, line 1, see Fig 3A, thus teaches m=6). Furthermore, since Lewis teaches the 4-arm PEG4SH of about 5000 D, thus teaches m is 2 and the number of ethylene glycol units (n) is about 30 (5000g/mol/4arms divided by about 44g/mol for each ethylene glycol unit). As stated supra, Tong teaches a hydrolysis-stable hydrogel (encapsulating matrix) comprising PEG-alpha-methacrylate and 4arm-PEG-SH (m=2).
However, Lewis, Sugaya, Souza, Kolhatkar, Tong and Nair do not specifically teach the multifunctional monomer of the encapsulating matrix material being a PEG-alpha-methacrylate with 8 PEG arms (m=6).
Tan teaches multi-arm PEG-based hydrogel for tissue engineering (title, abstract). Tan teaches the 8-arm PEG hydrogel shows a better stability in vitro than that of the 4-arm PEG hydrogel (p. 7, “conclusions” line 5, see Fig 7).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro tissue model comprising a microsphere composition and an encapsulating matrix material comprising a PEG-alpha-methacrylate monomer suggested by Lewis, Sugaya, Souza, Kolhatkar, Tong and Nair, by choosing an 8-arm PEG monomer as taught by Tan with a reasonable expectation of success. One of ordinary skill in the art would have had a reason to make this modification since Tan teaches the 8-arm PEG hydrogel shows a better stability in vitro than that of the 4-arm PEG hydrogel (p. 7, “conclusions” line 5), thus choosing an 8-arm PEG-alpha-methacrylate monomer in the hydrogel with the claimed number of ethylene glycol units would have improved the stability of the encapsulating matrix of Lewis, Tong and Nair.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 08/12/2025 are acknowledged and have been discussed above.
Claim 80 is rejected under 35 U.S.C. 103 as being unpatentable over Lewis et al (Biomater. Sci. 2015; 3: 821-832. Cited in IDS 11/17/2021) in view of Sugaya et al (2012 International Symposium on Micro-NanoMechatronics and Human Science (MHS), Nagoya, Japan, 2012, pp. 435-438, doi: 10.1109/MHS.2012.6492486. Prior art of record), Souza et al. (Nature Nanotechnology. 2010;5:291-296 and Supplementary p. 1-17), Kolhatkar et al (ACS Omega. 2017, 2, 8010−8019. Prior art of record), Tong et al (Journal of Polymer Science: Part A: Polymer Chemistry. 2011; 49: 1513–1516. Cited in IDS 03/25/2022) and Nair et al (Polymer. 2012; 53: 2429-2434. Prior art of record), as applied to claim 73 above, and further in view of Blakely et al (US PGPub 2012/0202263 A1. Prior art of record).
Claim 80 is directed to one degradable crosslinker comprising CGPQGIWGQGC peptide (SEQ ID NO:3).
As stated supra, Lewis teaches a crosslinker being an enzyme-degradable crosslinker di-cysteine peptide (KCGPQG↓IWGQCK) in the encapsulating matrix (p. 823, right col, “Thiol-ene hydrogel synthesis” para, line 1, see Fig 3A), in which the middle 8 amino acids GPQG↓IWGQ sequence is the matrix metalloproteinase (MMP) substrate and is the same as the middle 8 amino acid sequence in the instant claim.
However, Lewis, Sugaya, Souza, Kolhatkar, Tong and Nair do not teach an specific sequence of CGPQGIWGQGC.
Blakely teaches a PEG-based hydrogel comprising one bis-cysteine matrix metalloproteinase (MMP)-sensitive peptide (title, abstract). Blakely teaches a “highly degradable” (HD) peptide sequence CGPQGIWGQGCR (Blakely SEQ ID NO: 5) which comprises the amino acid sequence of instant SEQ ID NO:3 (see SCORE search 03/21/2023, .rag file, result #3). Blakely teaches “the MMP sensitive peptides are incorporated to allow for cells to naturally degrade and remodel the environment, which influences the cellular function in the scaffold” [0040].
Therefore it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro tissue model comprising a microsphere composition and an encapsulating matrix material comprising an enzyme degradable crosslinker di-cysteine peptide KCGPQGIWGQCK suggested by Lewis in view of Sugaya, Souza, Kolhatkar, Tong and Nair, by substituting with a CGPQGIWGQGC peptide taught by Blakely with a reasonable expectation of success. Since Lewis’s di-cysteine peptide comprises the same 8-amino acid MMP substrate, and Blakely teaches “the MMP sensitive peptides are incorporated to allow for cells to naturally degrade and remodel the environment, which influences the cellular function in the scaffold” [0040], one of ordinary skill in the art would have had a reason to make this modification in order to facilitate the remodeling of the encapsulating matrix and obtain a natural microenvironment for the encapsulated cells to improve the tissue model. Furthermore, unlike Lewis, Blakely demonstrates that the taught sequence can in fact be degraded and provides an enabling disclosure for doing, thereby establishing a reasonable expectation of success for the substitution for a proven sequence.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 08/12/2025 are acknowledged and have been discussed above.
Claim 81 is rejected under 35 U.S.C. 103 as being unpatentable over Lewis et al (Biomater. Sci. 2015; 3: 821-832. Cited in IDS 11/17/2021) in view of Sugaya et al (2012 International Symposium on Micro-NanoMechatronics and Human Science (MHS), Nagoya, Japan, 2012, pp. 435-438, doi: 10.1109/MHS.2012.6492486. Prior art of record), Souza et al. (Nature Nanotechnology. 2010;5:291-296 and Supplementary p. 1-17), Kolhatkar et al (ACS Omega. 2017, 2, 8010−8019. Prior art of record), Tong et al (Journal of Polymer Science: Part A: Polymer Chemistry. 2011; 49: 1513–1516. Cited in IDS 03/25/2022) and Nair et al (Polymer. 2012; 53: 2429-2434. Prior art of record), as applied to claim 73 above, and further in view of Ahmadi et al (Research in Pharmaceutical Sciences. 2015; 10(1): 1-16. Prior art of record).
Claim 81 is directed to the degradable crosslinker being a PEG photodegradable crosslinker with a SO3 and a thiol at the end.
As stated supra, Lewis teaches a photodegradable crosslinker, PEG di-photodegradable acrylate (PEGdiPDA) (p. 822, right col, last para, line 1, see Fig 2A), and teaches “the nitrobenzyl ether (NBE) groups in the PEGdiPDA crosslinker (yellow in Fig. 2A) are susceptible to cleavage by light, as depicted in Fig. 2C” (p. 825, right col, para 3, line 1), which are the same as the NBE groups in the instant Formula (II). Lewis teaches “the polymer network was formed via spontaneous base-catalyzed Michael addition between the thiol groups on PEG4SH and the acrylate groups on PEGdiPDA (Fig. 2A)” (p. 825, left col, last para, line 3).
However, Lewis, Sugaya, Souza, Kolhatkar, Tong and Nair do not teach the photodegradable crosslinker having a SO3 and a thiol group linked to the NBE group.
With respect to the SO3 group, Ahmadi teaches hydrogels are functionalized with hydrophilic groups to achieve high capacity for water absorption (p2, right col, para 1). Ahmadi teaches “hydrogel forming polymers have hydrophilic functional groups in their polymeric structure such as … sulphate (-SO3H). The hydrophilic groups enable the hydrogel to absorb water and watery fluids that results in hydrogel expansion and occupation of larger volume” (p2, right col, para 2, see Fig 1).
Therefore it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro tissue model comprising an encapsulating matrix material and a microsphere composition comprising a photodegradable crosslinker suggested by Lewis in view of Sugaya, Souza, Kolhatkar, Tong and Nair, by adding a SO3 group to the NBE group as taught by Ahmadi with a reasonable expectation of success. Since Ahmadi teaches the SO3 group is hydrophilic and enables the hydrogel to absorb water and watery fluids, one of ordinary skill in the art would have had a reason to make this modification in order to enhance the water/watery fluid absorption of the hydrogel to improve its function on cell culture.
With respect to the thiol group being linked at the end of the NBE group, as stated supra, Lewis teaches a thiol group linked to the PEG monomer and an acrylate group linked to the NBE group, thus the instant claim is a simple reversal of parts (i.e. the thiol group is linked to the NBE group instead of a PEG monomer).
Therefore, it would have been obvious to one having ordinary skill in the art at the time the invention was filed to do so since it has been held that rearranging parts of an invention involves only routine skill in the art. In re Japikse, 86 USPQ 70. Since Lewis teaches “the polymer network was formed via spontaneous base-catalyzed Michael addition between the thiol groups on PEG4SH and the acrylate groups on PEGdiPDA (Fig. 2A)” (p. 825, left col, last para, line 3), the swapping of the thiol group and the acrylate group will be predictably successful to form the polymer network via spontaneous reaction. One of ordinary skill in the art would have had a reason to make this modification in order to obtain photodegradable polymer as taught by Lewis.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 08/12/2025 are acknowledged and have been discussed above.
Claim 90 is rejected under 35 U.S.C. 103 as being unpatentable over Lewis et al (Biomater. Sci. 2015; 3: 821-832. Cited in IDS 11/17/2021) in view of Sugaya et al (2012 International Symposium on Micro-NanoMechatronics and Human Science (MHS), Nagoya, Japan, 2012, pp. 435-438, doi: 10.1109/MHS.2012.6492486. Prior art of record), Souza et al. (Nature Nanotechnology. 2010;5:291-296 and Supplementary p. 1-17), Kolhatkar et al (ACS Omega. 2017, 2, 8010−8019. Prior art of record), Tong et al (Journal of Polymer Science: Part A: Polymer Chemistry. 2011; 49: 1513–1516. Cited in IDS 03/25/2022) and Nair et al (Polymer. 2012; 53: 2429-2434. Prior art of record), as applied to claims 73 and 88 above, and further in view of Kloxin et al (Nat. Protoc. 2010, 5, 1867–1887. Prior art of record).
Claim 90 is directed to the crosslinker of the encapsulating matrix material comprising a non-degradable crosslinker and a degradable crosslinker wherein the method further comprises reducing the elastic modulus by degrading the degradable crosslinker.
As stated supra, Lewis and Tong teach the encapsulating matrix material comprises a non-degradable crosslinker PEG-SH (note that PEG-alpha-methacrylate can also be viewed as a non-degradable crosslinker) and a degradable crosslinker an enzyme-cleavable di-cysteine peptide (Lewis, p. 823, right col, “Thiol-ene hydrogel synthesis” para, line 1, see Fig 3A), thus teaches part of claim 90. With respect to the elastic modulus of the encapsulating matrix material, Lewis teaches the elastic modulus needs to be adjusted according to the modeling tissue (p. 827, left col, line 7), Tong teaches a hydrolysis-stable hydrogel suitable for long-term applications, Nair teaches a dual stage curing process that can increase the elastic modulus of the polymer.
However, Lewis, Sugaya, Souza, Kolhatkar, Tong and Nair do not specifically teach a step of reducing the elastic modulus after the dual stage curing process by degrading the degradable crosslinker.
Nevertheless, Lewis teaches degrading a degradable crosslinker allows “for softening of the gel” (p. 822, left col, last sentence – right col, first sentence). Regarding softening the encapsulating matrix, Lewis recites “previous work from our lab has demonstrated the versatility of PEG hydrogels for 3D culture of many primary cell types, in particular pioneering the use of the thiol–ene bio-click photoreaction between multi-arm PEGs functionalized with norbornene and cysteine-containing peptides (note that this refers to the encapsulating matrix hydrogel). Using a complementary photocleavage reaction, we also developed a PEG crosslinker that degrades upon exposure to single or two photon light, allowing for softening of the gel or its complete erosion on demand (ref. 45, 46)”, thus Lewis’ degrading the crosslinker for softening the gel encompasses the encapsulating matrix hydrogel mentioned before this statement.
Kloxin, being the reference #46 of Lewis recited above, teaches that “photodegradable hydrogels afford spatiotemporal control of modulus during 2D and 3D cell culture” (p. 1868, right col, para 2, see Fig 1a for light-induced reduction in polymer density of the encapsulating matrix, which is equivalent to the claimed reduction in elastic modulus), thus teaches degrading the crosslinker can be used to reduce the elastic modulus of the encapsulating matrix.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of culturing cells in an in vitro tissue model comprising a microsphere composition and an encapsulating matrix material comprising a non-degradable crosslinker and degradable crosslinker with greater elastic modulus after the dual stage curing process suggested by Lewis in view of Sugaya, Souza, Kolhatkar, Tong and Nair, by adding a step to soften the gel by degrading the degradable crosslinker as suggested by Lewis and taught by Kloxin with a reasonable expectation of success. Lewis teaches the elastic modulus needs to be adjusted according to the modeling tissue and teaches “the elastic modulus was ∼20 kPa, which is within the reported range of moduli for healthy lung tissue (i.e., 5–30 kPa) (p. 827, left col, para 1) and Nair teaches the dual stage curing process increases elastic modulus of the polymer, ranging from 2.5 to 18 fold (p. 2432, right col, para 1, Fig 3b), thus may exceed the range of elastic modulus of lung tissue. Since Lewis suggests degrading a degradable crosslinker allows “for softening of the gel”, including the encapsulating matrix (p. 822, right col, first sentence), and since Kloxin teaches light-induced reduction in polymer density of the encapsulating matrix “afford spatiotemporal control of modulus during 2D and 3D cell culture” (p. 1868, right col, para 2, see Fig 1a), one of ordinary skill in the art would have had a reason to add a degradation step as suggested by Lewis and taught by Kloxin following the dual stage curing process disclosed in the method of Lewis in view of Sugaya, Souza, Kolhatkar, Tong and Nair, in order to better mimic both the healthy and pathological conditions of lungs, which have different elastic moduli, for studying the biology of the lung epithelium, as well as screening potential therapeutics for treating diseases, such as lung fibrosis as taught by Lewis (p. 831, 1st para).
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 08/12/2025 are acknowledged and have been discussed above.
Conclusion
No claims are allowed.
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/JIANJIAN ZHU/Examiner, Art Unit 1631
/JAMES D SCHULTZ/ Supervisory Patent Examiner, Art Unit 1631