DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-3, 6-12 and 26-35 are pending in this application.
Applicant’s amendment to the claims filed 02/25/2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed on 02/25/2026 in response to the non-final rejection mailed on 11/25/2025 are acknowledged and have been fully considered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election
The elected subject matter is
Invention I, corresponding to claims 1-3, 6-12, 26-30 and 33-34, drawn to a method for forming a microscale cell-laden matrix using an aqueous two-phase system comprising a mixture of a first material and a second material having a phase boundary between the first and second materials, classified in CPC G01N 1/30,
elected without traverse in the reply filed 05/15/2024.
Claims 31-32 are withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed 05/15/2024.
New claim 35 is directed to an invention that is independent or distinct from the invention originally claimed for the following reasons:
Newly submitted independent claim 35 is drawn to a method of evaluating fibroplasia comprising generating a cell-laden fibrin microgel in a microplate comprising using an ATPS comprising a PEG phase and a DEX phase, wherein cells and fibrinogen are partitioned in the DEX phase and thrombin diffuses from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin, replacing the ATPS with growth media comprising serum, wherein the cells deposit collagen and the cell-laden fibrin microgel contracts into a cell-ECM spheroid, and measuring a projected area of the cell-ECM spheroid,
which is patentably distinct from the elected Invention I drawn to a method for forming a microscale cell-laden matrix using an aqueous two-phase system comprising a mixture of a first material and a second material having a phase boundary between the first and second materials. In the instant case, the elected Invention I and the invention of claim 35 are distinct methods comprising different active steps that have a materially different design and mode of operation, as the method steps and intermediates of the inventions are distinct from one another. Furthermore, the inventions as claimed do not encompass overlapping subject matter. Additionally, a serious burden would be required to co-examine each of these inventions together because (a) the inventions have acquired separate status in the art due to their divergent subject matter, (b) the inventions require a different field of search (for example, searching different classes/subclasses or electronic resources, or employing different search queries), and (c) the inventions are likely to raise different non-prior art issues under 35 U.S.C. 101 and/or 35 U.S.C. 112(a).
Since applicant has received an action on the merits for the originally presented invention, this invention has been constructively elected by original presentation for prosecution on the merits. Accordingly, claim 35 is withdrawn from consideration as being directed to a non-elected invention. See 37 CFR 1.142(b) and MPEP § 821.03.
To preserve a right to petition, the reply to this action must distinctly and specifically point out supposed errors in the restriction requirement. Otherwise, the election shall be treated as a final election without traverse. Traversal must be timely. Failure to timely traverse the requirement will result in the loss of right to petition under 37 CFR 1.144. If claims are subsequently added, applicant must indicate which of the subsequently added claims are readable upon the elected invention.
Should applicant traverse on the ground that the inventions are not patentably distinct, applicant should submit evidence or identify such evidence now of record showing the inventions to be obvious variants or clearly admit on the record that this is the case. In either instance, if the examiner finds one of the inventions unpatentable over the prior art, the evidence or admission may be used in a rejection under 35 U.S.C. 103 or pre-AIA 35 U.S.C. 103(a) of the other invention.
Claims 1-3, 6-12, 26-30 and 33-34 correspond to the elected invention and are being examined on the merits.
Information Disclosure Statement
The applicant has not submitted an Information Disclosure Statement.
Claim Interpretation
This application includes one or more claim limitations that do not use the word “means,” but are nonetheless being interpreted under 35 U.S.C. 112(f), because the claim limitation(s) uses a generic placeholder that is coupled with functional language without reciting sufficient structure to perform the recited function and the generic placeholder is not preceded by a structural modifier. Such claim limitation is: “the microscale high-throughput is configured for diagnostic and/or drug discovery applications” in claim 27.
Because this claim limitation is being interpreted under 35 U.S.C. 112(f), it is being interpreted to cover the corresponding structure described in the specification as performing the claimed function, and equivalents thereof.
If applicant does not intend to have this limitation interpreted under 35 U.S.C. 112(f), applicant may: (1) amend the claim limitation to avoid it being interpreted under 35 U.S.C. 112(f) (e.g., by reciting sufficient structure to perform the claimed function); or (2) present a sufficient showing that the claim limitation recites sufficient structure to perform the claimed function so as to avoid it being interpreted under 35 U.S.C. 112(f).
Claim Rejections - 35 USC § 112(b)
Claims 1-3, 6-12, 26-30 and 33-34 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
The instant rejection is maintained, and any newly recited portions are necessitated by claim amendment.
Claim 1 (claims 2-3, 6-12 and 34 dependent therefrom) is indefinite for the recitation of “the first material” and “the second material” in lines 10-11. There is insufficient antecedent basis for these limitations in the claims.
Claims 1 (claims 2-3, 6-12 and 34 dependent therefrom) and 26 (claims 27-30 and 33 dependent therefrom) are rejected for the recitation of “the cells are activated by the thrombin”. As the claims recite no particular cell, and the specification does not disclose or define the activation of cells by thrombin, one of ordinary skill in the art would be unable to determine the recited activation of said cells. Therefore the scope of the term “activated” is indefinite.
Claim 26 (claims 27-30 and 33 dependent therefrom) is indefinite for the recitation of “the cells” in line 16. There is insufficient antecedent basis for this limitation in the claims.
The limitation “the microscale high-throughput is configured for diagnostic and/or drug discovery applications” in claim 27 invokes 35 U.S.C. 112(f). However, the written description fails to disclose the corresponding structure, material, or acts for performing the entire claimed function and to clearly link the structure, material, or acts to the function, as the disclosure is devoid of any structure that performs the function in the claim. Therefore, the claim is indefinite and is rejected under 35 U.S.C. 112(b).
Applicant may:
(a) Amend the claim so that the claim limitation will no longer be interpreted as a limitation under 35 U.S.C. 112(f);
(b) Amend the written description of the specification such that it expressly recites what structure, material, or acts perform the entire claimed function, without introducing any new matter (35 U.S.C. 132(a)); or
(c) Amend the written description of the specification such that it clearly links the structure, material, or acts disclosed therein to the function recited in the claim, without introducing any new matter (35 U.S.C. 132(a)).
If applicant is of the opinion that the written description of the specification already implicitly or inherently discloses the corresponding structure, material, or acts and clearly links them to the function so that one of ordinary skill in the art would recognize what structure, material, or acts perform the claimed function, applicant should clarify the record by either:
(a) Amending the written description of the specification such that it expressly recites the corresponding structure, material, or acts for performing the claimed function and clearly links or associates the structure, material, or acts to the claimed function, without introducing any new matter (35 U.S.C. 132(a)); or
(b) Stating on the record what the corresponding structure, material, or acts, which are implicitly or inherently set forth in the written description of the specification, perform the claimed function. For more information, see 37 CFR 1.75(d) and MPEP §§ 608.01(o) and 2181.
Claim 33 is indefinite for the recitation of phrases “fetalclone III”, “cosmic calf serum”, and “fetalgo” which is understood to be “fetalgro”. These phrases are registered trademarks being used as limitations to identify particular materials or products, and according to MPEP 2173.05(u) if the trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of the 35 U.S.C. 112(b).
Response to Remarks: beginning on the bottom of page 1 of Applicant’s response to 112(b) rejections; Applicant in summary contends the phrase “microscale high-throughput configured for diagnostic and/or drug discovery applications” in claim 27 is not intended to be interpreted under 112(f) and refers to an intended use of the microscale high-throughput recited in the preamble of claim 26.
Applicant’s remarks are considered and found not convincing. As outlined in the rejection, Applicant may state on the record the corresponding structure, material, or acts, which are implicitly or inherently set forth in the written description of the specification, perform the claimed function. However, Applicant has not provided the corresponding structure, material or acts that perform the claimed function.
Claim Rejections - 35 USC § 112(a)
Claims 1-3, 6-12, 26-30 and 33-34 are newly rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, at the time the application was filed, had possession of the claimed invention. This is a new matter rejection and is necessitated by amendment.
MPEP § 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims.” See also MPEP 714.02. MPEP § 2163.II.A.3.(b) further states, “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112, para. 1, as lacking adequate written description.” According to MPEP § 2163.I.B, “While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure” and “The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117.”
Claim 1 (claims 2-3, 6-12 and 34 dependent therefrom) as currently amended recites “the cells are activated by thrombin”. According to Applicant’s instant remarks, there is no disclosure of support for the amendments to claim 1, and there is no apparent descriptive support for the limitation “the cells are activated by thrombin” in the original application as filed. Absent descriptive support, the noted limitation is considered to introduce new matter into the claims. Applicant is invited to show support for the limitation at issue.
Claim 26 (claims 27-30 and 33 dependent therefrom) as currently amended recites “the first phase being denser than the second phase”, “portioning controls timing of thrombin diffusion into the fibrinogen” and “the cells are activated by thrombin”. According to Applicant’s instant remarks, “Support is found throughout the Specification … for example … [at] para 0045 … [and] para 0080” [p 4, para 4]. However, there is no apparent descriptive support for the limitations “the first phase being denser than the second phase”, “portioning controls timing of thrombin diffusion into the fibrinogen” and “the cells are activated by thrombin” in the original application as filed. Absent descriptive support, the noted limitation is considered to introduce new matter into the claims. Applicant is invited to show support for the limitation at issue.
Claims 26-30 and 33 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, at the time the application was filed, had possession of the claimed invention. This is a new matter rejection and is maintained from the previous office action.
MPEP § 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims.” See also MPEP 714.02. MPEP § 2163.II.A.3.(b) further states, “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112, para. 1, as lacking adequate written description.” According to MPEP § 2163.I.B, “While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure” and “The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117.”
Claim 26 (claims 27-30 and 33 dependent therefrom) as previously amended recites “an imaging device comprising a cellular image library”. According to Applicant’s instant remarks, “The Specification provides ample support for imaging device comprising a cellular imaging library … [at] para 0057 … [and] para 0060” [p 2, [para 3]. However, there is no apparent descriptive support for the limitation “an imaging device comprising a cellular image library” in the original application as filed. Absent descriptive support, the noted limitation is considered to introduce new matter into the claims. Applicant is invited to show support for the limitation at issue.
Response to Remarks: beginning on page 2, para 3 of Applicant’s response to 112(a) rejections; Applicant in summary contends there is ample support throughout the specification for the claim 26 limitation “an imaging device comprising a cellular image library” specifically at paras 0057 and 0060.
Applicant’s remarks are considered and found not convincing.
As cited by Applicant, [para 0057] of the instant specification discloses “such imaging methods can be automated by implementing a cellular imaging library in combination with artificial intelligence or machine learning methods”, and [para 0060] of the instant application discloses “automated image processing and analysis utilized a library for computer vision, machine learning, and image processing”. However, there is no disclosure of any imaging device throughout paras 0057 and 0060 as cited by Applicant, and additionally there is no disclosure of an imaging device comprising a cellular imaging library. For this reason and those set forth in the section above, the new matter rejection is maintained.
Claim Rejections - 35 USC § 103
Claims 1-3, 6-12, 26-27 and 33-34 are rejected under 35 U.S.C. 103 as being unpatentable over Moraes et al. (Biomaterials, 2013, 34:9623; cited on the Form PTO-892 mailed 07/30/2024; herein Moraes) in view of Sell et al. (Biomed Mater, 2008, 3; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Sell), and evidentiary references Corning et al. (Corning and Falcon Microplates Selection Guide, 48 pages, 2015; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Corning) and Wallace et al. (Mol Reprod Dev, 2015, 82:630; cited on the Form PTO-892 mailed 11/25/2025; herein Wallace).
The instant rejection is maintained from the previous office action, any newly recited portion is necessitated by amendment.
Claim 1 (claims 2-3, 6-12 and 34 dependent therefrom) is drawn to a method comprising:
introducing into a first phase comprising thrombin and polyethylene glycol (PEG) a second phase comprising cells, fibrinogen and a dextran (DEX),
the phases comprising an aqueous two-phase system (ATPS),
controlling enzyme-mediated crosslinking of the fibrinogen with the thrombin utilizing the ATPS; and
generating an assay of a microscale cell-laden matrix comprising the crosslinked protein, and the cells;
wherein:
the first material is a first soluble polymer;
the second material is a second soluble polymer different than the first soluble polymer;
the cells are partitioned with the fibrinogen in the dextran phase of the ATPS;
the thrombin diffuses from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin; and
the cells are activated by the thrombin.
Claim 26 (claims 27 and 33 dependent therefrom) is drawn to a method of evaluating extracellular matrix (ECM) remodeling with a microscale high-throughput, label-free, in vitro phenotypic assays comprising:
generating an ECM in microplate-compatible assays having a volume from 0.5 µL to 300 µL;
imaging, via an imaging device comprising a cellular image library, a progression of one or more remodeling events of the ECM; and
detecting, via a detection system, the one or more remodeling events of the ECM;
wherein:
the ECM is a microgel of a cell-laden fibrin matrix;
the generating comprises using an aqueous two-phase system (ATPS) to separate fibrinogen from thrombin in two distinct immiscible aqueous phases so the thrombin must diffuse across a phase interface before crosslinking;
the ATPS allows partitioning of the fibrinogen into a first of the two phases while thrombin diffuses in from a second of the two phases, the first phase being denser than the second phase;
the thrombin contacts the cells and crosslinks the fibrinogen into fibrin;
partitioning controls timing of thrombin diffusion into fibrinogen;
the cells are activated by the thrombin; and
one of the one or more remodeling events is selected from the group consisting of matrix degradation, matrix deposition, matrix growth, matrix proliferation, matrix cell invasion, matrix cell contraction, matrix cell type, and matrix cell density.
Claims 1 and 26 will be discussed with their respective dependent claims when possible.
Moraes discusses aqueous two-phase printing of cell-containing contractile collagen [title], and discloses high-throughput methods to study in vitro cell-mediated contraction of collagen matrices that provide insights into biological processes to further the production of tissue-like structures for the purposes of tissue engineering and disease pathology [p 9623, col 1, para 1].
Regarding claim 1, Moraes teaches the mixing of an ATPS to print cell-containing contractile collagen microdroplets [abstract], wherein biomolecules are selectively partitioned to the one of the phases in order to position and maintain biomolecules on living cells, wherein the first and second materials of the ATPS are PEG and DEX [p 9624, col 1, para 3 to col 2, para 1]. Specifically, Moraes teaches NIH 3T3 cells are mixed with acidic collagen-DEX and dispensed into PEG-enriched medium [p 9626, col 1, para 3], which corresponds to the partitioning of cells in the DEX phase of the ATPS.
Regarding claim 1 and the controlling of enzyme-mediated crosslinking of a protein utilizing the ATPS, Moraes teaches the construction of the ATPS comprising collagen-DEX and PEG phases wherein the collagen was allowed to crosslink fully [p 9625, col 1, para 3].
Regarding claim 1 and the limitation of generating an assay of the microscale cell-laden matrix, Moraes teaches fluorescent labeling, imaging and analysis that includes viability assays of cells encapsulated in the collagen droplets [p 9625, col 1, para 3].
Moraes does not teach the use of thrombin and fibrinogen to polymerize fibrinogen into fibrin, the diffusion of thrombin from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin; and the activation of cells by the thrombin.
Sell discloses cross-linking methods for fibrinogen scaffolds used in tissue engineering applications [title], and discusses the construction of tissue engineering scaffolds that mimic native extracellular matrix (ECM) that would avoid an immune response and provide a framework for cellular proliferation using fibrinogen [p 1, col 1, para 1, and p 2, col 1 para 4].
Regarding claim 1, Sell teaches the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links [p 2, col 1, para 1]. As Sell teaches the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links, and Moraes teaches biomolecules are selectively partitioned to the one of the phases of an ATPS in order to position and maintain biomolecules, one of skill in the art would have been motivated to include the thrombin of Sell into the PEG phase of the ATPS of Moraes and the fibrinogen of Sell into the DEX phase of the ATPS of Moraes to facilitate the crosslinking of fibrin at the interphase of the ATPS in order to construct the ECM scaffold as taught by Sell.
Regarding claim 1 and the limitations of the diffusion of thrombin from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin and the activation of cells by the thrombin, each of these limitations are considered to be effects of the recited active step of generating the ATPS. Put another way, as the combined method of Moraes and Sell performs the active steps of the method to produce an ATPS that shares substantial structural similarity to the ATPS recited in the claims, one of ordinary skill in the art would reasonably expect that the ATPS of the prior art would have the functional activity of allowing diffusion of thrombin from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin (MPEP 2112.01). In view of the indefiniteness of the phrase “the cells are activated by the thrombin”, as the combined method of Moraes and Sell puts thrombin into contact with cells, the cells are considered to be activated as recited in the claim.
In view of Moraes and Sell, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Moraes by adding the protein fibrinogen and the enzyme thrombin, as taught by Sell, to arrive at the method of claim 1. One of ordinary skill in the art would have been motivated to modify the method of Moraes by using the enzyme and protein of Sell because Sell teaches the construction of tissue engineering scaffolds using fibrinogen can mimic native extracellular matrix (ECM) that would avoid an immune response and provide a framework for cellular proliferation. One of ordinary skill in the art would have had a reasonable expectation of success because both Moraes and Sell relate to methods of producing tissue-like structures comprising cross-linked protein for the proliferation of cells.
Regarding claim 2, Moraes discloses simulations that suggest the diffusion of protein throughout an ATPS droplet over time interpreted to encompass diffusion between phases [Figure 6A], which are corroborated by experiments showing increased collagen contraction in ATPS droplets with increased exposure time to said protein as a result of the suggested large molecule diffusion [Figure 6C], wherein the diffusion of the protein of Moraes is considered to correspond to the diffusion of thrombin across the phase interphase of the claimed ATPS.
Regarding claim 3, Moraes teaches the method of producing an ATPS comprising cells in 1 µL droplets [p 9626, col 2, para 2].
Regarding claim 6, Moraes teaches the method of generating an ATPS comprising HEK 293 cells [p 9624, col 2, para 3], which are understood in the art to be epithelial cells.
Regarding claims 7 and 9, Moraes teaches that after polymerization and rinsing of the microgels, the microgels were exposed to 1 ng/mL TGF-β1 for short bursts of time [p 9628, col 2, para 2], wherein the TGF-β1 was provided as a supplement (i.e., an additive) to cell growth medium [p 9625, col 1, para 3]. As the post-polymerization microgels of Moraes correspond to the formed cell-laden matrix, the rinsing of said microgels is considered to encompass the removal of the first and second polymers.
Regarding claims 8 and 34, Moraes teaches addition of TGF-β1 [p 9628, col 2, para 2] and the imaging of a cell-laden matrix [Figure 4B].
Regarding claim 10, while the instant specification discloses a digestive agent can be “agents, such as, for example, plasminogen, plasmin, serine proteases, or other suitable digestive enzymes” [para 0054], it does not specifically define a digestive agent. Sell teaches the use of thrombin to cleave fibrinogen protein to cells [p 2, col 1, para 1] and in view of the disclosure of the specification, thrombin is considered to be a “digestive agent” in claim 10.
Regarding claim 11, Moraes teaches the imaging of a cell-laden matrix [Figure 4B].
Regarding claim 12, Moraes teaches the method of using an ATPS to detect matrix cell contraction [p 9627, col 2, para 2].
Regarding claim 26 and the limitations regarding the formation of the microgel of a cell-laden matrix that is the ECM, as discussed above Moraes teaches the mixing of an ATPS to print cell-containing contractile collagen microdroplets [abstract], wherein biomolecules are selectively partitioned to the one of the phases in order to position and maintain biomolecules on living cells, wherein the first and second materials of the ATPS are poly(ethylene) glycol (PEG) and dextran [p 9624, col 1, para 3 to col 2, para 1]. Specifically, Moraes teaches NIH 3T3 cells are mixed with acidic collagen-DEX and dispensed into PEG-enriched medium [p 9626, col 1, para 3]. Moraes further teaches the construction of the ATPS comprising collagen-DEX and PEG phases wherein the collagen was allowed to crosslink fully [p 9625, col 1, para 3].
Regarding claim 26 and the limitation of generating an ECM in microplate-compatible assays having a volume of 0.5-300 µL, Moraes teaches the formation of ATPS microdroplets is carried out using a 48-, 96- or 384-well plate, wherein collagen-DEX solutions were pipetted directly into PEG-enriched media using pipet tip magazine loaded with 96 sterile 25 µL pipet tips [p 9625, col 1, para 3], wherein the use of the 96-well plate to carry out the method is understood to correspond to the working volume of 50-200 µL per well as evidenced by Corning [p 8, green box at top of page].
Regarding claim 26 and limitation of detection, via a detection system, of ECM remodeling events, and the limitation of imaging, via an imaging device comprising a cellular image library, a progression of one or more remodeling events of the ECM, Moraes teaches the method of using an ATPS to detect matrix cell contraction [p 9627, col 1, para 2], wherein contraction can be visualized by brightfield images shown in Figure 4, which is considered to correspond to the detection via a detection system of ECM remodeling events, as well as the imaging of ECM remodeling events. Moraes additionally teaches the use of a confocal microscope to show cell spreading within the matrix, wherein confocal microscopes are understood in the art to accumulate, and therefore store digital images as evidenced by Wallace [p 2, final paragraph], which corresponds to the limitation of an imaging device comprising an image library. Therefore, the use of a confocal microscope to visualize cell spreading within a matrix is considered to correspond to the limitation of imaging via an imaging device comprising a cellular image library.
Regarding claim 26, Sell teaches the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links [p 2, col 1, para 1] as discussed above. As Sell teaches the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links, and Moraes teaches biomolecules are selectively partitioned to the one of the phases of an ATPS in order to position and maintain biomolecules, one of skill in the art would have been motivated to include the thrombin of Sell into the PEG phase of the ATPS of Moraes and the fibrinogen of Sell into the DEX phase of the ATPS of Moraes to facilitate the crosslinking of fibrin at the interphase of the ATPS in order to construct the ECM scaffold as taught by Sell.
Regarding claim 26 and the limitations of the fibrinogen phase being denser than the thrombin phase, partitioning controlling the timing of thrombin diffusion into the fibrinogen, and the cells being activated by thrombin, each of these limitations are considered to be effects of the recited active step of generating the ATPS. Put another way, as the combined method of Moraes and Sell performs the active steps of the method to produce an ATPS that shares substantial structural similarity to the ATPS recited in the claims, one of ordinary skill in the art would reasonably expect that the ATPS of the prior art would have the functional activity of the different phase densities, and the control of diffusion via partitioning (MPEP 2112.01). In view of the indefiniteness of the phrase “the cells are activated by the thrombin”, as the combined method of Moraes and Sell puts thrombin into contact with cells, the cells are considered to be activated as recited in the claim.
In view of Moraes and Sell, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Moraes by adding the protein fibrinogen and the enzyme thrombin, as taught by Sell, to arrive at the method of claim 26. One of ordinary skill in the art would have been motivated to modify the method of Moraes by using the enzyme and protein of Sell because Sell teaches the construction of tissue engineering scaffolds using fibrinogen can mimic native extracellular matrix (ECM) that would avoid an immune response and provide a framework for cellular proliferation. One of ordinary skill in the art would have had a reasonable expectation of success because both Moraes and Sell relate to methods of producing tissue-like structures comprising cross-linked protein for the proliferation of cells.
Regarding claim 27, the combined method of forming ATPS of Moraes and Sell is considered to encompass microprinting the microgel. Regarding the limitation of controlling enzymatic crosslinking, the use of the ATPS of Moraes to facilitate the crosslinking of fibrin is considered to encompass a controlled exposure of the fibrinogen of one phase to the thrombin of the other phase to facilitate crosslinking at the interphase of the ATPS. Regarding the limitation of using fibroblasts, Moraes teaches the use of NIH 3T3 cells [p 9626, col 1, para 3] which are understood in the art to be embryonic mouse fibroblasts. Regarding the limitation of the microscale high-throughput configured for diagnostic and/or drug delivery applications, Moraes teaches a method of evaluating cell-mediated contraction of matrices, wherein such assays are important to the investigation of disease pathologies [p 9623, col 1, para 1].
Regarding claim 33, Moraes teaches the method of using an ATPS to detect matrix cell contraction [p 9627, col 1, para 2], and the use of a confocal microscope in Figure 4, which is considered to correspond to a fluorescence detection system.
Therefore, the invention of claims 1-3, 6-12, 26-27 and 33-34 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 28 is rejected under 35 U.S.C. 103 as being unpatentable over Moraes and Sell as applied to claims 1-3, 6-12, 26-27 and 33-34 above, and further in view of Ignotz et al. (J Biol Chem, 1987, 262:6443; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Ignotz).
The instant rejection is maintained from the previous office action, and any newly recited portion is necessitated by claim amendment.
Claim 28 is drawn to the method of claim 26, further comprising stimulating the microgel with pro-fibrotic cytokines to evaluate mRNA expression and ECM deposition via the detecting.
The teachings of Moraes and Sell as applied to claims 1-3, 6-12, 26-27 and 33-34 are discussed above and include the detection of ECM deposition by Moraes [Figures 2A and 2B] and the addition of TGF-β1 by Moraes [p 9628, col 2, para 2], wherein TGF-β1 is disclosed as a pro-fibrotic stimulating molecule in the specification at [para 00112]. These references do not teach evaluating mRNA expression.
Ignotz discusses the regulation of mRNA levels by TGF-β1 [title], and discloses that TGF-β1 is part of a larger family of polypeptide factors controlling tissue development [p 6443, col 2, para 1] and is known to increase accumulation of ECM proteins such as fibronectin and collagen [abstract].
Regarding claim 28, Ignotz teaches the modulation of ECM components such as collagen is associated with TGF-β1, as it controls the corresponding levels of mRNA of each component [p 6443, col 1, para 3], and therefore teaches a method of treating fibroblast samples with TGF-β1 and analyzing resulting mRNA levels [p 6443, col 2, final para, and Figure 1].
In view of Ignotz, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Moraes and Sell to analyze mRNA expression, as taught by Ignotz, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Moraes and Sell by analyzing mRNA, because Ignotz teaches the modulation of ECM components is associated with TGF-β1 and its control of the mRNA levels of ECM component. One of ordinary skill in the art would have had a reasonable expectation of success because both Moraes and Ignotz relate to methods of treating cells with TGF-β1 to and analyzing the resulting ECM.
Therefore, the invention of claim 28 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 29 is rejected under 35 U.S.C. 103 as being unpatentable over Moraes and Sell as applied to claims 1-3, 6-12, 26-27 and 33-34 above, and further in view of Pins et al. (J Invest Dermatol, 2000, 114:647; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Pins).
The instant rejection is maintained from the previous office action, and any newly recited portion is necessitated by claim amendment.
Claim 29 is drawn to the method of claim 26, further comprising introducing plasmin to the cell-laden fibrin matrix, wherein the detecting comprises detecting matrix degradation.
The teachings of Moraes and Sell as applied to claims 1-3, 6-12, 26-27 and 33-34 are discussed above. These references do not teach the addition of plasmin.
Pins discusses plasmin and the rapid contraction and degradation of fibroblast populated collagen lattices [title], and discloses ECM degradation is critical to tissue remodeling for the removal of damaged tissue and provisional matrix, the facilitation of cell migration and guiding of angiogenesis during wound healing and tissue repair [p 647, col 1, para 1].
Regarding claim 29, Pins teaches a method of examining the role of plasmin in a cell-mediated remodeling of the dermis during wound repair [p 647, col 2, final para] comprising the addition of plasmin to fibroblast-populated collagen lattices [abstract] and subsequent detection of matrix degradation [Figure 1A].
In view of Pins, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Moraes and Sell to add plasmin to the cell matrix, as taught by Pins, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Moraes and Sell by adding plasmin, because Pins teaches ECM degradation is critical to tissue remodeling. One of ordinary skill in the art would have had a reasonable expectation of success because both Moraes and Pins relate to methods of treating cell-laden ECM with biomolecules that effect ECM structure.
Therefore, the invention of claim 29 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 30 is rejected under 35 U.S.C. 103 as being unpatentable over Moraes in view of Sell and Pins as applied to claims 1-3, 6-12, 26-27, 29 and 33-34 above, and further in view of Collen et al. (Blood, 1991, 78:3114; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Collen).
The instant rejection is maintained from the previous office action, and any newly recited portion is necessitated by claim amendment.
Claim 30 is drawn to the method of claim 26, wherein fibrinolysis through cell-mediated activation of exogenous plasminogen degrades the cell-laden fibrin matrix, and the detecting comprises detecting matrix degradation.
The teachings Moraes, Sell and Pins as applied to claims 1-3, 6-12, 26-27, 29 and 33-34 are discussed above. These references do not teach fibrinolysis through cell-mediated activation of exogenous plasminogen degrades the cell-laden fibrin matrix.
Collen discusses basic and clinical aspects of fibrinolysis [title], and discloses one of the main functions of the enzymatic fibrinolytic system is the dissolution of fibrin clots in the blood vessels [p 3114, col 1, para 1].
Regarding claim 30, Pins teaches the role of plasmin in a cell-mediated remodeling of the dermis during wound repair [p 647, col 2, final para] leading to matrix degradation [Figure 1A], and Collen teaches the fibrinolytic system comprises the proenzyme plasminogen that is converted to the active plasmin by plasminogen activators found in tissues [p 3114, col 1, para 1].
In view of Collen, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Moraes, Sell and Pin to add plasminogen, as taught by Collen, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Moraes, Sell and Pins by adding plasminogen, because Collen teaches one of the main functions of the enzymatic fibrinolytic system is the dissolution of fibrin clots that is facilitated by the activation of plasminogen. One of ordinary skill in the art would have had a reasonable expectation of success because both Moraes and Pins relate to methods of treating cell-laden ECM with biomolecules that effect ECM structure, and both Pins and Collen relate to methods of plasmin degradation of cell matrices.
Therefore, the invention of claim 30 would have been obvious to one of ordinary skill in the art before the effective filing date.
Response to Remarks: beginning at the bottom of page 3 of Applicant’s response to rejections under 35 USC 103; Applicant in summary contends the prior art does not teach cell activation having the thrombin, and that said thrombin-activated cells in combination with very small fibrin microstructures induce the behaviors described in the Application; Applicant further contends the prior art does not teach the critical ATPS structure of fibrinogen and cells in a DEX phase partitioned from thrombin in a PEG phase wherein thrombin diffuses into the DEX phase to contact the cells and fibrinogen, as Moraes does not teach a cell-laden fibrin matrix or thrombin-mediated crosslinking, and uses collagen rather than fibrin so there is no teaching of an enzyme that diffuses into a cell-containing phase during matrix formation, and Sell doesn’t teach an ATPS system, cells partitioned with fibrinogen in a DEX phase, or diffusion of thrombin from a PEG phase to a DEX phase to contact cells; Applicant further contends there is no motivation to modify Moraes as the ATPS of Moraes does not rely on enzymatic crosslinking, therefore one of skill in the art would not be motivated to replace collagen with fibrinogen and introduce thrombin into a separate phase.
Applicant’s remarks are considered and found not convincing.
Regarding the assertion that thrombin-activated cells in combination with very small fibrin microstructures induce the behaviors described in the Application, it is unclear to what behavior described in the Application the Applicant is referring. As the combined method of Moraes and Sell generates an ATPS wherein thrombin contacts cells and fibrinogen as stated in the rejection above, it is considered to activate said cells in a fibrin microstructure.
Regarding the assertion that the prior art does not teach the critical ATPS structure of fibrinogen and cells in a DEX phase partitioned from thrombin in a PEG phase wherein thrombin diffuses into the DEX phase to contact the cells and fibrinogen:
The teachings of the prior art are discussed in the section above. Briefly, Moraes teaches the construction of an ATPS with PEG and DEX, the DEX phase comprising cells and a protein as the basis of a system to study biological processes and produce tissue-like structures. Moraes further teaches the use of an ATPS to selectively partition biomolecules into phases to position and maintain them on cells.
As cited by Applicant, the crosslinking in the ATPS is driven by pH and not by enzyme, as Moraes teaches collagen polymerization is pH-sensitive, thermally driven, and relatively slow [p 9624, col 1, para 2]. Moraes discloses in the same section regarding pH sensitive collagen polymerization that once collagen solutions are neutralized, collagen solutions begin a gelation process with varying times. Moraes further teaches the ATPS formation comprises mixing acidic collagen-DEX phase with a PEG-enriched in culture medium [p 9626, col 1, para 3], that the collagen was selectively partitioned to the interface of the DEX and PEG phases of the ATPS [p 9625, col 2, para 3], and that droplets of acidic collagen-DEX were neutralized when contacted with the PEG-enriched media [p 9626, col 1, para 3]. While this last disclosure by Moraes is in reference to neutralizing pH to prevent collagen crosslinking before contact with cells, it underlines the point that the crosslinking is pH sensitive, and that the PEG phase of the ATPS of Moraes functions to control crosslinking initiation.
While Moraes does not teach the thrombin-fibrinogen interaction recited in the claims, Sell teaches the use of this interaction of thrombin initiating crosslinking of fibrinogen in tissue engineering applications to mimic native ECM scaffolds. While Sell does not teach the inclusion of thrombin into the PEG phase of Moraes, Moraes already teaches the partitioning of cells and a protein to be crosslinked (e.g., collagen) into a DEX phase of an ATPS and the use of a PEG layer to control crosslinking initiation at the interphase of an ATPS, therefore as Sell teaches the introduction of thrombin to crosslink fibrinogen, one of skill in the art would have been motivated to include thrombin in the PEG phase of the ATPS of Moraes that functions for controlling initiation of crosslinking, and fibrinogen (e.g., the protein to be crosslinked) with the cells in the DEX phase of the ATPS of Moraes to facilitate the crosslinking of fibrin in order to construct the ECM scaffold as taught by Sell, via utilizing the control and partitioning aspects of the ATPS as taught by Moraes. Therefore as the combined method of Moraes and Sell carries out all of the active steps of the claimed methods to produce an ATPS with substantial structural identity to the substantial structural similarity to the ATPS recited in the claims, one of ordinary skill in the art would reasonably expect that the ATPS of the prior art would have the functional activities of the ATPS as recited by the claims.
Double Patenting
Claims 1-3, 6-12, 26-27 and 33-34 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 6 and 10 of U.S. Patent No. 9,244,057 (cited on the Form PTO-892 mailed 03/07/2025; herein “patent”) in view of Moraes and Sell and evidentiary references Corning and Wallace.
The instant rejection is maintained from the previous office action, any newly recited portion is necessitated by amendment.
Regarding instant claim 1, claim 6 of the patent recites a method comprising contacting a solid support with a PEG solution comprising cells, wherein the solid support comprises dehydrated DEX (understood to be dextran), wherein the PEG solution rehydrates the dehydrated DEX and the PEG and DEX solution form an ATPS, and claim 10 of the patent recites the cells are used in an assay.
The claims of the patent do not recite thrombin in the PEG phase, fibrinogen and cells in the PEG phase, controlling enzyme-mediated crosslinking of the fibrinogen with the thrombin utilizing the ATPS, an assay of a microscale cell-laden matrix comprising the crosslinked protein, and the cells, thrombin diffusion from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin; and the activation of cells by thrombin.
Moraes discusses aqueous two-phase printing of cell-containing contractile collagen [title], and discloses high-throughput methods to study in vitro cell-mediated contraction of collagen matrices that provide insights into biological processes to further the production of tissue-like structures for the purposes of tissue engineering and disease pathology [p 9623, col 1, para 1].
Regarding claim 1, Moraes discloses the mixing of an ATPS to print cell-containing contractile collagen microdroplets [abstract], wherein biomolecules are selectively partitioned to the one of the phases in order to position and maintain biomolecules on living cells, wherein the first and second materials of the ATPS are PEG and DEX [p 9624, col 1, para 3 to col 2, para 1]. Specifically, Moraes discloses NIH 3T3 cells are mixed with acidic collagen-DEX and dispensed into PEG-enriched medium [p 9626, col 1, para 3], which corresponds to the partitioning of cells in the DEX phase of the ATPS.
Regarding claim 1 and the controlling of enzyme-mediated crosslinking of a protein utilizing the ATPS, Moraes discloses the construction of the ATPS comprising collagen-DEX and PEG phases wherein the collagen was allowed to crosslink fully [p 9625, col 1, para 3].
Regarding claim 1 and the limitation of generating an assay of the microscale cell-laden matrix, Moraes discloses fluorescent labeling, imaging and analysis that includes viability assays of cells encapsulated in the collagen droplets [p 9625, col 1, para 3].
Sell discloses cross-linking methods for fibrinogen scaffolds used in tissue engineering applications [title], and discusses the construction of tissue engineering scaffolds that mimic native extracellular matrix (ECM) that would avoid an immune response and provide a framework for cellular proliferation using fibrinogen [p 1, col 1, para 1, and p 2, col 1 para 4].
Regarding instant claim 1, Sell discloses the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links [p 2, col 1, para 1]. As Sell discloses the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links, and Moraes discloses biomolecules are selectively partitioned to the one of the phases of an ATPS in order to position and maintain biomolecules, one of skill in the art would have been motivated to include the thrombin of Sell into the PEG phase of the ATPS of Moraes and the fibrinogen of Sell into the DEX phase of the ATPS of Moraes to facilitate the crosslinking of fibrin at the interphase of the ATPS in order to construct the ECM scaffold as disclosed by Sell.
Regarding instant claim 1 and the limitations of the diffusion of thrombin from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin and the activation of cells by the thrombin, each of these limitations are considered to be effects of the recited active step of generating the ATPS. Put another way, as the combined method of the patent, Moraes and Sell performs the active steps of the method to produce an ATPS that shares substantial structural similarity to the ATPS recited in the claims, one of ordinary skill in the art would reasonably expect that the ATPS of the prior art would have the functional activity of allowing diffusion of thrombin from the PEG phase into the DEX phase to contact the cells and crosslink the fibrinogen into fibrin (MPEP 2112.01). In view of the indefiniteness of the phrase “the cells are activated by the thrombin”, as the combined method of the patent, Moraes and Sell puts thrombin into contact with cells, the cells are considered to be activated as recited in the claim.
In view of Moraes and Sell, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by using the ATPS of Moraes and the thrombin and fibrinogen of Sell to evaluate ECM remodeling as disclosed by Moraes to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by using the ATPS of Moraes and the thrombin and fibrinogen of Sell to evaluate ECM remodeling because Moraes discloses the use of crosslinked collagen in ATPSs to form a cell-laden matrix in high-throughput methods to study in vitro cell-mediated contraction of collagen matrices that provide insights into biological processes to further the production of tissue-like structures for the purposes of tissue engineering and disease pathology, and Sell discloses the construction of tissue engineering scaffolds using fibrinogen can mimic native ECM that would avoid an immune response and provide a framework for cellular proliferation. One of ordinary skill in the art would have had a reasonable expectation of success because the patent and Moraes relate to construction of ATPSs, and Moraes and Sell relate to methods of producing tissue-like structures comprising cross-linked protein for the proliferation of cells.
Regarding instant claim 2, Moraes discloses simulations that suggest the diffusion of protein throughout an ATPS droplet over time interpreted to encompass diffusion between phases [Figure 6A], which are corroborated by experiments showing increased collagen contraction in ATPS droplets with increased exposure time to said protein as a result of the suggested large molecule diffusion [Figure 6C], wherein the diffusion of the protein of Moraes is considered to correspond to the diffusion of thrombin across the phase interphase of the claimed ATPS.
Regarding instant claim 3, Moraes discloses the method of producing an ATPS comprising cells in 1 µL droplets [p 9626, col 2, para 2].
Regarding instant claim 6, Moraes discloses the method of generating an ATPS comprising HEK 293 cells [p 9624, col 2, para 3], which are understood in the art to be epithelial cells.
Regarding instant claims 7 and 9, Moraes discloses that after polymerization and rinsing of the microgels, the microgels were exposed to 1 ng/mL TGF-β1 for short bursts of time [p 9628, col 2, para 2], wherein the TGF-β1 was provided as a supplement (i.e., an additive) to cell growth medium [p 9625, col 1, para 3]. As the post-polymerization microgels of Moraes correspond to the formed cell-laden matrix, the rinsing of said microgels is considered to encompass the removal of the first and second polymers.
Regarding instant claims 8 and 34, Moraes discloses addition of TGF-β1 [p 9628, col 2, para 2] and the imaging of a cell-laden matrix [Figure 4B].
Regarding instant claim 10, while the instant specification discloses a digestive agent can be “agents, such as, for example, plasminogen, plasmin, serine proteases, or other suitable digestive enzymes” [para 0054], it does not specifically define a digestive agent. Sell discloses the use of thrombin to cleave fibrinogen protein to cells [p 2, col 1, para 1] and in view of the disclosure of the specification, thrombin is considered to be a “digestive agent” in claim 10.
Regarding instant claim 11, Moraes discloses the imaging of a cell-laden matrix [Figure 4B].
Regarding instant claim 12, Moraes discloses the method of using an ATPS to detect matrix cell contraction [p 9627, col 2, para 2].
Regarding instant claim 26 and the limitations regarding the formation of the microgel of a cell-laden matrix that is the ECM, as discussed above Moraes discloses the mixing of an ATPS to print cell-containing contractile collagen microdroplets [abstract], wherein biomolecules are selectively partitioned to the one of the phases in order to position and maintain biomolecules on living cells, wherein the first and second materials of the ATPS are poly(ethylene) glycol (PEG) and dextran [p 9624, col 1, para 3 to col 2, para 1]. Specifically, Moraes discloses NIH 3T3 cells are mixed with acidic collagen-DEX and dispensed into PEG-enriched medium [p 9626, col 1, para 3]. Moraes further discloses the construction of the ATPS comprising collagen-DEX and PEG phases wherein the collagen was allowed to crosslink fully [p 9625, col 1, para 3].
Regarding instant claim 26 and the limitation of generating an ECM in microplate-compatible assays having a volume of 0.5-300 µL, Moraes discloses the formation of ATPS microdroplets is carried out using a 48-, 96- or 384-well plate, wherein collagen-DEX solutions were pipetted directly into PEG-enriched media using pipet tip magazine loaded with 96 sterile 25 µL pipet tips [p 9625, col 1, para 3], wherein the use of the 96-well plate to carry out the method is understood to correspond to the working volume of 50-200 µL per well as evidenced by Corning [p 8, green box at top of page].
Regarding instant claim 26 and limitation of detection, via a detection system, of ECM remodeling events, and the limitation of imaging, via an imaging device comprising a cellular image library, a progression of one or more remodeling events of the ECM, Moraes discloses the method of using an ATPS to detect matrix cell contraction [p 9627, col 1, para 2], wherein contraction can be visualized by brightfield images shown in Figure 4, which is considered to correspond to the detection via a detection system of ECM remodeling events, as well as the imaging of ECM remodeling events. Moraes additionally discloses the use of a confocal microscope to show cell spreading within the matrix, wherein confocal microscopes are understood in the art to accumulate, and therefore store digital images as evidenced by Wallace [p 2, final paragraph], which corresponds to the limitation of an imaging device comprising an image library. Therefore, the use of a confocal microscope to visualize cell spreading within a matrix is considered to correspond to the limitation of imaging via an imaging device comprising a cellular image library.
Regarding instant claim 26, Sell discloses the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links [p 2, col 1, para 1] as discussed above. As Sell discloses the enzyme thrombin is used in the coagulation cascade in the bloodstream to cleave fibrinogen leading to the formation of the polymer fibrin that is stabilized by covalent cross-links, and Moraes discloses biomolecules are selectively partitioned to the one of the phases of an ATPS in order to position and maintain biomolecules, one of skill in the art would have been motivated to include the thrombin of Sell into the PEG phase of the ATPS of Moraes and the fibrinogen of Sell into the DEX phase of the ATPS of Moraes to facilitate the crosslinking of fibrin at the interphase of the ATPS in order to construct the ECM scaffold as disclosed by Sell.
Regarding instant claim 26 and the limitations of the fibrinogen phase being denser than the thrombin phase, partitioning controlling the timing of thrombin diffusion into the fibrinogen, and the cells being activated by thrombin, each of these limitations are considered to be effects of the recited active step of generating the ATPS. Put another way, as the combined method of Moraes and Sell performs the active steps of the method to produce an ATPS that shares substantial structural similarity to the ATPS recited in the claims, one of ordinary skill in the art would reasonably expect that the ATPS of the prior art would have the functional activity of the different phase densities, and the control of diffusion via partitioning (MPEP 2112.01). In view of the indefiniteness of the phrase “the cells are activated by the thrombin”, as the combined method of Moraes and Sell puts thrombin into contact with cells, the cells are considered to be activated as recited in the claim.
In view of Moraes and Sell, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by using the ATPS of Moraes and the thrombin and fibrinogen of Sell to evaluate ECM remodeling as disclosed by Moraes to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by using the ATPS of Moraes and the thrombin and fibrinogen of Sell to evaluate ECM remodeling because Moraes discloses the use of crosslinked collagen in ATPSs to form a cell-laden matrix in high-throughput methods to study in vitro cell-mediated contraction of collagen matrices that provide insights into biological processes to further the production of tissue-like structures for the purposes of tissue engineering and disease pathology, and Sell discloses the construction of tissue engineering scaffolds using fibrinogen can mimic native ECM that would avoid an immune response and provide a framework for cellular proliferation. One of ordinary skill in the art would have had a reasonable expectation of success because the patent and Moraes relate to construction of ATPSs, and Moraes and Sell relate to methods of producing tissue-like structures comprising cross-linked protein for the proliferation of cells.
Regarding instant claim 27, the combined method of forming ATPS of Moraes and Sell is considered to encompass microprinting the microgel. Regarding the limitation of controlling enzymatic crosslinking, the use of the ATPS of Moraes to facilitate the crosslinking of fibrin is considered to encompass a controlled exposure of the fibrinogen of one phase to the thrombin of the other phase to facilitate crosslinking at the interphase of the ATPS. Regarding the limitation of using fibroblasts, Moraes discloses the use of NIH 3T3 cells [p 9626, col 1, para 3] which are understood in the art to be embryonic mouse fibroblasts. Regarding the limitation of the microscale high-throughput configured for diagnostic and/or drug delivery applications, Moraes discloses a method of evaluating cell-mediated contraction of matrices, wherein such assays are important to the investigation of disease pathologies [p 9623, col 1, para 1].
Regarding instant claim 33, Moraes discloses the method of using an ATPS to detect matrix cell contraction [p 9627, col 1, para 2], and the use of a confocal microscope in Figure 4, which is considered to correspond to a fluorescence detection system.
Claim 28 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 6 and 10 of U.S. Patent No. 9,244,057 in view of Moraes and Sell as applied to claims 1-3, 6-12, 26-27 and 33-34 above, and further in view of Ignotz.
The instant rejection is maintained from the previous office action, any newly recited portion is necessitated by amendment.
The claims of the patent and the disclosures of Moraes and Sell as applied to instant claims 1-3, 6-12, 26-27 and 33-34 are discussed above and include the detection of ECM deposition by Moraes [Figures 2A and 2B] and the addition of TGF-β1 by Moraes [p 9628, col 2, para 2], wherein TGF-β1 is disclosed as a pro-fibrotic stimulating molecule in the specification at [para 00112]. The claims of the patent do not recite evaluating mRNA expression.
Ignotz discusses the regulation of mRNA levels by TGF-β1 [title], and discloses that TGF-β1 is part of a larger family of polypeptide factors controlling tissue development [p 6443, col 2, para 1] and is known to increase accumulation of ECM proteins such as fibronectin and collagen [abstract].
Regarding instant claim 28, Ignotz discloses the modulation of ECM components such as collagen is associated with TGF-β1, as it controls the corresponding levels of mRNA of each component [p 6443, col 1, para 3], and therefore discloses a method of treating fibroblast samples with TGF-β1 and analyzing resulting mRNA levels [p 6443, col 2, final para, and Figure 1].
In view of Ignotz, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by analyzing mRNA expression, as disclosed by Ignotz, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by analyzing mRNA, because Ignotz discloses the modulation of ECM components is associated with TGF-β1 and its control of the mRNA levels of ECM component. One of ordinary skill in the art would have had a reasonable expectation of success because the patent and Moraes relate to methods comprising the construction of ATPSs, and Moraes and Ignotz relate to methods of treating cells with TGF-β1 to and analyzing the resulting ECM.
Claim 29 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 6 and 10 of U.S. Patent No. 9,244,057 in view of Moraes and Sell as applied to claims 1-3, 6-12, 26-27 and 33-34 above, and further in view of Pins.
The instant rejection is maintained from the previous office action, any newly recited portion is necessitated by amendment.
The claims of the patent and the disclosures of Moraes and Sell as applied to instant claims 1-3, 6-12, 26-27 and 33-34 are discussed above. The claims of the patent do not recite the addition of plasmin.
Pins discusses plasmin and the rapid contraction and degradation of fibroblast populated collagen lattices [title], and discloses ECM degradation is critical to tissue remodeling for the removal of damaged tissue and provisional matrix, the facilitation of cell migration and guiding of angiogenesis during wound healing and tissue repair [p 647, col 1, para 1].
Regarding instant claim 29, Pins discloses a method of examining the role of plasmin in a cell-mediated remodeling of the dermis during wound repair [p 647, col 2, final para] comprising the addition of plasmin to fibroblast-populated collagen lattices [abstract] and subsequent detection of matrix degradation [Figure 1A].
In view of Pins, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent to add plasmin to the cell matrix, as disclosed by Pins, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by adding plasmin, because Pins discloses ECM degradation is critical to tissue remodeling. One of ordinary skill in the art would have had a reasonable expectation of success because the patent and Moraes relate to methods comprising the construction of ATPSs, and Moraes and Pins relate to methods of treating cell-laden ECM with biomolecules that effect ECM structure.
Claim 30 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 6 and 10 of U.S. Patent No. 9,244,057 in view of Moraes, Sell and Pins as applied to claims 1-3, 6-12, 26-27, 29 and 33-34 above, and further in view of Collen.
The instant rejection is maintained from the previous office action, any newly recited portion is necessitated by amendment.
The claims of the patent and the disclosures of Moraes, Sell and Pins as applied to instant claims 1-3, 6-12, 26-27, 29 and 33-34 are discussed above. The claims of the patent do not recite fibrinolysis through cell-mediated activation of exogenous plasminogen degrades the cell-laden fibrin matrix.
Collen discusses basic and clinical aspects of fibrinolysis [title], and discloses one of the main functions of the enzymatic fibrinolytic system is the dissolution of fibrin clots in the blood vessels [p 3114, col 1, para 1].
Regarding instant claim 30, Pins discloses the role of plasmin in a cell-mediated remodeling of the dermis during wound repair [p 647, col 2, final para] leading to matrix degradation [Figure 1A], and Collen discloses the fibrinolytic system comprises the proenzyme plasminogen that is converted to the active plasmin by plasminogen activators found in tissues [p 3114, col 1, para 1].
In view of Collen, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent to add plasminogen, as disclosed by Collen, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by adding plasminogen, because Collen discloses one of the main functions of the enzymatic fibrinolytic system is the dissolution of fibrin clots that is facilitated by the activation of plasminogen. One of ordinary skill in the art would have had a reasonable expectation of success because the patent and Moraes relate to methods comprising the construction of ATPSs, Moraes and Pins relate to methods of treating cell-laden ECM with biomolecules that effect ECM structure, and both Pins and Collen relate to methods of plasmin degradation of cell matrices.
Response to remarks: beginning on the bottom of page 2 of Applicant’s response to double patenting rejections; Applicant does not explicitly address the double patenting rejections of record, however the bona fide attempt of Applicant to reply to the non-final office action mailed 11/25/2025, in particular the rejections under 35 USC 103, is considered to correspond to a response to the double patenting rejections of record as well.
As the Applicant’s response to 35 USC 103 rejections is considered to correspond to the double patenting rejections of record as well, Applicant’s remarks are considered and found not convincing. The response to the remarks is stated above and is repeated here.
Regarding the assertion that thrombin-activated cells in combination with very small fibrin microstructures induce the behaviors described in the Application, it is unclear to what behavior described in the Application the Applicant is referring. As the combined method of the claims of the patent, Moraes and Sell generates an ATPS wherein thrombin contacts cells and fibrinogen as stated in the rejection above, it is considered to activate said cells in a fibrin microstructure.
Regarding the assertion that the prior art does not teach the critical ATPS structure of fibrinogen and cells in a DEX phase partitioned from thrombin in a PEG phase wherein thrombin diffuses into the DEX phase to contact the cells and fibrinogen:
The teachings of the prior art are discussed in the section above. Briefly, Moraes teaches the construction of an ATPS with PEG and DEX, the DEX phase comprising cells and a protein as the basis of a system to study biological processes and produce tissue-like structures. Moraes further teaches the use of an ATPS to selectively partition biomolecules into phases to position and maintain them on cells.
As cited by Applicant, the crosslinking in the ATPS is driven by pH and not by enzyme, as Moraes teaches collagen polymerization is pH-sensitive, thermally driven, and relatively slow [p 9624, col 1, para 2]. Moraes discloses in the same section regarding pH sensitive collagen polymerization that once collagen solutions are neutralized, collagen solutions begin a gelation process with varying times. Moraes further teaches the ATPS formation comprises mixing acidic collagen-DEX phase with a PEG-enriched in culture medium [p 9626, col 1, para 3], that the collagen was selectively partitioned to the interface of the DEX and PEG phases of the ATPS [p 9625, col 2, para 3], and that droplets of acidic collagen-DEX were neutralized when contacted with the PEG-enriched media [p 9626, col 1, para 3]. While this last disclosure by Moraes is in reference to neutralizing pH to prevent collagen crosslinking before contact with cells, it underlines the point that the crosslinking is pH sensitive, and that the PEG phase of the ATPS of Moraes functions to control crosslinking initiation.
While Moraes does not teach the thrombin-fibrinogen interaction recited in the claims, Sell teaches the use of this interaction of thrombin initiating crosslinking of fibrinogen in tissue engineering applications to mimic native ECM scaffolds. While Sell does not teach the inclusion of thrombin into the PEG phase of Moraes, Moraes already teaches the partitioning of cells and a protein to be crosslinked (e.g., collagen) into a DEX phase of an ATPS and the use of a PEG layer to control crosslinking initiation at the interphase of an ATPS, therefore as Sell teaches the introduction of thrombin to crosslink fibrinogen, one of skill in the art would have been motivated to include thrombin in the PEG phase of the ATPS of Moraes that functions for controlling initiation of crosslinking, and fibrinogen (e.g., the protein to be crosslinked) with the cells in the DEX phase of the ATPS of Moraes to facilitate the crosslinking of fibrin in order to construct the ECM scaffold as taught by Sell, via utilizing the control and partitioning aspects of the ATPS as taught by Moraes. Therefore as the combined method of the claims of the patent, Moraes and Sell carries out all of the active steps of the claimed methods to produce an ATPS with substantial structural identity to the substantial structural similarity to the ATPS recited in the claims, one of ordinary skill in the art would reasonably expect that the ATPS of the prior art would have the functional activities of the ATPS as recited by the claims.
Conclusion
Status of the Application:
Claims 1-3, 6-12 and 26-35 are pending.
Claims 31-32 and 35 are withdrawn.
Claims 1-3, 6-12, 26-30 and 33-34 are rejected.
No claim is in condition for allowance.
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 JOSEPH SPANGLER whose telephone number is (571)270-0314. The examiner can normally be reached M-F 7:30 am - 4:30 pm.
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/JOSEPH R SPANGLER/
Examiner
Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656
Prosecution Insights