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 .
This action is in response to the papers filed on September 24, 2025.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 09/24/2025 has been entered.
The examiner acknowledges receiving an executed Declaration under 37 C.F.R. § 1.132 executed by Dr. Michiya Matsusaki on February 06, 2025 (“Matsusaki Declaration ”), and filed on 02/07/2025.
The examiner acknowledges receiving an executed Declaration under 37 C.F.R. § 1.132 executed by Dr. Michiya Matsusaki on September 24, 2025 (“ 2nd Matsusaki Declaration ”), and filed on 09/24/2025.
Claim 12 is amended and 28-31 have been newly added as set forth in Applicant’s amendments filed on 02/07/2025.
No claims were canceled. Claim 1 is independent.
Claims 1, 4-6, 8-21 and 28-31 are pending in the application and examined on the merits.
Priority
The present application is a Continuation of a national stage filing of International Application No. PCT/JP2017/006691 filed 02/22/2017
Applicant’s claim for the foreign benefit of Japanese Application 2016-030916 filed 02/22/2016 is acknowledged.
Thus, the earliest possible priority for the instant application is February 22, 2016.
Maintained objections/ Rejections in response to Applicants’ arguments or amendments
Claim Rejections - 35 USC § 103
Claim 1, 4-13, 16-18, and 22 remain rejected and claims 28-31 are newly rejected under 35 U.S.C. 103 as being unpatentable over Nishiguchi (Jan 2015. Macromol. Biosci., 15, 312–317; IDS Reference filed on 08/20/2018; Applicant’s own work) in view of Suri (Tissue Engineering Part A 16.5 (2010): 1703-1716).
This rejection has been modified in light of the amendments and arguments filed 09/24/25.
Regarding claim 1, Nishiguchi teaches L929 mouse fibroblasts and normal human dermal fibroblasts which are coated in fibronectin (i.e. ECM component) and heparin (i.e. polymeric electrolyte/polyelectrolyte/glycosaminoglycan) with a washing step in between alternating the solutions of heparin and fibronectin (Figure 1b; p. 314, Section 2.1) via the layer-by-layer method. The solution of heparin comprises Tris-HCL (i.e. cationic substance) (p. 314, Section 2.1). Thereby contacting the cells with an ECM component, polymeric electrolyte, and cationic substance. Centrifugation was performed on the mixture to remove the supernatant (i.e. portion of the liquid) thereby obtaining aggregates (1st aggregate) (p. 314, Section 2.1, 3.1). The aggregates of cells coated in FN-Hep (1st aggregate) (fibronectin-heparin) were then centrifuged in media (i.e. suspended) and repeatedly was coated in four coating/centrifuging/washing steps, thereby forming first, second, third, and fourth aggregates, the cell precipitates being stackable in form of a plurality of layers of cell precipitates…. (pg 314, col. 1, Section 2.1; Figure 4; pg 316, col. 2, Section 3.4, “then cultured for 24h”). The washing step to exchange solutions fulfills the claimed removing step. Nishiguchi teaches culturing the 3D tissue construct comprising the FN-Hep-coated cells (e.g. Figure 4; pg 316, col. 2, Section 3.4, “then cultured for 24h”). Each first, second, third, and fourth cell aggregate would necessarily be “stackable in form…”. The precipitated cell pellet after each coating/centrifuging/washing steps is itself a 3D tissue (per “the precipitating to construct a 3D tissue” (p. 314, Section 2.3).
However, while Nishiguchi teaches an ECM component, cationic solution and polymer electrolyte, Nishiguchi does not teach that all of these components are in solution together as there is a wash step between the alternating of the Hep-Tris solution and the fibronectin.
Suri teaches a cell-laden hydrogel construct comprising collagen (i.e. ECM component) and hyaluronic acid (HA) (i.e. GAG; polyelectrolyte) referenced as ColHA (Table 1, Figure 1). In the fabrication of the hydrogel, the cells are prepared in a mixture of both the collagen solution and the HA (in the form of GMHA) with HEPES (i.e. cationic substance) wherein the entirety is in solution and the cell suspension is added to the prepolymer solution to achieve the desired cell concentration in the final pre-gel solution (Figure 1, p. 1705, Cell encapsulation).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to combine all of the solution components of Nishiguchi (Heparin-Tris, Fibronectin and cells) into an initial pre-gel solution in the same manner of Suri (Collagen-HEPES, HA and cells) to produce a cell-laden hydrogel construct with a reasonable expectation of success. Both Nishiguchi and Suri utilize the claimed components of polymeric electrolytes, ECM components and cationic substances with Suri initially combining the components successfully in a solution to generate a cell-laden hydrogel construct. Therefore, an artisan would be motivated to combine all of the components of Nishiguchi at the start of the method as it is known in the art as evidenced by Suri in cell-laden hydrogels. The solutions are both made of the same components and therefore the treatment of Suri’s components is applicable to that of Nishiguchi’s wherein the solutions do not need to be separated to be added to the centrifuge tubes with the purpose of generating a cell-laden hydrogel construct.
Regarding claim 4, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches that the 1st aggregate coated in nanofilms have a high viscosity and is in a gel-like solution (i.e. slurry) form (Figure 3, Figure 1d).
Regarding claims 5 and 6, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches that the precipitating and removal of liquid/supernatant is via centrifugal separation (p. 314, 1st column).
Regarding claim 8 and 10, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches utilizing heparin (i.e. a glycosaminoglycan) in Tris-HCl buffer (Figure 1b; p. 314, Section 2.1).
Regarding claim 9, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches utilizing fibronectin as an ECM component (p. 314, Section 2.1).
Regarding claim 11-13, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches utilizing heparin at 0.2 mg/ml and fibronectin at 0.2 mg/ml at a 1:1 ratio. However, Nishiguchi does not teach utilizing these components in amounts between 0.05 and 0.1 mg/mL as required in claim 12.
MPEP 2144.05 states that generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. Bantchev teaches utilizing coatings minimally and optimizing the concentrations to eliminate the washing procedure. Therefore the optimization of the concentration of the substances at a 1:1 ratio would be obvious to one of ordinary skill in the art to reach the desired range of 0.05 to 1.0 mg/mL.
Regarding claim 13, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches a heparin to fibronectin ratio of 1:1 (p. 314, Section 2.1).
Regarding claims 16 and 17, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches that the 3D tissue has 25 layers and a thickness of 130 micrometers (p. 316, Section 3.4).
Regarding claim 18, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Nishiguchi teaches Figure 4 as shown below. The red box indicates a 100micrometer thickness and a 50micrometer width according to the scale of Nishiguchi. The blue stained cells are about 40 and therefore less than 70 cells within the measurement area.
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Regarding claims 22, as the kit only recites a polymeric electrolyte, cationic substance and extracellular matrix component to carry out the method of claim 1 which has been rejected as obvious, the references above utilize a polymeric electrolyte, cationic substance and extracellular matrix component in a method of producing 3D cell tissue and therefore would implicitly have the claimed kit.
Regarding claims 28-29, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, although the cell layers per 10 micrometers or cell thickness are not calculated, each and every limitation of the method of claim 1 is taught. Therefore, the same method steps would yield the same predictable results of the same product with a reasonable expectation of success.
Regarding claim 30, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, as interpreted below in a new claim objection a typographical error in light of the specification where the units should be mM rather than nM, Nishiguichi teaches Tris-HCl buffer at 50mM (p. 214, 1st column).
Regarding claim 30, the combination of Nishiguchi and Suri render obvious method of claim 1. Moreover, Suri teaches utilizing a collagen I solution as an ECM component and hyaluronic acid (a glycosaminoglycan) (p. 1704, 1st column).
Therefore the invention would have been prima facie obvious at the time of the effective filing date.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Nishiguchi (supra) in view of Suri (supra) as applied to claim 1 above and in further view of Nakamura et al. (2014. J Periodont Res 49: 363- 370)
As discussed in the above 103 rejection, the combined references of Nishiguchi and Suri teach a method of mixing cells, collagen (i.e. ECM component), Tris Buffer (i.e. cationic solution) and heparin (i.e. polymeric electrolyte in a solution, and forming cell aggregates in order to form 3D tissue structures.
However, regarding claim 19, these references do not teach culturing the cell aggregates in the presence of a ROCK inhibitor.
Nakamura et al. examined a Rock inhibitor’s, Y-27632, effects on the detachment of MSC cell sheets during multilayer growth, and the differentiation potential of the cells contained in the sheets (Abstract). Y-27632 is known as a highly selective ROCK inhibitor, releases cell contractions, and maintains the pluripotency of stem cells (Abstract, p. 364). Contractile force detaches cell sheets from the conventional tissue culture dishes and the detachment of cell sheets decreases their metabolic activity and the growth of cells in the sheets goes into arrest. (p. 364).
It would have been obvious to culture the microspheres and cells in the teachings of Nishiguchi and Suri in the presence of a Rock inhibitor such as Y-27632 as taught by Nakamura et al. with a reasonable expectation of success. An artisan would be motivated to do so as Y-27632 inhibits the contractile forces which detach cell sheets from tissue culture dishes and shrink the sheets resulting in a decrease in metabolic activity and thus maintains cell growth (Nakamura et al. p. 364, 369).
Therefore, the invention would have been prima facie obvious at the time of the effective filing date.
Claims 14, 15, 20 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Nishiguchi (supra) in view of Suri (supra) as applied to claim 1 above, and in further view of Nishiguchi2 (2011. Adv. Mater. 23, 3506–3510; Applicant’s own work).
As discussed above, Nishiguchi and Suri teach culturing fibroblasts coated in FN-Hep via layer-by-layer techniques which are centrifuged, aggregated and cultured in order to form 3D layered tissue. Nishiguchi additionally teaches that two types of fibroblasts (i.e. plurality of types of cells) are capable of being utilized in the layer-by-layer method (p. 314).
However, regarding claims 14, 15 and 20, these references do not disclose a plurality of types of cells within the culturing process and in the 3D tissue itself.
Nishiguchi2 teaches utilizing layer-by-layer technology to coat hFCs and HUVECs (i.e. a plurality of cell types; fibroblasts, endothelial cells) (p. 3509, Experimental Section). The coated HFCs were coated and inserted into the cell culture insert and incubated for one day to construct the 3D tissues, then HUVECs coated in the same nanofilms via layer-by-layer were seeded into the tissues (p. 3509, Experimental section; a plurality of cells in the tissue).
Based on such teachings, it would be obvious to one of skill in the art to utilize HUVECs as taught by Nishiguchi2 in addition to the fibroblasts utilized in Nishiguchi with a reasonable expectation of success in order to produce vascularized 3D tissues with a plurality of cells. Nishiguchi teaches that an inherent feature of tissues with HUVECs that has been established in previous papers is that the cells result in vascularization of the tissue via capillary networks and their 3D tissues involving coated hFC and HUVECs exhibited the same (p. 3507, last paragraph; p. 3509, Experimental Section). An artisan would be motivated to utilize these cells because Nishiguchi2 teaches that the resulting vascularized 3D tissue would provide a benefit for the surrounding cells, because cells in living tissues require endothelial tube networks within a perimeter of 100–150 μm to supply oxygen (p. 3509, 1st paragraph).
Regarding claim 21, the above combination of Nishiguchi, Suri and Nishiguchi2 provides 3D cell tissues with coated HUVECs and hFCs. Nishiguchi teaches that an inherent feature of tissues with HUVECs that has been established in previous papers is that the cells result in vascularization of the tissue via capillary networks and their 3D tissues involving coated hFC and HUVECs exhibited the same (p. 3507, last paragraph; p. 3509, Experimental Section).
Therefore, the structures of Nishiguchi, Suri, and Nishiguchi2 would have vasculature with a reasonable expectation of success.
In response to the arguments, declaration and amendments regarding the 103 rejections over Nishiguchi and Suri,
Applicant’s arguments and amendments regarding the 103 rejections have been considered, however they are not persuasive.
The Declaration under 37 CFR 1.132 filed 09/24/25 is insufficient to overcome the rejections based upon Nishiguchi and Suri as set forth in the last Office action because: the calculations are concerning Suri alone and not in combination with Nishiguchi. Suri is a secondary reference and is only utilized for the teaching of combinations and other components, not for the layers. The layers are taught in the primary reference in Nishiguchi’s data.
Applicant argues unexpectedly good tissue results regarding thickness and favorable low cell density made by the combination of all three components. Applicant further states that specifically this is achieved by utilizing Tris-HCL buffer as the cationic solution (p. 8 of Applicant’s remarks).
However, Examiner points out that the characteristics are based on a specific buffer which is not recited in the independent claim but rather the dependent claim 10 in a list of catonic substances. Thus, the scope of the invention does not match applicant’s claimed unexpected results.
Applicant additionally states that Suri does not teach the adequate thickness and cell number of the present invention as calculated in the Declaration filed 09/24/25. However, Examiner states that the rejection is based on not only the teachings of Suri but more so the teachings of components and processes by Nishiguchi in combination.
Examiner reiterates in response to applicant's argument that secondary references do not remedy or detail steps not found in the primary reference, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). Suri suggests the combination of all components in a solution therefore the process as recited presently is taught.
Applicant states that there is a difference between Nishiguchi’s components and the Collagen I and II utilized in their Tables which provide unexpected results. Furthermore, with respect to applicants' argument that, "Tables 3 and 4 of the present application indicate the difference between Applicant's Collagen I and II and fibronectin used as an extracellular matrix component, with combination of the polymeric electrolyte. For example, when the heparin which is the glycosaminoglycan (Paragraph 0025) was used as the polymeric electrolyte, the thickness of the three-dimensional cell tissue including the fibronectin is thinner than that including the Collagen I or IL Specifically, the thickness of the three-dimensional cell tissue including the fibronectin was 1.8-2.0 times (Table 3) or 2.2-2.8 times (Table 4) of a cell without the extracellular matrix component and the polymeric electrolyte while the thickness of the three-dimensional cell tissue including the collagen I or II was 3.5-5.5 times (Table 3) or 4.9-7.2 times of the cell without the extracellular matrix component and the polymeric electrolyte” being unexpected results, is not found persuasive because it is noted that the features upon which applicant relies (i.e., specific combinations of Tris, Heparin and Collagen I or II to construct a three-dimensional cell tissue according to the instant specification, Tables 3 and 4) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26USPQ2d 1057 (Fed. Cir. 1993). This is the case here. The claims do not recite the specific combinations of Tris, Heparin and Collagen I or II and their concentrations as taught in the specification. Again, claim 1 broadly recites any ECM component and any polymeric electrolyte. This is not in commensurate with the scope of the unexpected results of the invention. Instead, this is only recited in dependent claim 31.
Additionally, prior to the filing of the current claim set, another claim set was proposed on 07/28/2025 which recited specific concentrations. If these ranges additionally contribute to the overall unexpected results, they also should be amended into the independent claim to obtain the specific unexpected results.
New grounds of objections/ Rejections in response to Applicants’ arguments or amendments
Claim Objections
Claim 30 is objected to because of the following informalities:
Claim 30 recites units of nM, this is a typographical error as the units are mM in the specification where the concentration of cationic solutions is indicated by Applicant in Applicant’s remarks (para. 0023 of the instant application).
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 4-6, 8-21 and 28-31 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The term “a portion” in line 7 of claim 1 is indefinite. The metes and bounds of “a portion” is not defined by the claims nor the specification. The amount could be 1% to 99% of the total amount of the liquid in order to obtain an aggregate.
As all claims depend on independent claim 1, they too are rendered indefinite.
Conclusion
No claims are allowed.
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/ALEXANDRA F CONNORS/Examiner, Art Unit 1634 /MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634