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 .
Priority
The instant application is a Continuation of PCT/JP2023/000554 (filed 01/12/2023). Acknowledgement is made of Applicants’ claim for priority to foreign Application No. JP 2022-005014 (filed 01/17/2022).
Claim Interpretation
The following comments are made to establish broadest reasonable interpretation for the record.
Regarding claims 1, 5: These claims are each directed to a method of producing three-dimensional cellular tissues, wherein a thickness of the three-dimensional cellular tissue remains greater than 50 µm for at least 3 days after culturing the three-dimensional cellular tissue in the second medium comprising ascorbic acid and Transforming Growth Factor-β. The Specification of the instant disclosure defines the term three-dimensional cellular tissue as a three-dimensional cell aggregate (pg. 6; lines 7-8).
Further guidance per the Specification:
Specifically, the thickness of the three-dimensional cellular tissue can remain greater than 50 µm for at least 3 days after culturing in a medium containing ascorbic acid and TGF-β. In other words, the thickness of the three-dimensional cellular tissue can be maintained at greater than 50 µm for at least 3 days after the start of culturing in a medium containing ascorbic acid and TGF-β.
The thickness of a three-dimensional cellular tissue as described herein refers to the thickness of a slice obtained by cutting the three-dimensional cellular tissue along a line passing through the center of gravity in a direction perpendicular to the top of the three-dimensional cellular tissue, which is the thickness of a slice taken at substantially the center part of the three-dimensional cellular tissue. (pg. 4; lines 19-26)
…three-dimensional cellular tissue was removed from the cell culture insert, embedded in paraffin, and cut along a line passing through the center of gravity in a direction perpendicular to the top of the three-dimensional cellular tissue (top of the cell culture insert) to prepare a 6-mm thin slice. Then, the thin slice was stained with hematoxylin-eosin (HE), and the HE-stained thin slice was imaged using an optical microscope (MX51, Olympus Corporation) to measure a maximum thickness of the three-dimensional cellular tissue using ImageJ. (pg. 23, lines 12-18; and pg. 29, lines 1-7)
Therefore, keeping the above definition and guidance in mind, the thickness of the three-dimensional cellular tissue is interpreted as referring to the horizontal “slice” of the middle of the three-dimensional (3D) cellular tissue. Additionally, under broadest reasonable interpretation, the limitation which recites for at least 3 days after culturing the three-dimensional cellular tissue in the second medium is interpreted as for at least 3 days after the start of culturing in the second medium; this is based on the bolded guidance set forth above, as well as the “Measurement of Thickness of Three-Dimensional Cellular Tissues” of Experimental Examples 1 and 4 (pgs. 23, 29).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4 are rejected under 35 U.S.C. 103 as being unpatentable over Hiraoka (JP 2020-202814) in view of Fujie, et al. (JP 2011-155945), as evidenced by Lu, et al. (Cold Spring Harb Perspect Biol. 2011).
Hiraoka teaches a method for culturing a three-dimensional cell tissue (par. 0001).
Fujie, et al. teaches a method for producing a three-dimensional tissue culture (Abstract).
Regarding claim 1: Hiraoka teaches a method for culturing a three-dimensional (3D) cell tissue (par. 0014), comprising: (A) mixing cells with a cationic substance, an extracellular matrix component, and a polymer electrolyte to obtain a mixture; (B) collecting cells from the obtained mixture and forming a cell aggregate on a substrate; and (C) culturing cells in a medium (e.g., DMEM, RPMI-1640) to obtain a 3D cell tissue (pars. 0019, 0040). In an embodiment, step (A) further comprises removing a liquid portion from the obtained mixture to obtain the cell aggregate (par. 0022); the cells may be immune cells, vascular endothelial cells, lymphatic endothelial cells, fibroblasts (par. 0033). Hiraoka teaches an embodiment wherein the thickness of the 3D cell tissue is 60 µm or more (par. 0008). This reads on:
the method of producing three-dimensional cellular tissues, comprising:
mixing stromal cells with a cationic substance, an extracellular matrix component and a polyelectrolyte;
removing a liquid portion from a mixture of the stromal cells, the cationic substance, the extracellular matrix component and the polyelectrolyte such that a cell aggregate is obtained;
culturing the cell aggregate in a first medium such that a three-dimensional cellular tissue is obtained limitations recited in claim 1.
Hiraoka does not explicitly teach the thickness as remaining greater than 50 µm for at least 3 days after culturing in the second medium, nor does it teach culturing in a second medium comprising ascorbic acid and Transforming Growth Factor-β, as required by the remaining limitations of claim 1.
Regarding the medium limitation:
Hiraoka teaches the thickness of 3D cell tissue gradually becomes thinner over time (par. 0006); the thickness can decrease due to suppression of cell proliferation as well as necrosis of the cells (par. 0016). As evidenced by Lu, et al., the extracellular matrix (ECM) is a major component of the microenvironment of a cell and modulates basic cell behaviors including proliferation and cell death (pg. 1; par. 1). Further, Fujie, et al. teaches TGF-β1, TGF-β3, and ascorbic acid promote the production and secretion of ECM (par. 0055).
Therefore, it would have been prima facie obvious to a person having ordinary skill in the art to have modified the method of Hiraoka by exchanging the culture medium of step (C), after the 3D cell tissue is obtained, with a medium comprising ascorbic acid and TGF-β1 and -β3. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’. While the Hiraoka disclosure teaches the method thereof significantly suppresses a decrease in volume of the 3D cell tissue (par. 0009), the skilled artisan would nevertheless be motivated to further include culturing the 3D tissue in medium comprising ascorbic acid and TGF-β for the production and secretion of ECM (Fujie, et al.; par. 0055), to achieve an additive effect of suppressing volume loss via the modulation of proliferation and cell death by ECM (Valastyan; pg. 1). Further, as use of ascorbic acid and TGF-β in culture media is well known in the art, as evidenced by the Fujie, et al. disclosure, one would have more than a reasonable expectation of success. This renders obvious:
the culturing the three-dimensional cellular tissue in a second medium comprising ascorbic acid and Transforming Growth Factor-β limitation recited in claim 1.
Regarding the thickness limitation:
It is set forth above Hiraoka teaches an embodiment wherein the thickness of the 3D cell tissue is 60 µm or more (par. 0008). Hiraoka does not explicitly teach the thickness remains greater than 50 µm for at least 3 days of culturing; however, it does teach an embodiment wherein the 3D cell tissue is cultured in DMEM medium comprising fibroblast growth factor 2 (FGF2) for up to 168 hours (par. 0062). Thus, it stands to reason the disclosure implicitly teaches a 3D cell tissue with a thickness of 60 µm after culturing for 168 hours (i.e., 7 days).
The Patent and Trademark Office is not equipped to conduct experimentation in order to determine whether or not the thickness of the 3D cell tissue of Hiraoka remains greater than 50 µm for said culture period. Therefore, the burden of establishing novelty by objective evidence is shifted to Applicants.
Thus, the modified method of Hiraoka set forth above further renders obvious:
the wherein a thickness of the three-dimensional cellular tissue remains greater than 50 µm for at least 3 days after culturing the three-dimensional cellular tissue in the second medium comprising ascorbic acid and Transforming Growth Factor-β limitations recited in claim 1.
Regarding claim 2: Following the above discussion, Hiraoka teaches the ECM may be collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, or proteoglycan (par. 0030). This reads on:
the wherein the extracellular matrix component includes at least one component selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, and proteoglycan limitations recited in claim 2.
Regarding claims 3-4: Following the above discussion, Hiraoka teaches the polymer electrolyte may be glycosaminoglycans, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, or polyacrylic acid (par. 0028). This reads on:
the wherein the polyelectrolyte includes at least one selected from the group consisting of glycosaminoglycan, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, and polyacrylic acid limitations recited in claims 3 and 4.
Claims 5-11 are rejected under 35 U.S.C. 103 as being unpatentable over Hiraoka (JP 2020-202814) in view of Fujie, et al. (JP 2011-155945), further in view of Valastyan and Weinberg (Cell. 2011); as evidenced by Lu, et al. (Cold Spring Harb Perspect Biol. 2011).
The teachings of Hiraoka and Fujie, et al. are set forth above.
Valastyan and Weinberg (hereinafter Valastyan) teaches metastases as the end products of the multi-step cell-biological process termed the invasion-metastasis cascade (Abstract).
Regarding claims 5-6: Following the above discussion, Hiraoka teaches an embodiment wherein steps (A) and (B) are repeated to construct a 3D cell tissue having a plurality of layers, using a plurality of cell types, and wherein the thickness of the final 3D cell tissue is 250 µm or more (par. 0044). Further disclosed is an embodiment wherein the cells used to construct the 3D cell tissue are cancer cells (par. 0034).
Hiraoka does not explicitly teach the limitations of the instant claim (i.e., construction of a first 3D cellular tissue comprising stromal cells; disposing a target cell population on said 3D cellular tissue; construction and subsequent disposal of a second 3D cellular tissue comprising stromal cells on the first 3D cellular tissue and target cell population). Rather, Hiraoka discloses layering a plurality of cell aggregates to obtain a 3D cell tissue.
However, Valastyan teaches carcinomas metastasize via the invasion-metastasis cascade, whereby the epithelial cells of primary tumors initiate metastasis by invading locally through surrounding ECM and stromal cell layers ("The Invasion-Metastasis Cascade"; pg. 275).
Therefore, it would have been prima facie obvious to a person having ordinary skill in the art to have further modified the method of Hiraoka by constructing a first 3D cell tissue comprising the immune cells, vascular endothelial cells, lymphatic endothelial cells, or fibroblasts; layering carcinoma cells on said first 3D cell tissue; constructing a second 3D cell tissue comprising immune cells, vascular endothelial cells, lymphatic endothelial cells, or fibroblasts; and layering said second 3D cell tissue on the carcinoma cells. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’. One would be motivated to do so in order to generate a comprehensive invasion-metastasis cascade tissue model wherein the two stromal 3D cell tissues sandwich the layer of carcinoma cells, to recapitulate a primary tumor wherein the epithelial carcinoma cells invade the surrounding ECM and stromal cell layers, as taught by Valastyan (pg. 275). As more than 90% of cancer mortality is attributable to metastasis (Valastyan; pg. 275, par. 1), the skilled artisan would understand such a tissue model is an invaluable tool; e.g., furthering understanding of invasion-metastasis processes, target validation, drug screens. Further, as Hiraoka discloses a 3D cell tissue with multiple layers of multiple cell types, as well as the use of cancer cells in the 3D cell tissue, one would have more than a reasonable expectation of success.
Thus, the modified method set forth above renders obvious:
the method of producing three-dimensional cellular tissues, comprising:
mixing stromal cells with a cationic substance, an extracellular matrix component and a polyelectrolyte such that a first mixture is obtained;
removing a liquid portion from the first mixture of the stromal cells, the cationic substance, the extracellular matrix component and the polyelectrolyte such that a first cell aggregate is obtained;
culturing the first cell aggregate in a first medium such that a first three-dimensional cellular tissue is obtained;
disposing target cells on the first three-dimensional cellular tissue;
mixing stromal cells with a cationic substance, an extracellular matrix component and a polyelectrolyte such that a second mixture is obtained;
removing a liquid portion from the second mixture of the stromal cells, the cationic substance, the extracellular matrix component and the polyelectrolyte such that a second cell aggregate is obtained;
disposing the second cell aggregate such that the second cell aggregate is in contact with target cells; and
culturing the second cell aggregate in a second medium comprising ascorbic acid and Transforming Growth Factor-β such that a second three-dimensional cellular tissue is obtained,
wherein a total thickness of the first three-dimensional cellular tissue, the target cells and the second three-dimensional cellular tissue remains greater than 50 µm for at least 3 days after culturing the second three-dimensional cellular tissue in the medium containing ascorbic acid and Transforming Growth Factor-β limitations recited in claim 5; and
the wherein the target cells are cancer cells limitation recited in claim 6.
Regarding claims 7, 9: Following the above discussion, Hiraoka teaches the ECM may be collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, or proteoglycan (par. 0030). This reads on:
the wherein the extracellular matrix component includes at least one component selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, and proteoglycan limitations recited in claims 7 and 9.
Regarding claims 8, 10-11: Following the above discussion, Hiraoka teaches the polymer electrolyte may be glycosaminoglycans, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, or polyacrylic acid (par. 0028). This reads on:
the wherein the polyelectrolyte includes at least one selected from the group consisting of glycosaminoglycan, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, and polyacrylic acid limitations recited in claims 8, 10, and 11.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GINA PRONZATI whose telephone number is (571)270-5725. The examiner can normally be reached Monday - Friday 9:00a - 5:00p ET.
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/GINA PRONZATI/Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633
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