Fibrin gel sheet for cell transplantation

Abstract

The purpose of the present invention is to provide: a fibrin gel sheet for cell transplantation, in which cells are enclosed uniformly dispersed while at the same time the sheet has a larger size than heretofore available; and a method for producing this sheet. The present invention provides a fibrin gel sheet for cell transplantation, in which cells are enclosed uniformly dispersed in a fibrin gel sheet, wherein the sheet has a size with a surface area for one side of at least 2.25 cm2 and has a thickness of not more than 1 mm. The present invention also provides a method for producing this fibrin gel sheet for cell transplantation.

Description

Fibrin gel sheet for cell transplantation

[0001] The present invention relates to a fibrin gel sheet for cell transplantation, which has cells uniformly dispersed and encapsulated in the fibrin gel sheet and has an unprecedentedly large size, and a method for producing the same.

[0002] [Background of the Invention] Functional differentiated cells induced from pluripotent stem cells such as induced pluripotent cells and embryonic stem cells (ES cells) are expected to serve as a cell source for transplantation regenerative medicine. Currently, various structures in which such functional differentiated cells are encapsulated in biodegradable gels have been developed, and research into cell transplantation therapy using these structures is underway.

[0003] For example, Non-Patent Document 1 discloses a sheet-like structure formed by sandwiching pancreatic islet cells encapsulated in a fibrin gel between Vicryl (registered trademark) meshes measuring 7 mm x 7 mm, and describes that the structure was transplanted into a site of a recipient diabetic model rat that had previously been subjected to angiogenesis treatment.

[0004] Non-Patent Document 2 discloses a sheet-like structure formed by sandwiching pancreatic islet cells encapsulated in a hydrogel containing polyvinyl alcohol (PVA) between polyethylene terephthalate (PET) meshes measuring 20 mm x 15 mm, and describes that the structure was transplanted into a recipient diabetic model rat.

[0005] Patent Document 1 discloses a sheet-like structure formed by sandwiching pancreatic islet cells encapsulated in a hydrogel containing PVA between circular PET meshes with a diameter of 15 mm, and describes that the structure was transplanted into recipient diabetic model mice.

[0006] WO2018 / 155622

[0007] Hirotake Komatsu et al., Transplant International 2020;33:806-818. Meirigeng Qi et al., Biomaterials. 2004 December;25(27):5885-92.

[0008] On the other hand, previously reported sheet-like structures in which functional differentiated cells are encapsulated in a biodegradable gel were intended for transplantation into diabetic model animals (rats and mice), but their size was sometimes insufficient for use in large mammals such as humans, and the number of cells that could be loaded was also insufficient. Therefore, there has been a strong demand in this field for the development of such structures that are larger in size and contain more cells, making them usable in humans, etc.

[0009] To solve this problem, the inventors attempted to fabricate larger-sized constructs using fibrin gel. However, they found that when larger amounts of fibrin gel were prepared to fabricate larger-sized constructs and then spread to the desired size to form a sheet, they gelled (solidified) before the desired size could be formed, making it difficult to form the desired size. Furthermore, when attempting to form a sheet of the desired size by continuously injecting a large amount of fibrin gel, the cells in the solution precipitated, resulting in a gradual decrease in cell content from the beginning to the end of the continuous injection, resulting in the fabrication of constructs with uneven cell density. Non-uniform cell density is undesirable because it may reduce therapeutic efficacy and affect the long-term engraftment of transplanted cells.

[0010] Therefore, an object of the present invention is to solve these problems found by the present inventors, and to provide a sheet-like fibrin gel structure (i.e., a fibrin gel sheet) that has a larger size than ever before and contains a larger number of cells uniformly dispersed therein.

[0011] As a result of intensive research to solve the above problems, the present inventors have found that, in forming a fibrin gel, by adjusting the amounts of fibrinogen and thrombin and / or the temperature during gelation (solidification), it is possible to adjust the gelation (solidification) rate of the fibrin gel being formed, and to spread and mold the fibrin gel into a fibrin gel sheet having a desired larger size.

[0012] Furthermore, in forming a fibrin gel, it was found that by adding and suspending biodegradable particles to a fibrinogen solution in which cells are suspended, or by adding and dissolving a biodegradable gelling agent, it is possible to prevent cells from settling in the fibrinogen solution and maintain the cells in a uniformly dispersed and suspended state, and by gelling (solidifying) this, a fibrin gel sheet with a uniform cell density can be obtained even when molded by continuous injection.

[0013] The present invention is based on these new findings and includes the following inventions: [1] A fibrin gel sheet in which cells are uniformly dispersed and encapsulated, with a surface area of ​​2.25 cm 2 [2] A fibrin gel sheet for cell transplantation having a size of 1.5 x 10 cells or more and a thickness of 1 mm or less. 6 pieces / cm 2 [3] The fibrin gel sheet of [1], wherein the cells are in the form of spheroids. [4] The fibrin gel sheet of any of [1] to [3], wherein the cells are iPS cell-derived pancreatic islet cells. [5] The fibrin gel sheet of any of [1] to [4], further comprising a biodegradable gelling agent. [6] The fibrin gel sheet of [5], wherein the biodegradable gelling agent is 0.2 to 2 w / v %. [7] The fibrin gel sheet of [5] or [6], wherein the biodegradable gelling agent is collagen. [8] The fibrin gel sheet of any of [1] to [4], further comprising biodegradable particles. [9] The fibrin gel sheet of [8], wherein the biodegradable particles are of a size that can pass through an opening of 100 μm to 1000 μm.

[10] The fibrin gel sheet of [8] or [9], which contains biodegradable particles in an amount of 10 to 30 w / v %.

[11] The fibrin gel sheet of any of [8] to

[10] , wherein the biodegradable particles are gelatin gel particles.

[12] The fibrin gel sheet of any of [1] to

[11] , further comprising a support.

[13] The fibrin gel sheet of any of [1] to

[11] , wherein cells are uniformly dispersed and encapsulated in the fibrin gel sheet, and wherein one surface area of ​​the fibrin gel sheet is 2.25 cm. 2A method for producing a fibrin gel sheet for cell transplantation having a size of 2.25 cm or more and a thickness of 1 mm or less, comprising reacting a fibrinogen solution in which cells are suspended with thrombin to produce a fibrin gel sheet having a surface area of ​​2.25 cm or more. 2

[14] A method for producing a fibrin gel sheet, the method comprising the steps of: forming the fibrin gel sheet into a sheet having a size of 1.5 × 10 or more and a thickness of 1 mm or less, and gelling the sheet. 6 pieces / cm 2

[13] The manufacturing method of

[15] or

[14] , wherein the cells are in the form of spheroids.

[16] The manufacturing method of any of

[13] to

[15] , wherein the cells are iPS cell-derived pancreatic islet cells.

[17] The manufacturing method of any of

[13] to

[16] , wherein the fibrinogen solution in which the cells are suspended further contains a biodegradable gelling agent.

[18] The manufacturing method of

[17] , wherein the fibrinogen solution in which the cells are suspended contains the biodegradable gelling agent in an amount of 0.2 to 2 w / v %.

[19] The manufacturing method of

[17] or

[18] , wherein the biodegradable gelling agent is collagen.

[20] The manufacturing method of any of

[17] to

[19] , wherein the cells are formed into a sheet by coating on a support.

[21] The manufacturing method of

[20] , wherein the cells are coated on a support by bioprinting.

[22] The manufacturing method of

[20] or

[21] , wherein the support contains thrombin.

[23] The manufacturing method of any one of

[13] to

[16] , wherein the fibrinogen solution in which the cells are suspended further contains biodegradable particles.

[24] The manufacturing method of

[23] , wherein the biodegradable particles are of a size that can pass through an opening of 100 μm to 1000 μm.

[25] The manufacturing method of

[23] or

[24] , wherein the fibrinogen solution in which the cells are suspended contains the biodegradable particles in an amount of 10 to 30 w / v %.

[26] The manufacturing method of any one of

[23] to

[25] , wherein the biodegradable particles are gelatin gel particles.

[27] The manufacturing method of any one of

[23] to

[26] , wherein the fibrinogen solution is formed into a sheet by coating on a support.

[28] The manufacturing method of

[27] , wherein the fibrinogen solution is coated on a support by bioprinting.

[29] The manufacturing method of

[27] or

[28] , wherein the support contains thrombin.

[30] The manufacturing method of any one of

[13] to

[16] , wherein thrombin is allowed to act in an amount that results in a compounding ratio of 0.4 U or less per 1 mg of fibrinogen.

[31] The manufacturing method of

[30] , which comprises molding into a sheet under cooling at 2 to 8° C.

[32] The manufacturing method of

[30] or

[31] , which further comprises a step of integrating a support with the fibrin gel sheet.

[33] A fibrin gel sheet laminate for cell transplantation, which is formed by laminating a plurality of fibrin gel sheets according to any one of [1] to

[12] .[1a] Cells are uniformly dispersed and encapsulated in a fibrin gel sheet, with a surface area of ​​2.25 cm. 2 [2a] A fibrin gel sheet for use in a cell transplantation therapy method, having a size of 1.5 x 10 cells or more and a thickness of 1 mm or less. 6 pieces / cm 2 [3a] The fibrin gel sheet of [1a] or [2a], wherein the cells are in the form of spheroids. [4a] The fibrin gel sheet of any of [1a] to [3a], wherein the cells are iPS cell-derived pancreatic islet cells. [5a] The fibrin gel sheet of any of [1a] to [4a], further comprising a biodegradable gelling agent. [6a] The fibrin gel sheet of [5a], wherein the biodegradable gelling agent is 0.2 to 2 w / v %. [7a] The fibrin gel sheet of [5a] or [6a], wherein the biodegradable gelling agent is collagen. [8a] The fibrin gel sheet of any of [1a] to [4a], further comprising biodegradable particles. [9a] The fibrin gel sheet of [8a], wherein the biodegradable particles are of a size that can pass through an opening of 100 μm to 1000 μm. [10a] The fibrin gel sheet of [8a] or [9a], containing biodegradable particles in an amount of 10 to 30 w / v %. [11a] The fibrin gel sheet of any of [8a] to [10a], wherein the biodegradable particles are gelatin gel particles. [12a] The fibrin gel sheet of any of [1a] to [10a], further comprising a support. [33a] A fibrin gel sheet laminate for use in a cell transplantation therapy method, comprising a plurality of fibrin gel sheets of any of [1a] to [12a] laminated together. This specification includes the contents of the specification and / or drawings of Japanese Patent Application No. 2022-113546, filed July 14, 2022, from which the present application claims priority. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0014] According to the present invention, it is possible to provide a fibrin gel sheet that has a larger size than ever before and contains a larger number of cells uniformly dispersed therein, as well as a method for producing the same.

[0015] FIG. 1 is a photograph showing the appearance of (A) a fibrin gel clot containing iPIC and (B) a fibrin gel sheet containing iPIC. Scale bar: 10 mm. FIG. 2 is a graph showing the analytical results of measuring blood human c-peptide concentrations over time by ELISA in nude rats into which an iPIC-containing fibrin gel clot or an iPIC-containing fibrin gel sheet was subcutaneously implanted. FIG. 3 is a photograph showing the results of histological analysis of the implantation site 10 days and 6 weeks after implantation in nude rats into which (A) an iPIC-containing fibrin gel clot or (B) an iPIC-containing fibrin gel sheet was subcutaneously implanted. Scale bar: 1 mm. FIG. 4 is a graph showing the results of analyzing the relationship between thrombin concentration and fibrin gel gel time. Figure 5 is a photograph showing (A) the appearance of a fibrin gel sheet encapsulating large animal / human-sized iPICs produced by a method using a cooled metal mold, and (B) the results of observing positions 1 to 4 of the sheet using a phase-contrast microscope. Figure 6 is a photograph showing the state of iPICs suspended in (A) a fibrinogen solution and (B) a fibrinogen + gelatin microsphere solution and allowed to stand for a predetermined period of time. The arrowhead in (A) indicates the precipitation of iPIC. Figure 7 is a graph showing the analytical results of time-dependent ELISA measurements of blood human c-peptide concentrations in nude rats subcutaneously implanted with an iPIC-encapsulated fibrin gel sheet or an iPIC-encapsulated fibrin + gelatin microsphere gel sheet. Figure 8 shows photographs (A) of the appearance and cross-section of fibrin + gelatin microsphere gel sheets containing large animal- and human-sized iPICs prepared by manual pipette injection and using a 3D printer, the results of calculating the standard deviation by image analysis of the brightness distribution of each sheet (B), and a graph (C) showing the results of measuring the number of cells contained in four arbitrary locations (1 to 4) of the fibrin + gelatin microsphere gel sheet prepared using a 3D printer. Figure 9 is a graph showing the results of measuring the number of cells (concentration) in a fibrinogen + collagen solution discharged from a syringe.Figure 10 is a graph showing the analytical results of measuring the blood human c-peptide concentration by ELISA in nude rats subcutaneously implanted with a fibrin+gelatin microsphere gel sheet containing iPIC or a fibrin+collagen gel sheet containing iPIC. Figure 11 is a graph showing the results of measuring the number of cells contained in six arbitrary locations (1 to 6) of a fibrin+collagen gel sheet containing large animal / human-sized iPIC fabricated using a 3D printer.

[0016] 1. Terms The terms used in this specification are explained below.

[0017] As used herein, "about" refers to a value that varies by plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%, respectively, from the reference value. Preferably, the term "about" refers to a range of plus or minus 15%, 10%, 5%, or 1%, respectively, from the reference value.

[0018] As used herein, "comprise(s)" or "comprising" means the inclusion, but not limitation, of the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements.

[0019] As used herein, the phrase "consist(s) of" or "consisting of" means inclusive of and limited to all elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that other elements are substantially absent.

[0020] As used herein, "feeder cell-free" means that feeder cells are not essentially contained, and that a medium preconditioned by culturing feeder cells is not used. Therefore, the medium does not contain substances such as growth factors and cytokines secreted by feeder cells.

[0021] The term "feeder cells" or "feeders" refers to cells that are co-cultured with other types of cells and provide an environment in which the cells can grow and support them. Feeder cells may be derived from the same or a different species as the cells they support. For example, human dermal fibroblasts or human embryonic stem cells may be used as feeders for human cells, as well as primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. Feeder cells can be inactivated by irradiation or mitomycin C treatment, for example.

[0022] As used herein, "adherent" refers to cells that are attached to a container, e.g., cells that are attached to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate culture medium. Some cells cannot be maintained or grown in culture unless they are attached to a cell culture container. In contrast, non-adherent cells can be maintained and grown in culture without being attached to a container.

[0023] As used herein, "culture" refers to maintaining, growing, and / or differentiating cells in an in vitro environment. "Culturing" refers to maintaining, expanding, and / or differentiating cells in a tissue or outside the body, for example, in a cell culture dish or flask. Culture includes two-dimensional culture (plate culture) and three-dimensional culture (suspension culture).

[0024] As used herein, "purify" and "purification" refer to the removal of impurities from a composition, such as a cellular composition, to render the composition pure for a particular component. "Purified," when used to describe a cellular composition, refers to a cellular composition in which the amount of impurities is reduced compared to the proportion of such component in the cellular composition prior to purification, thereby increasing the purity of the particular component. For example, a cellular composition can be purified for a target cell type, such that the proportion of the target cell type is increased compared to the proportion of target cells present in the cellular composition prior to purification. A cellular composition can also be purified for a target cell type by cell selection and sorting methods known in the art. A cellular composition can also be purified by a specific sorting or selection process described herein. In certain embodiments of the invention, the method of purifying target cells can result in target cell purity of at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or no detectable impurities (including contaminating cells).

[0025] As used herein, a "growth factor" refers to an endogenous protein that promotes the differentiation and / or proliferation of specific cells. Examples of "growth factors" include epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), transferrin, various interleukins (e.g., IL-1 to IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ, etc.), and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0026] As used herein, the term "ROCK inhibitor" refers to a substance that inhibits Rho kinase (ROCK: Rho-associated, coiled-coil containing protein kinase), and may be a substance that inhibits either ROCK I or ROCK II. The ROCK inhibitor is not particularly limited as long as it has the above-mentioned function, and examples thereof include N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (sometimes referred to as Y27632 in this specification), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1α-carboxamide (Wf-536), N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4PER[( Examples include N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]oxy}benzamide (GSK269962A), and N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A). ROCK inhibitors are not limited to these, and antisense oligonucleotides and siRNAs against ROCK mRNA, antibodies that bind to ROCK, dominant-negative ROCK mutants, and the like can also be used as ROCK inhibitors, and these are commercially available or can be synthesized according to known methods.

[0027] As used herein, a "GSK3β inhibitor" refers to a substance that has inhibitory activity against GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a type of serine / threonine protein kinase and is involved in many signal pathways related to glycogen production, apoptosis, stem cell maintenance, and the like. GSK3 exists in two isoforms, α and β. The "GSK3β inhibitor" used in the present invention is not particularly limited as long as it has GSK3β inhibitory activity, and may be a substance that has both GSK3β inhibitory activity and GSK3α inhibitory activity.

[0028] GSK3β inhibitors include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidine), (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), Examples of such an alkyl ester include 3-(2,4-dichlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy]phenol), Kenpaullone, 1-azakempaullone, SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), AR-AO144-18, CT99021, CT20026, BIO, BIO-acetoxime, pyridocarbazole-cyclopentadienyl ruthenium complex, OTDZT, alpha-4-dibromoacetophenone, and lithium. GSK3β inhibitors are not limited to these, and antisense oligonucleotides and siRNAs against GSK3β mRNA, antibodies that bind to GSK3β, dominant-negative GSK3β mutants, etc. can also be used as GSK3β inhibitors, and these are commercially available or can be synthesized according to known methods.

[0029] As used herein, "serum replacement" refers to, for example, KnockOut™ Serum Replacement (KSR: Thermo Fisher Scientific), StemSure® Serum Replacement (Wako), B-27 supplement, N2-supplement, albumin (e.g., lipid-rich albumin), insulin, transferrin, fatty acids, collagen precursors, trace elements (e.g., zinc, selenium (e.g., sodium selenite)), 2-mercaptoethanol, 3'-thiolglycerol, or mixtures thereof (e.g., ITS-G). Preferred serum replacements are B-27 supplement, KSR, StemSure® Serum Replacement, and ITS-G. When a serum substitute is added to a medium, the concentration in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight. In the present invention, it is preferable to use a "serum substitute" instead of serum.

[0030] As used herein, "factors having CDK8 / 19 inhibitory activity" refers to any substance that has inhibitory activity against CDK8 / 19. In contrast to other proteins in the same CDK family, CDK8 is not required for cell proliferation, and inhibition of CDK8 does not have a significant effect under normal conditions. CDK19 and CDK8 are similar, and CDK8 inhibition is usually accompanied by inhibition of CDK19 as well. Conventionally known "factors having CDK8 / 19 inhibitory activity" can be used, and such CDK8 / 19 inhibitors can be found in patent literature or non-patent literature. For example, among the compounds described in US2012 / 0071477, WO2015 / 159937, WO2015 / 159938, WO2013 / 116786, WO2014 / 0038958, WO2014 / 134169, JP2015 / 506376, US2015 / 0274726, US2016 / 0000787, WO2016 / 009076, WO2016 / 0016951, WO2016 / 018511, WO2016 / 100782, and WO2016 / 182904, compounds having CDK8 / 19 inhibitory activity or salts thereof can be used as the "factor having CDK8 / 19 inhibitory activity" in the present invention. For example, in the present invention, diethyl (E)-(4-(3-(5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamido)benzyl)phosphonate, 2-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide ...4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-(4-(2,3 (Oxin-6-yl)-1H-pyrazol-3-yl)benzene-1,3-diol, 3-(2-(imidazo[1,2-b]pyridazin-6-ylthio)ethyl)-4-(naphthalen-1-ylsulfonyl)-3,4-dihydroquinoxalin-2(1H)-one, and (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide or a salt thereof can be used as the "factor having CDK8 / 19 inhibitory activity."Factors having CDK8 / 19 inhibitory activity are not limited to these, and antisense oligonucleotides and siRNAs against CDK8 / 19 mRNA, antibodies that bind to CDK8 / 19, dominant-negative CDK8 / 19 mutants, etc. can also be used as factors having CDK8 / 19 inhibitory activity, and these are commercially available or can be synthesized according to known methods.

[0031] 2. Fibrin Gel Sheet for Cell Transplantation The present invention relates to a fibrin gel sheet for cell transplantation in which cells are uniformly dispersed and encapsulated in a fibrin gel sheet (hereinafter, sometimes simply referred to as the "fibrin gel sheet of the present invention").

[0032] The fibrin gel sheet of the present invention is characterized by having a size that could not be achieved with conventional fibrin gel sheets for cell transplantation, and has a surface area of ​​2.25 cm2 on one side of the sheet (i.e., either of the main surfaces of the sheet, or also referred to as one side of the sheet). 2 More than 4cm, preferably 2 More than 9cm, preferably 2 More preferably, 16 cm 2 More preferably, 25 cm 2 Above 36 cm, especially preferably 2 More than 49 cm, especially more preferably 2 More preferably, 64 cm or more 2 More preferably, 81 cm or more 2 More than 100 cm, particularly preferably 2 The upper limit is not particularly limited and can be appropriately selected depending on the size and shape of the transplant site. For example, 2 It can have the following sizes:

[0033] The range of the surface area of ​​the fibrin gel sheet of the present invention can be expressed using two numerical values ​​selected from the lower and upper limits. For example, the surface area of ​​the fibrin gel sheet of the present invention is 2.25 cm 2 More than 400cm 2 Less than 4 cm, preferably2 More than 400cm 2 Less than 9cm, more preferably 2 More than 400cm 2 Less than 16 cm, more preferably 2 More than 400cm 2 Less than 25 cm, even more preferably 2 More than 400cm 2 Below, particularly preferably 36 cm 2 More than 400cm 2 Below, especially preferably 49 cm 2 More than 400cm 2 Below, particularly more preferably 64 cm 2 More than 400cm 2 Below, especially more preferably 81 cm 2 More than 400cm 2 Particularly preferably 100 cm or less 2 More than 400cm 2 The size can be selected appropriately from the following ranges.

[0034] The shape of the above-mentioned one surface of the fibrin gel sheet of the present invention is not particularly limited and can be appropriately selected depending on the size and shape of the transplantation site. Examples include, but are not limited to, polygons (e.g., triangles, rectangles, pentagons, hexagons, octagons, etc.), rounded polygons, circles, ellipses, etc.

[0035] For example, when the fibrin gel sheet of the present invention has a rectangular shape, its longitudinal length is 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, even more preferably 40 mm or more, even more preferably 50 mm or more, particularly preferably 60 mm or more, especially more preferably 70 mm or more, especially more preferably 80 mm or more, especially even more preferably 90 mm or more, and particularly preferably 100 mm or more, with no particular upper limit, but for example, 200 mm or less.Further, the fibrin gel sheet of the present invention has a transverse length of 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, even more preferably 40 mm or more, even more preferably 50 mm or more, especially preferably 60 mm or more, especially more preferably 70 mm or more, especially more preferably 80 mm or more, especially even more preferably 90 mm or more, and particularly preferably 100 mm or more, with no particular upper limit, but for example, 200 mm or less.

[0036] The ranges of the vertical length and horizontal length can be expressed using two numerical values ​​selected from the above-mentioned lower and upper limits, respectively. For example, the size of the fibrin gel sheet of the present invention (vertical length × horizontal length (or horizontal length × vertical length)) is 15 mm to 200 mm × 15 mm to 200 mm, preferably 20 mm to 200 mm × 20 mm to 200 mm, more preferably 30 mm to 200 mm × 30 mm to 200 mm, even more preferably 40 mm to 200 mm × 40 mm to 200 mm, and even more preferably The size can be appropriately selected from the range of preferably 50 mm to 200 mm x 50 mm to 200 mm, particularly preferably 60 mm to 200 mm x 60 mm to 200 mm, particularly more preferably 70 mm to 200 mm x 70 mm to 200 mm, particularly even more preferably 80 mm to 200 mm x 80 mm to 200 mm, particularly even more preferably 90 mm to 200 mm x 90 mm to 200 mm, and particularly preferably 100 mm to 200 mm x 100 mm to 200 mm. For example, the size of the fibrin gel sheet of the present invention (vertical length × horizontal length (or horizontal length × vertical length)) is 15 mm or more × 15 mm or more, preferably 20 mm × 20 mm, 20 mm × 30 mm, or 30 mm × 30 mm, more preferably 40 mm × 40 mm, 40 mm × 50 mm, 50 mm × 50 mm, 40 mm × 60 mm, or 60 mm × 60 mm, and even more preferably 70 mm × 70 mm, 80 mm × 80 mm, 90 mm × 90 mm, 100 mm × 100 mm, or 200 mm × 200 mm, but is not limited to these.

[0037] The thickness of the fibrin gel sheet of the present invention can be appropriately set depending on factors such as the size and quantity of cells encapsulated and the size of the transplant site, but is preferably 1 mm or less. A thickness of 1 mm or less of the fibrin gel sheet of the present invention allows for efficient supply of nutrients and oxygen to the cells encapsulated in the fibrin gel sheet and also allows for rapid degradation of the fibrin gel after transplantation, which is preferable. The thickness of the fibrin gel sheet of the present invention can be, for example, 1 mm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, or 400 μm or less. The lower limit is not particularly limited, but can be, for example, 100 μm or more, 200 μm or more, or 300 μm or more. The thickness range of the fibrin gel sheet of the present invention can be expressed using two numerical values ​​selected from the upper and lower limits. For example, the thickness of the fibrin gel sheet of the present invention can be 100 μm to 1 mm, 100 μm to 800 μm, 200 μm to 600 μm, or 300 μm to 400 μm, and can be appropriately selected from these ranges. For example, the thickness of the fibrin gel sheet of the present invention is approximately 400 μm. The thickness of the fibrin gel sheet of the present invention does not need to be uniform throughout the entire sheet; it is sufficient that the thickness at the thickest point is within the above range.

[0038] As used herein, "uniformly dispersed and encapsulated" cells means that the seeded cells are not confined to any particular site on the fibrin gel sheet of the present invention but are widely distributed throughout the fibrin gel sheet. While it is not necessary for all cells to be entirely embedded in the fibrin gel sheet, a cell culture seeded on the surface of a fibrin gel and cultured as an adherent culture results in cells being confined to the surface of the fibrin gel sheet, and therefore differs from an embodiment in which cells are "dispersed and encapsulated." More specifically, as used herein, "uniformly dispersed and encapsulated" cells means that there is little variation in the number of cells (cell density) contained within a given range at any number of locations (e.g., two, four, or six locations) on the fibrin gel sheet of the present invention, and preferably means that the value of each cell number is within ±20%, preferably ±15%, and more preferably ±10% of the average value. Furthermore, as used herein, the phrase "uniformly dispersed and encapsulated" refers to the positional relationship between the seeded cells. For example, when cells seeded on or within a fibrin gel are cultured and proliferated, resulting in contact / bonding between cell populations of different origins, the cells cannot be said to be dispersed among the seeded cells, and therefore the state does not fall under the category of "uniformly dispersed and encapsulated." Examples of such contact / bonding between seeded cells include cell cultures (e.g., cell sheets) formed by seeding and culturing on or within a fibrin gel, but such embodiments are not intended to be included in the present invention. On the other hand, the term "uniformly dispersed and encapsulated" refers to, for example, cell clusters or cell aggregates in which cells aggregate and aggregate together, i.e., spheroids, or the proliferation of seeded cells, as long as the seeded cells are not in such a state of contact / bonding between them.

[0039] In the present invention, "cells" refer to cells transplanted into patients in need of cell therapy. They can be appropriately selected depending on the patient's symptoms. Examples include, but are not limited to, cells that constitute organs and tissues such as the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lung, thyroid, pancreas, liver, muscle, skeleton, heart, blood vessels, spleen, kidney, and blood, or cells with functions equivalent to or similar to their progenitor cells. The "cells" may be used singly or in combination. The morphology of the cells in the fibrin gel sheet of the present invention is not particularly limited. They may be isolated from each other (single cells), aggregated or clumped together to form cell clusters or cell aggregates, i.e., spheroids, or suspension-cultured cells. Examples of spheroid-form cells include, but are not limited to, pancreatic islet cells, hepatocytes, and cardiomyocytes.

[0040] In the present invention, the "cells" may be cells collected from a living body, cultured cells, or frozen-thawed cells. Preferably, the "cells" in the present invention are pluripotent stem cell-derived cells obtained by inducing differentiation from pluripotent stem cells.

[0041] As used herein, "pluripotency" refers to the ability to differentiate into tissues and cells with a variety of different morphologies and functions, and to differentiate into cells of any lineage of the three germ layers. "Pluripotency" cannot differentiate into the germ disc and therefore does not have the ability to form an individual, and is therefore distinguished from "totipotency," which can differentiate into any tissue of the body, including the germ disc.

[0042] As used herein, "multipotency" refers to the ability to differentiate into cells of multiple, limited number of lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent but not pluripotent.

[0043] As used herein, "pluripotent stem cells" refers to embryonic stem cells (ES cells) and cells that have the same pluripotency, i.e., the potential to differentiate into various tissues in the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification). Preferably, in the present invention, the pluripotent stem cells are human pluripotent stem cells.

[0044] As "ES cells," available mouse ES cells include various mouse ES cell lines established by inGenious, RIKEN, and the like, and available human ES cells include various human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis, Inc. For example, available ES cell lines include NIH strains CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28, WiCell Research Institute strains H1 and H9, and RIKEN strains KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3.

[0045] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), and Nanog-iPS cells established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106.), and iPS cells established by introducing six virus-free factors (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31(3): 458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (JP 2008-307007 A), and the like can also be used.

[0046] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholeer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7,795-797), or patents (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008 / 2336610, US 2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852) known in the art can be used.

[0047] As induced pluripotent cell lines, various iPS cell lines established by the NIH, the Institute of Physical and Chemical Research (RIKEN), Kyoto University, and the like, are available. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain; Kyoto University's Ff-WJ-18 strain, Ff-I01s01 strain, Ff-I01s02 strain, Ff-I01s04 strain, Ff-I01s06 strain, Ff-I14s03 strain, Ff-I14s04 strain, QHJI01s01 strain, QHJI01s04 strain, QHJI14s03 strain, QHJI14s04 strain, 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain; and CDI's MyCell iPS Examples of such strains include MyCell iPS Cells (21525.102.10A) strain, MyCell iPS Cells (21526.101.10A) strain, and the like.

[0048] Pluripotent stem cell-derived cells that can be used in the present invention include, but are not limited to, iPS cell-derived pancreatic islet cells (hereinafter sometimes referred to as "iPIC"). iPIC includes insulin-producing cells and / or pancreatic β cells. Insulin-producing cells are cells characterized by the expression of at least one marker, insulin and NK6 homeobox 1 (NKX6.1). Pancreatic β cells are more mature than insulin-producing cells and are characterized by the expression of at least one marker, MAFA, UCN3, and IAPP. "Marker" refers to a cell antigen or its gene that is specifically expressed by a specific cell type, such as a "marker protein" or "marker gene." Preferably, the marker is a cell surface marker, which enables the enrichment, isolation, and / or detection of viable cells. The marker may be a positive selection marker or a negative selection marker.

[0049] Marker proteins can be detected using immunological assays (ELISA, immunostaining, flow cytometry, etc.) using antibodies specific to the marker proteins. Marker genes can be detected using nucleic acid amplification methods and / or nucleic acid detection methods (RT-PCR, microarrays, biochips, etc.) known in the art. For example, a marker protein being "positive" means that it is detected as positive by flow cytometry, and "negative" means that it is below the detection limit by flow cytometry. Furthermore, a marker gene being "positive" means that it is detected by RT-PCR, and "negative" means that it is below the detection limit by RT-PCR.

[0050] "Expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter within a cell.

[0051] iPICs can be obtained by inducing differentiation from pluripotent stem cells in accordance with known techniques (WO 2009 / 012428, WO 2016 / 021734, Stem Cell Research (2015) 14, 185-197). That is, iPICs can be obtained using the following differentiation induction steps: Step 1) Inducing differentiation from pluripotent stem cells into definitive endoderm cells; Step 2) Inducing differentiation from definitive endoderm cells into primitive gut cells; Step 3) Inducing differentiation from primitive gut cells into posterior foregut cells; Step 4) Inducing differentiation from posterior foregut cells into pancreatic precursor cells; Step 5) Inducing differentiation from pancreatic precursor cells into endocrine precursor cells; Step 6) Inducing differentiation from endocrine precursor cells into iPICs. Each step will be described below, but the differentiation induction into each cell type is not limited to these techniques.

[0052] Step 1) Differentiation into Definitive Endoderm Cells Pluripotent stem cells are first differentiated into definitive endoderm cells. Methods for inducing definitive endoderm from pluripotent stem cells are already known, and any of these methods may be used. Preferably, pluripotent stem cells are cultured in a medium containing activin A, more preferably in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, to differentiate into definitive endoderm cells. The number of cells at the start of culture is not particularly limited, and may be 22,000 to 150,000 cells / cm. 2 , preferably 22,000 to 100,000 cells / cm 2 , more preferably 22,000 to 80,000 cells / cm 2 The culture period is 1 to 4 days, preferably 1 to 3 days, and particularly preferably 3 days.

[0053] The culture temperature is not particularly limited, but is preferably 30 to 40°C (e.g., 37°C). The carbon dioxide concentration in the culture vessel is, for example, about 5%. Culture may be performed by either two-dimensional culture or three-dimensional culture.

[0054] The medium used in this step may be a basal medium used for culturing mammalian cells, such as RPMI 1640 medium, MEM medium, iMEM medium, DMEM / F12 medium, Improved MEM Zinc Option medium, Improved MEM / 1% B-27 / Penisilin Streptomycin medium, or MCDB131 / 20 mM Glucose / NaHCO3 / FAF-BSA / ITS-X / GlutaMAX™ / Ascorbic Acid / Penisilin Streptomycin medium.

[0055] The concentration of activin A in the medium is usually 30 to 200 ng / mL, preferably 50 to 150 ng / mL, more preferably 70 to 120 ng / mL, and particularly preferably about 100 ng / mL.

[0056] In another embodiment, activin A can be included in the medium at a low dose, for example, at an amount of 5 to 100 ng / mL, preferably 5 to 50 ng / mL, more preferably 5 to 10 ng / mL.

[0057] In yet another embodiment, the concentration of activin A in the medium is about 0.1 to 100 ng / mL, preferably about 1 to 50 ng / mL, more preferably about 3 to 10 ng / mL.

[0058] The concentration of the GSK3β inhibitor in the medium is appropriately set depending on the type of GSK3β inhibitor used. For example, when CHIR99021 is used as the GSK3β inhibitor, the concentration is usually 2 to 5 μM, preferably 2 to 4 μM, and particularly preferably about 3 μM.

[0059] The concentration of the ROCK inhibitor in the medium is appropriately set depending on the type of ROCK inhibitor used. For example, when Y27632 is used as the ROCK inhibitor, the concentration is usually 5 to 20 μM, preferably 5 to 15 μM, and particularly preferably about 10 μM.

[0060] The medium may further contain insulin. Insulin may be contained in the medium in an amount of 0.01 to 20 μM, preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM. The concentration of insulin in the medium may be, but is not limited to, the concentration of insulin contained in the added B-27 supplement.

[0061] In a specific embodiment, after culturing for one day in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, the cells are further cultured for two days with the medium being changed every day in a medium containing only activin A. Alternatively, pluripotent stem cells can be produced by first culturing in a medium containing 0.01 to 20 μM insulin in the presence of a low dose of activin A, followed by second culturing in a medium without insulin.

[0062] Step 2) Differentiation into primitive gut cells The definitive endoderm cells obtained in step 1) are further cultured in a medium containing growth factors to induce differentiation into primitive gut cells. The culture period is 2 to 8 days, preferably about 4 days.

[0063] The culture temperature is not particularly limited, but is preferably 30 to 40°C (e.g., 37°C). The carbon dioxide concentration in the culture vessel is, for example, about 5%. Culture may be performed by either two-dimensional culture or three-dimensional culture.

[0064] The medium may be a basal medium used for culturing mammalian cells, as in step 1. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be added to the medium as appropriate.

[0065] As the growth factor, EGF, KGF, and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is even more preferred.

[0066] The concentration of growth factor in the medium is set appropriately depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / mL (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (i.e., about 0.6 to 58 nM). For example, when KGF is used as the growth factor, the concentration is usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0067] Step 3) Differentiation into Posterior Foregut Cells The primitive gut cells obtained in step 2) are further cultured in a medium containing growth factors, cyclopamine, noggin, etc., to induce differentiation into posterior foregut cells. The culture period is 1 to 5 days, preferably about 2 days. Culture may be performed in either two-dimensional culture or three-dimensional culture.

[0068] The culture temperature is not particularly limited, but is preferably 30 to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0069] The medium may be a basal medium used for culturing mammalian cells, as in step 1. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be added to the medium as appropriate.

[0070] As the growth factor, EGF, KGF, and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is even more preferred.

[0071] The concentration of growth factor in the medium is set appropriately depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / mL (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (i.e., about 0.6 to 58 nM). For example, when KGF is used as the growth factor, the concentration is usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0072] The concentration of cyclopamine in the medium is not particularly limited, but is usually 0.5 to 1.5 μM, preferably 0.3 to 1.0 μM, and particularly preferably about 0.5 μM.

[0073] The concentration of Noggin in the medium is not particularly limited, but is usually 10 to 200 ng / mL, preferably 50 to 150 ng / mL, and particularly preferably about 100 ng / mL.

[0074] Step 4) Differentiation into Pancreatic Progenitor Cells The posterior foregut cells obtained in step 3) are cultured in a medium further containing a factor having CDK8 / 19 inhibitory activity, preferably a medium containing a factor having CDK8 / 19 inhibitory activity and a growth factor, to induce differentiation into pancreatic progenitor cells. The culture period is 2 to 10 days, preferably about 5 days. Culture may be performed in either two-dimensional or three-dimensional culture.

[0075] In the case of two-dimensional culture, according to a previous report (Toyoda et al., Stem Cell Research (2015) 14, 185-197), the posterior foregut cells obtained in step 3) are treated with a 0.25% trypsin-EDTA solution and dispersed in the solution by pipetting to obtain a cell dispersion, the resulting dispersion is centrifuged, the recovered cells are resuspended in a small amount of fresh medium, and the cell suspension is replated in fresh medium in step 4).

[0076] The medium may be a basal medium used for culturing mammalian cells, as in step 1. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be added to the medium as appropriate.

[0077] The factor having CDK8 / 19 inhibitory activity can be any of the various compounds or salts thereof described above, and the amount added to the medium is determined appropriately depending on the compound or salt thereof used, but is usually about 0.00001 μM to 5 μM, preferably 0.00001 μM to 1 μM. The concentration of the factor having CDK8 / 19 inhibitory activity in the medium is preferably a concentration that achieves 50% or more inhibitory activity against CDK8 / 19.

[0078] As the growth factor, EGF, KGF, and FGF10 are preferred, KGF and / or EGF are more preferred, and KGF and EGF are even more preferred.

[0079] The concentration of growth factor in the medium is set appropriately depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / mL (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (i.e., about 0.6 to 58 nM). For example, when KGF and EGF are used as growth factors, the concentrations of EGF are usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL, and of KGF are usually 10 to 200 ng / mL, preferably 50 to 150 ng / mL, and particularly preferably about 100 ng / mL.

[0080] In step 4), the first day of culture may be carried out in the presence of a ROCK inhibitor, and thereafter culture may be carried out in a medium that does not contain a ROCK inhibitor.

[0081] The medium may also contain a protein kinase C (PKC) activator. Examples of PKC activators include, but are not limited to, PDBu (PKC activator II) and TPB (PKC activator V). The PKC activator is added at a concentration of about 0.1 to 100 ng / mL, preferably about 1 to 50 ng / mL, and more preferably about 3 to 10 ng / mL.

[0082] The medium may also contain dimethyl sulfoxide and / or activin (1 to 50 ng / mL).

[0083] In any step, in addition to the above-mentioned components, serum substitutes (e.g., B-27 supplement, ITS-G) may be added to the medium. Furthermore, if necessary, amino acids, L-glutamine, GlutaMAX (product name), non-essential amino acids, vitamins, nicotinamide, antibiotics (e.g., antibiotic-antimycotic, penicillin, streptomycin, or a mixture thereof), antibacterial agents (e.g., amphotericin B), antioxidants, pyruvic acid, buffers, inorganic salts, etc. may be added. When antibiotics are added to the medium, the concentration in the medium is usually 0.01 to 20% by weight, preferably 0.1 to 10% by weight. Culture may be performed in either two-dimensional or three-dimensional culture.

[0084] In the case of two-dimensional cell culture, adherent culture is performed without using feeder cells. Culture vessels such as dishes, flasks, microplates, and cell culture sheets such as OptiCell (product name) (Nunc) are used during culture. The culture vessels are preferably surface-treated to improve cell adhesion (hydrophilicity) or coated with a cell adhesion substrate such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel (e.g., BD Matrigel (Becton Dickinson Japan)), or vitronectin. Culture vessels coated with type I collagen, Matrigel, fibronectin, vitronectin, or poly-D-lysine are preferred, with Matrigel or poly-D-lysine being more preferred.

[0085] The culture temperature is not particularly limited, but is preferably 30 to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0086] The pancreatic progenitor cells obtained in step 4) can be further purified using a known surface marker such as glycoprotein 2 (GP2). The purification can be carried out by a known method, for example, using beads on which anti-GP2 antibodies are immobilized.

[0087] Step 5) Differentiation into Endocrine Precursor Cells The pancreatic precursor cells obtained in step 4) are further cultured in a medium containing growth factors to induce differentiation into endocrine precursor cells. Culture may be performed in either two-dimensional or three-dimensional culture. In the case of two-dimensional culture, the pancreatic precursor cells obtained in step 4) are treated with a 0.25% trypsin-EDTA solution and dispersed in the solution by pipetting to obtain a cell dispersion. The resulting dispersion is centrifuged, and the recovered cells are resuspended in a small amount of fresh medium, and the cell suspension is replated in the fresh medium in step 5). The culture period is 2 to 3 days, preferably about 2 days.

[0088] The medium may be a basal medium used for culturing mammalian cells, as in step 1. According to a previous report (Nature Biotechnology 2014; 32: 1121-1133), the medium may contain SANT1, retinoic acid, ALK5 inhibitor II, T3, and LDN, and may further contain a Wnt inhibitor, a ROCK inhibitor, FGF (preferably FGF2), a serum substitute, vitamins, antibiotics, and the like, as appropriate.

[0089] The culture is performed in a non-adherent culture without using feeder cells. During the culture, dishes, flasks, microplates, multi-hole plates (Nunc), or bioreactors are used. The culture vessels are preferably surface-treated to reduce cell adhesion.

[0090] The culture temperature is not particularly limited, but is preferably 30 to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0091] The endocrine precursor cells obtained in step 5) can be further purified using a known surface marker such as glycoprotein 2 (GP2). The purification can be carried out by a known method, for example, using beads on which anti-GP2 antibodies are immobilized.

[0092] Step 6) Differentiation into iPICs The endocrine precursor cells obtained in step 5) are further cultured in a medium containing growth factors to induce differentiation into iPICs. The culture period is 10 to 30 days, preferably about 10 to 20 days.

[0093] As in step 1), the medium may be a basal medium used for culturing mammalian cells. According to a previous report (Nature Biotechnology 2014; 32: 1121-1133), the medium may contain ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor XX, γ-secretase inhibitor RO, N-cysteine, AXL inhibitor, and ascorbic acid, and may further contain a Wnt inhibitor, a ROCK inhibitor, FGF (preferably FGF2), a serum substitute, vitamins, antibiotics, and the like, as appropriate. For example, the medium may be supplemented with ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, and ascorbic acid, or may be supplemented with T3, ALK5 inhibitor II, ZnSO 4 , heparin, N-acetylcysteine, Trolox, and R428.

[0094] Culture may be performed in either two-dimensional or three-dimensional culture. No feeder cells are used for culture. Three-dimensional culture is performed as a non-adherent culture. During culture, dishes, flasks, microplates, multi-hole plates (Nunc), or bioreactors are used. It is preferable that the culture vessels are surface-treated to reduce cell adhesion.

[0095] The culture temperature is not particularly limited, but is preferably 30 to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0096] The fibrin gel sheet of the present invention is characterized by having a larger number of cells (high density of cells) than has been achieved with conventional fibrin gel sheets for cell transplantation, and the cells can be transplanted to a density of 1 cm 2 Per 1.5 x 10 6 More than 10, preferably 2 x 10 6 or more, more preferably 2.5 × 10 6 or more, more preferably 3 × 10 6 or more, and even more preferably 4.5 x 10 6 or more, particularly preferably 5 × 10 6 The upper limit is not particularly limited, but for example, 10 × 10 6 8 or less, preferably 7 × 10 6 The range of the number of cells encapsulated in the fibrin gel sheet of the present invention can be expressed using two numerical values ​​selected from the above-mentioned lower and upper limits. For example, the fibrin gel sheet of the present invention can contain 1.5 × 10 cells or less. 6 More than 10×10 6 pieces / cm 2 , preferably 2 × 10 6 More than 10×10 6 pieces / cm 2 , more preferably 2.5 × 10 6 More than 10×10 6 pieces / cm 2 , and more preferably 3 × 10 6 More than 10×10 6 pieces / cm 2 , and even more preferably 4.5×10 6 More than 10×10 6 pieces / cm 2 , particularly preferably 5 × 10 6 More than 10×10 6 pieces / cm 2 The amount can be selected appropriately from the range.

[0097] The above-mentioned iPICs are usually present in the form of cell clusters having a size of about 100 μm to 200 μm, but in the fibrin gel sheet of the present invention, the number of iPICs is 1 cm 2The fibrin gel sheet of the present invention contains more than 3,000 iPICs (cell clusters), preferably 4,000 or more, more preferably 5,000 or more, even more preferably 6,000 or more, even more preferably 9,000 or more, and particularly preferably 10,000 or more iPICs per cm, and the upper limit is not particularly limited, but can be, for example, 20,000 or less, preferably 14,000 or less. The range of the number of iPICs (cell clusters) contained in the fibrin gel sheet of the present invention can be expressed using two numerical values ​​selected from the above-mentioned lower and upper numerical values, and for example, the fibrin gel sheet of the present invention contains more than 3,000 iPICs (cell clusters) per cm to 20,000 or less iPICs (cell clusters) per cm. 2 , preferably 4000 or more to 20000 or less / cm 2 , more preferably 5,000 to 20,000 particles / cm 2 , and more preferably 6,000 to 20,000 particles / cm 2 , and even more preferably 9,000 or more to 20,000 or less / cm 2 , and particularly preferably 10,000 or more to 20,000 or less / cm 2 The amount can be selected appropriately from the range.

[0098] In the fibrin gel sheet of the present invention, the fibrin gel is formed by reacting fibrinogen with thrombin in an appropriate solvent (e.g., water, physiological saline, etc.). The amounts of fibrinogen and thrombin used to form the fibrin gel and the method for mixing them can be appropriately determined based on the method for producing the fibrin gel sheet of the present invention, which will be described in detail below.

[0099] In one embodiment, the fibrin gel sheet of the present invention further contains a biodegradable material in particulate form (hereinafter, sometimes referred to as "biodegradable particles").

[0100] In the present invention, "biodegradable particles" are added to and suspended in a fibrinogen solution together with cells in the method for producing a fibrin gel sheet of the present invention, which will be described in detail below, and serve to prevent the cells from settling in the solution and maintain their dispersion, thereby enabling the cells to be dispersed and encapsulated in the fibrin gel sheet that is finally obtained.

[0101] The biodegradable material constituting the "biodegradable particles" may be any substance that can be decomposed in vivo by hydrolysis or the like, and is not particularly limited. Examples of the biodegradable material include polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polyethylene glycol (PEG), polyhydroxyethyl methacrylate, polyester, polyglycolic acid (PGA), polylactic acid-co-glycolic acid (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(ethylene-co-vinyl acetate) (PEVA), polyacrylamide, polyethylene oxide, polyethyleneamine, polyhydroxybutyric acid, poly(N Examples of suitable biodegradable materials include polyvinylpyrrolidone, polyvinyl alcohol, polypropylene fumarate, polyacrylic acid, poly-e-caprolactone, polymethacrylic acid, polyvinylidene difluoride (PVDF), pectinic acid, hyaluronic acid, heparin sulfate, chondroitin sulfate, heparan sulfate proteoglycan, heparin, chitin, chitosan, xanthan, carboxymethylcellulose, carboxymethylchitosan, alginic acid, alginate ester, collagen, cellulose, silk fibroin, keratin, gelatin, fibrin, pullulan, laminin, gellan, silicone, urethane, elastin, and modified forms thereof, and combinations thereof. Preferably, the biodegradable particles are composed of a gel-like biodegradable material selected from the group consisting of gelatin, collagen, hyaluronic acid, carboxymethylcellulose, PLA, PGA, PLGA, and modified forms thereof, and combinations thereof.

[0102] The shape of the "biodegradable particles" may be any shape that can suppress cell precipitation and maintain dispersion in the solution, and may be any shape such as block, bead, pellet, sheet, gel, etc., and may be solid or porous. The shape of the "biodegradable particles" is preferably three-dimensional, and examples thereof include, but are not limited to, spheres, polyhedra such as tetrahedrons and hexahedrons, cylinders, prisms, cones, truncated cones, pyramids, truncated pyramids, tori, disks, ellipsoids, and modified solids thereof.

[0103] The size of the "biodegradable particles" may be any size that can suppress cell precipitation and maintain dispersion in the solution, and that does not interfere with the formation of the fibrin gel sheet of the present invention, and can be a size that can pass through an opening of 100 μm to 1000 μm, preferably 100 μm to 800 μm, more preferably 150 μm to 400 μm, and even more preferably 200 μm.

[0104] In one embodiment of the present invention, the biodegradable particles are gelatin gels and have the shape of spheres that can pass through a 200 μm opening.

[0105] The amount of biodegradable particles contained in the fibrin gel sheet of the present invention may be any amount that can suppress cell precipitation and maintain dispersion in the fibrinogen solution, and that does not interfere with the formation of the fibrin gel sheet of the present invention. For example, the biodegradable particles can be contained in the fibrinogen solution in an amount of 10 to 30 w / v %, preferably 15 to 25 w / v %, more preferably 18 to 22 w / v %, and even more preferably 20 w / v %.

[0106] In the fibrin gel sheet of the present invention, the biodegradable particles may maintain their shape and form, or may be partially or entirely integrated with the fibrin gel.

[0107] In yet another embodiment, the fibrin gel sheet of the present invention further comprises a biodegradable gelling agent.

[0108] In the present invention, the "biodegradable gelling agent" is added to and dissolved in a fibrinogen solution together with cells in the method for producing a fibrin gel sheet of the present invention, which will be described in detail below, and imparts viscosity to the solution, thereby inhibiting precipitation of the cells in the solution and enabling them to remain dispersed, thereby enabling the cells to be dispersed and encapsulated in the fibrin gel sheet that is finally obtained.

[0109] The "biodegradable gelling agent" is not particularly limited as long as it is a substance that can be decomposed in vivo by hydrolysis or other means, can impart a viscosity to the solution that can inhibit cell precipitation and maintain dispersion, and does not interfere with the formation of the fibrin gel sheet of the present invention. For example, the biodegradable materials listed above can be used (with the exception of fibrin). Preferably, the biodegradable gelling agent is a biodegradable material selected from the group consisting of collagen, hyaluronic acid, heparin sulfate, chondroitin sulfate, heparan sulfate proteoglycan, carboxymethylcellulose, alginic acid, cellulose, silk fibroin, keratin, gelatin, dextran, PEG, etc., and modified forms thereof, as well as combinations thereof.

[0110] The amount of biodegradable gelling agent contained in the fibrin gel sheet of the present invention may be any amount that can suppress cell precipitation and maintain dispersion in the fibrinogen solution, and that does not interfere with the formation of the fibrin gel sheet of the present invention. For example, the biodegradable gelling agent can be contained in the fibrinogen solution in an amount of 0.2 to 2 w / v%, preferably 0.4 to 1.5 w / v%, more preferably 0.5 to 1 w / v%, and even more preferably 0.75 w / v%.

[0111] The fibrin gel sheet of the present invention can further include a support. The support supports the fibrin gel sheet of the present invention during its production and / or use and can contribute to improving the operability of the fibrin gel sheet. The support is made of the aforementioned biodegradable or non-biodegradable materials, such as polyesters (PEs), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), nylon, or silk (but is not limited to these), and can have any shape (e.g., sheet, mesh, plate, woven fabric, nonwoven fabric, etc.) that can support the fibrin gel sheet of the present invention. The support may be adhered to the surface of the fibrin gel sheet of the present invention, or may be partially or entirely fixed within the fibrin gel sheet of the present invention.

[0112] In one aspect of the present invention, the fibrin gel sheet of the present invention comprises a mesh-shaped support made of polyester or polylactic-co-glycolic acid.

[0113] The fibrin gel sheet of the present invention may be provided in the form of a laminate in which a plurality of the fibrin gel sheets are stacked together. The laminate may be composed of fibrin gel sheets in which all the fibrin gel sheets contain the same cells, or may be composed of a combination of fibrin gel sheets in which some or all of the fibrin gel sheets contain different cells.

[0114] 3. Method for Producing a Fibrin Gel Sheet of the Present Invention The present invention also relates to a method for producing the fibrin gel sheet of the present invention (hereinafter, may be referred to as the "method of the present invention"), which comprises reacting a fibrinogen solution containing suspended cells with thrombin to form a fibrin gel sheet having a surface area of ​​2.25 cm². 2 The method includes a step of forming the fibrin gel into a sheet having a size of 1 mm or less and a thickness of 1 mm or less, followed by a step of gelling (solidifying) the fibrin gel. The fibrin gel can be obtained by combining fibrinogen and thrombin in an appropriate solvent (e.g., water, physiological saline, etc.), followed by gelling (solidifying).

[0115] The cells are uniformly dispersed and suspended in the fibrinogen solution, and the amount of cells can be determined based on the fibrin gel sheet after molding. 2 Per 1.5 x 10 6 More than 10, preferably 2 x 10 6 or more, more preferably 2.5 × 10 6 or more, more preferably 3 × 10 6 or more, and even more preferably 4.5 x 10 6 or more, particularly preferably 5 × 10 6 The upper limit of the amount of 10 or more is not particularly limited, but for example, 10 × 10 6 8 or less, preferably 7 × 10 6The range of the number of cells suspended in the fibrinogen solution can be expressed using two numerical values ​​selected from the above-mentioned lower and upper limits. For example, the number of cells suspended in the fibrinogen solution can be 1.5 × 10 or less on the fibrin gel sheet. 6 More than 10×10 6 pieces / cm 2 , preferably 2 × 10 6 More than 10×10 6 pieces / cm 2 , more preferably 2.5 × 10 6 More than 10×10 6 pieces / cm 2 , and more preferably 3 × 10 6 More than 10×10 6 pieces / cm 2 , and even more preferably 4.5×10 6 More than 10×10 6 pieces / cm 2 , particularly preferably 5 × 10 6 More than 10×10 6 pieces / cm 2 The suspension can be prepared in an amount selected appropriately from the range in which

[0116] In one embodiment of the method of the present invention, when the cells are iPIC, the fibrinogen solution contains iPIC in a quantity equivalent to the number of cell clusters per 1 cm of fibrin gel sheet. 2 The fibrinogen solution can be suspended in an amount that will give more than 3,000 cells, preferably 4,000 or more cells, more preferably 5,000 or more cells, even more preferably 6,000 or more cells, even more preferably 9,000 or more cells, and particularly preferably 10,000 or more cells per cm of the fibrin gel sheet, and the upper limit is not particularly limited, but for example, the amount will give 20,000 or less cells, preferably 14,000 or less cells. The range of the number of iPICs (cell clusters) suspended in the fibrinogen solution can be expressed using two numerical values ​​selected from the above-mentioned lower and upper numerical values, and for example, the ... gel sheet can be expressed using two numerical values ​​selected from the above-mentioned lower and upper numerical values, and for example, the range of the number of iPICs (cell clusters) suspended in the fibrinogen solution can be expressed using two numerical values ​​selected from the above-mentioned lower and upper numerical values, and for example, the range of the number of iPICs (cell clusters) suspended in the fibrinogen solution can be expressed using two numerical values ​​selected from the above-mentioned lower and upper numerical values, and for example, the range of the number of iPICs (cell clusters) 2 , preferably 4000 or more to 20000 or less / cm 2, more preferably 5,000 to 20,000 particles / cm 2 , and more preferably 6,000 to 20,000 particles / cm 2 , and even more preferably 9,000 or more to 20,000 or less / cm 2 , and particularly preferably 10,000 or more to 20,000 or less / cm 2 The suspension can be prepared in an amount selected appropriately from the range in which

[0117] The fibrin gel sheet was formed by molding a sheet having a surface area of ​​2.25 cm2 on one side of the sheet (i.e., either of the main surfaces of the sheet, or also referred to as one side of the sheet). 2 More than 4cm, preferably 2 More than 9cm, preferably 2 More preferably, 16 cm 2 More preferably, 25 cm 2 Above 36 cm, especially preferably 2 More than 49 cm, especially more preferably 2 More preferably, 64 cm or more 2 More preferably, 81 cm or more 2 More than 100 cm, particularly preferably 2 The upper limit is not particularly limited and can be appropriately selected depending on the size and shape of the transplant site. For example, 2 The following size can be used:

[0118] The range of the surface area of ​​the fibrin gel sheet obtained by molding can be expressed using two numerical values ​​selected from the lower and upper limits, respectively. For example, the surface area of ​​the fibrin gel sheet obtained by molding is 2.25 cm 2 More than 400cm 2 Less than 4 cm, preferably 2 More than 400cm 2 Less than 9cm, more preferably 2 More than 400cm 2 Less than 16 cm, more preferably 2 More than 400cm 2 Less than 25 cm, even more preferably 2More than 400cm 2 Below, particularly preferably 36 cm 2 More than 400cm 2 Below, especially preferably 49 cm 2 More than 400cm 2 Below, particularly more preferably 64 cm 2 More than 400cm 2 Below, especially more preferably 81 cm 2 More than 400cm 2 Particularly preferably 100 cm or less 2 More than 400cm 2 The size can be selected appropriately from the following ranges.

[0119] The shape of the surface of the fibrin gel sheet obtained by molding is not particularly limited and can be appropriately selected depending on the size and shape of the transplantation site. Examples include, but are not limited to, polygons (e.g., triangles, rectangles, pentagons, hexagons, octagons, etc.), rounded polygons, circles, ellipses, etc.

[0120] When the fibrin gel sheet obtained by molding has a rectangular shape, the longitudinal length is 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, even more preferably 40 mm or more, still more preferably 50 mm or more, particularly preferably 60 mm or more, particularly more preferably 70 mm or more, particularly more preferably 80 mm or more, particularly even more preferably 90 mm or more, and particularly preferably 100 mm or more; the upper limit is not particularly limited, but can be, for example, 200 mm or less; and the lateral length is 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, even more preferably 40 mm or more, still more preferably 50 mm or more, particularly preferably 60 mm or more, particularly more preferably 70 mm or more, particularly more preferably 80 mm or more, particularly even more preferably 90 mm or more, and particularly preferably 100 mm or more; the upper limit is not particularly limited, but can be, for example, 200 mm or less.

[0121] The ranges of the vertical length and horizontal length can be expressed using two numerical values ​​selected from the above-mentioned lower limit and upper limit numerical values, respectively. For example, the size of the fibrin gel sheet obtained by molding (vertical length × horizontal length (or horizontal length × vertical length)) is 15 mm or more to 200 mm or less × 15 mm or more to 200 mm or less, preferably 20 mm or more to 200 mm or less × 20 mm or more to 200 mm or less, more preferably 30 mm or more to 200 mm or less × 30 mm or more to 200 mm or less, even more preferably 40 mm or more to 200 mm or less × 40 mm or more to 200 mm or less, and even more preferably Preferably, the size is appropriately selected from the range of 50 mm to 200 mm x 50 mm to 200 mm, particularly preferably 60 mm to 200 mm x 60 mm to 200 mm, particularly more preferably 70 mm to 200 mm x 70 mm to 200 mm, particularly even more preferably 80 mm to 200 mm x 80 mm to 200 mm, particularly even more preferably 90 mm to 200 mm x 90 mm to 200 mm, particularly preferably 100 mm to 200 mm x 100 mm to 200 mm. For example, the size of the fibrin gel sheet obtained by molding (vertical length × horizontal length (or horizontal length × vertical length)) may be 15 mm or more × 15 mm or more, preferably 20 mm × 20 mm, 20 mm × 30 mm, or 30 mm × 30 mm, more preferably 40 mm × 40 mm, 40 mm × 50 mm, 50 mm × 50 mm, 40 mm × 60 mm, or 60 mm × 60 mm, and even more preferably 70 mm × 70 mm, 80 mm × 80 mm, 90 mm × 90 mm, 100 mm × 100 mm, or 200 mm × 200 mm, but is not limited to these.

[0122] The thickness of the fibrin gel sheet obtained by molding can be appropriately set depending on factors such as the size and amount of cells to be encapsulated and the size of the transplant site, but is preferably 1 mm or less. A thickness of 1 mm or less of the fibrin gel sheet obtained by molding allows for efficient supply of nutrients and oxygen to the cells encapsulated in the fibrin gel sheet and also enables rapid decomposition of the fibrin gel after transplantation, which is preferable. The thickness of the fibrin gel sheet obtained by molding can be, for example, 1 mm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, or 400 μm or less; the lower limit is not particularly limited, but can be, for example, 100 μm or more, 200 μm or more, or 300 μm or more. The thickness range of the fibrin gel sheet obtained by molding can be expressed using two numerical values ​​selected from the upper and lower limits described above. For example, the thickness of the fibrin gel sheet obtained by molding can be 100 μm to 1 mm, 100 μm to 800 μm, 200 μm to 600 μm, or 300 μm to 400 μm, and can be selected appropriately from these ranges. For example, the thickness of the fibrin gel sheet obtained by molding can be approximately 400 μm. The thickness of the fibrin gel sheet obtained by molding does not need to be uniform throughout the sheet; it is sufficient that the thickness at the thickest point is within the above range.

[0123] In one embodiment of the method of the present invention, the gelling (solidification) rate of the formed fibrin gel can be adjusted by adjusting the amounts of fibrinogen and thrombin and / or the molding temperature, making it possible to spread and mold the fibrin gel into a fibrin gel sheet of a desired size.

[0124] In this embodiment, the fibrinogen solution contains fibrinogen in an amount of 5 to 150 mg / mL, preferably 10 to 80 mg / mL, and more preferably 20 mg / mL, and thrombin in an amount of 0.4 U or less, preferably 0.3 U or less or 0.25 U or less, more preferably 0.2 U or less or 0.15 U or less, and even more preferably 0.1 U or less or 0.08 U or less per 1 mg of fibrinogen, with the lower limit not being particularly limited, but being 0.04 U or more. If the amount of fibrinogen and / or thrombin added exceeds the above range, gelation (solidification) proceeds too quickly, making it difficult or impossible to spread and mold into a sheet of the desired size. On the other hand, if the amount of fibrinogen and / or thrombin added is less than the above range, gelation (solidification) may not occur sufficiently, making it difficult or impossible to mold into a sheet of the desired size. Thrombin can be allowed to act on fibrinogen in any form. It may be added directly to the fibrinogen solution, or it may be dissolved in a suitable solvent (e.g., water, saline, etc.) in advance to form a thrombin solution, which may then be mixed with the fibrinogen solution.

[0125] Any means can be used to mold the fibrinogen solution reacted with thrombin into a sheet of desired size and thickness, for example, one or a combination of any conventional molding means known in the field of polymer sheet production (e.g., mold molding, extrusion molding, calendar molding, inflation molding, etc.), preferably mold molding. In this embodiment, the fibrinogen solution reacted with thrombin can be pressed and molded into a sheet of desired size and thickness, and mold molding, i.e., can be easily carried out by pouring the fibrinogen solution into a sheet mold of desired size and thickness and, as necessary, pressing and molding using a spatula, trowel, etc.

[0126] The fibrinogen solution reacted with thrombin is molded under cooling. By placing the fibrin gel solution under cooling, the gelation (solidification) rate is slowed, allowing it to be molded into a sheet of the desired size and thickness, more specifically, spread and molded into a sheet. The cooling temperature is not particularly limited as long as it allows the fibrin gel solution to be molded into a sheet of the desired size and thickness, and is, for example, 2 to 8°C, preferably 2 to 6°C, and more preferably 4°C. The cooling temperature is sufficient as long as it can cool the fibrin gel solution, and may be the fibrin gel solution itself, or the molding means (e.g., a mold frame) that comes into contact with the fibrin gel solution.

[0127] If necessary, the above-mentioned support may be integrated with the fibrin gel sheet before the cast fibrin gel solution gels (solidifies). The support may be attached to the surface of the fibrin gel solution, or may be partially or entirely immersed in the fibrin gel solution. By gelling (solidifying) the resulting sheet, a fibrin gel sheet having the support integrated therewith can be obtained.

[0128] In another aspect of the method of the present invention, by further adding and suspending "biodegradable particles" or by adding and dissolving a "biodegradable gelling agent" to the fibrinogen solution in which cells are suspended, the precipitation of cells in the fibrinogen solution is suppressed, and the cells are maintained in a uniformly dispersed and suspended state in the fibrinogen solution, making it possible to produce a fibrin gel sheet in which cells are dispersed and encapsulated.

[0129] The "biodegradable particles" can be those defined above, and can be added and suspended in an amount that can suppress cell precipitation and maintain cell dispersion in a fibrinogen solution containing suspended cells, and that does not interfere with the formation of the fibrin gel sheet of the present invention. For example, the biodegradable particles are added and suspended in a fibrinogen solution containing suspended cells in an amount of 10 to 30 w / v%, preferably 15 to 25 w / v%, more preferably 18 to 22 w / v%, and even more preferably 20 w / v%. In one embodiment of this aspect, the biodegradable particles are gelatin gel particles that have a spherical shape that can pass through a 200 μm mesh, and are added and suspended in a fibrinogen solution containing suspended cells in an amount of 20 w / v%.

[0130] The "biodegradable gelling agent" can be one defined above, and can be added and dissolved in an amount that can suppress cell precipitation and maintain cell dispersion in the fibrinogen solution in which the cells are suspended, and that does not interfere with the formation of the fibrin gel sheet of the present invention. For example, the biodegradable gelling agent is added and dissolved in the fibrinogen solution in an amount of 0.2 to 2 w / v%, preferably 0.4 to 1.5 w / v%, more preferably 0.5 to 1 w / v%, and even more preferably 0.75 w / v%. In one embodiment of this aspect, the biodegradable gelling agent is collagen, and is added and dissolved in the fibrinogen solution in an amount of 0.75 w / v%.

[0131] In this embodiment, the fibrinogen solution contains fibrinogen in an amount of 5 to 150 mg / mL, preferably 20 to 100 mg / mL, and more preferably 80 mg / mL. Thrombin may be added in an amount sufficient to gel (solidify) the fibrinogen solution, for example, 0.5 to 5 U, preferably 1 to 4 U, and more preferably 1.5 to 3.5 U per 1 mg of fibrinogen. Thrombin can be added to fibrinogen in any form, including directly to the fibrinogen solution; by dissolving thrombin in a suitable solvent (e.g., water, saline, etc.) beforehand to form a thrombin solution and then adding it to the fibrinogen solution; by incorporating thrombin into a "substrate" (described in detail below); or by a combination of these methods. When the substrate contains thrombin, fibrinogen and thrombin can be combined by applying the fibrinogen solution to the substrate.

[0132] Any means can be used to mold the fibrinogen solution containing suspended cells into a sheet of the desired size and thickness, for example, by applying the fibrin gel solution to a suitable substrate, or by one or a combination of any of the molding means conventionally known in the field of polymer sheet production (e.g., formwork molding, extrusion molding, calendar molding, inflation molding, etc.), preferably by applying the fibrin gel solution to a suitable substrate. This embodiment makes it possible to maintain a uniform dispersion of the suspended cells, and can easily mold the fibrinogen solution containing suspended cells into a sheet of uniformly dispersed cells not only when the fibrinogen solution containing suspended cells is injected all at once, but also when the fibrinogen solution is injected continuously (over a long period of time from the start to the end of injection).

[0133] The "substrate" may be any one having a flat surface on which a sheet can be formed, and is not particularly limited, but preferably the above-mentioned supports can be used as the substrate. The fibrinogen solution in which cells are suspended can be applied to the substrate by any means, for example, by using one or more of injection, a spatula, a trowel, a spray, a bioprinter (2D or 3D printer), etc., manually and / or mechanically, and preferably mechanically using a bioprinter.

[0134] The fibrin gel sheet of the present invention produced by the method of the present invention can be stored until use in a cell culture medium such as, but not limited to, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, MEM medium, CMRL medium, etc., or a buffer solution such as, but not limited to, phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), etc., or an isotonic solution such as, but not limited to, physiological saline.

[0135] 4. Uses of the Fibrin Gel Sheet of the Present Invention The fibrin gel sheet of the present invention, depending on the above-described cells dispersed and encapsulated therein, has functions equivalent to or similar to those of cells or their precursor cells that constitute organs or tissues, such as (but not limited to) the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lung, thyroid, pancreas, liver, muscle, skeleton, heart, blood vessels, spleen, kidney, and blood, and can be used in cell therapy methods to enhance or maintain the functions of these organs or tissues, or to support or supplement the functions of these organs or tissues that have been reduced or lost due to disease, disorder, or the like, and can be transplanted into patients in need thereof.

[0136] In one embodiment of the present invention, when the fibrin gel sheet of the present invention contains iPIC derived from pluripotent stem cells, the fibrin gel sheet of the present invention can improve and / or maintain blood glucose levels in transplanted patients through the action of insulin and glucagon secreted by the iPIC, thereby controlling blood glucose levels to normal levels. Therefore, the fibrin gel sheet of the present invention containing iPIC can be used to treat or prevent diseases, disorders, or symptoms that require improvement and / or maintenance of blood glucose levels. Such diseases, disorders, or symptoms include, but are not limited to, diabetes (type 1 diabetes, type 2 diabetes), altered fasting and postprandial glucose levels, and hypoglycemia (e.g., hypoglycemia caused by insulin administration in diabetic patients). "Treatment" refers to the treatment, cure, prevention, or improvement of remission of a disease, disorder, or symptom, or the reduction in the rate of progression of a disease, disorder, or symptom. "Prevention" refers to reducing the likelihood or risk of onset of a disease, disorder, or symptom, or delaying the onset of a disease, disorder, or symptom.

[0137] The subjects for transplantation of the fibrin gel sheet of the present invention are patients who require it for cell therapy, and examples thereof include mammals such as mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, and humans (but are not limited to these), preferably large mammals, and particularly preferably humans.

[0138] When the fibrin gel sheet of the present invention is transplanted, the fibrin gel is rapidly degraded, allowing the encapsulated cells to quickly come into contact with the host tissue and receive oxygen, nutrients, etc. Therefore, the fibrin gel sheet of the present invention can be transplanted into tissues with poor blood vessels (e.g., subcutaneous tissue), and prior angiogenesis treatment is not necessary or essential for transplantation.

[0139] The fibrin gel sheet of the present invention can be transplanted in one or more sheets depending on the patient's symptoms and the size of the transplantation site. Furthermore, due to the thinness of the fibrin gel sheet of the present invention, multiple sheets may be stacked and transplanted as needed.

[0140] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0141] - Production of iPIC iPIC was prepared by inducing differentiation from human iPS cells (Ff-I14-s04 strain) according to the above steps 1)-6) and previously reported (Stem Cell Research (2015) 14, 185-197; Nature Biotechnology 2014; 32: 1121-1133).

[0142] Specifically, pancreatic progenitor cells prepared by inducing differentiation from human iPS cells were cultured for 2 days in a differentiation induction medium (Improved MEM / 1% B-27 / Penisilin Streptomycin medium) containing ALK5iII (10 μM) together with differentiation factors (SANT1, retinoic acid, T3, LDN, a Wnt inhibitor, a ROCK inhibitor, and FGF2), to induce differentiation into endocrine precursor cells.

[0143] Next, the cells were cultured for 7 days in a differentiation induction medium (Improved MEM / 1% B-27 / Penisilin Streptomycin medium) containing ALK5iII (10 μM) together with differentiation factors (T3, LDN, γ-secretase inhibitor RO, FGF receptor 1 inhibitor PD-166866), and then cultured in a differentiation induction medium (MCDB131 / 20 mM Glucose / NaHCO3 / FAF-BSA / ITS-X / Glutamax / Penisilin medium) containing ALK5iII (10 μM) together with T3, LDN, γ-secretase inhibitor RO, N-acetylcysteine, AXL inhibitor R428, ascorbic acid, ROCK inhibitor, ZnSO4, heparin, and Trolox. The cells were cultured in a streptomycin-free medium for 4 days to induce differentiation into iPICs.

[0144] The obtained iPIC had the form of an aggregate (spheroid) (diameter: approximately 150 μm) of approximately 500 cells.

[0145] Example 1 Evaluation of implant shape <Method> Preparation of fibrin gel clot iPIC 3x10 6The cells (approximately 6,000 cells in aggregate) were placed in a 1.5 mL tube and centrifuged. The medium supernatant was removed, and 25 μL of fibrinogen solution (80 mg / mL) was added to suspend the cells. 25 μL of thrombin solution (125 U / mL) was then added and the cells were left to stand for 5 minutes to form a gel, producing a fibrin gel clot (approximately 5 mm in diameter) containing iPIC.

[0146] ・Preparation of fibrin gel sheet iPIC 3x10 6 The cells were separated into 1.5 mL tubes and centrifuged. The supernatant was removed, and 25 μL of fibrinogen solution (80 mg / mL) was added to suspend the cells. A thin layer of the fibrinogen solution containing iPIC was applied to a polyester mesh (opening ratio 69%, 10 × 10 mm) impregnated with 12.5 μL of thrombin solution (125 U / mL). 25 μL of thrombin solution was then applied to the entire surface and allowed to stand for 5 minutes to gel, producing a thin fibrin gel sheet containing iPIC.

[0147] In vivo evaluation: A fibrin gel clot containing iPIC and a fibrin gel sheet containing iPIC were implanted subcutaneously into anesthetized nude rats. After implantation, blood was collected from the tail vein, plasma was separated, and human c-peptide concentration was measured by ELISA.

[0148] <Results> The appearance of the fibrin gel clot and fibrin gel sheet is shown in Figure 1. In both cases, iPIC was retained within the gel, and no cell leakage was observed.

[0149] Figure 2 shows the results of measuring the blood human c-peptide concentration over time in nude rats into which a fibrin gel clot containing iPIC or a fibrin gel sheet containing iPIC was subcutaneously implanted, and Figure 3 shows the results of histological analysis of each implantation site 10 days and 6 weeks after implantation.

[0150] The results in FIG. 2 show that the blood human c-peptide concentration was higher when the fibrin gel containing iPIC was implanted in the form of a sheet than when it was implanted in the form of a block.

[0151] Furthermore, the histological analysis results 10 days after implantation, shown in Figure 3, showed that in the block-form, only the fibrin gel in the peripheral area was degraded, and the iPIC in that area was in contact with the host tissue, but most of the fibrin gel remained, and some iPIC was not in contact with the host tissue. On the other hand, in the sheet-form, almost all of the fibrin gel was rapidly degraded, and the implanted iPIC was in contact with the host tissue.

[0152] Six weeks after transplantation, all gels had decomposed even when transplanted in block form, but the blood human c-peptide concentration was higher when the gel was transplanted in sheet form. Therefore, even when a biodegradable gel is used, the rapid degradation of the gel in the early stages of transplantation, allowing the transplanted cells to come into contact with the host and receive a supply of oxygen, nutrients, etc., is important for the survival of the transplanted cells and also affects the long-term efficacy of the drug thereafter, demonstrating the usefulness of transplanting in thin sheet form.

[0153] Example 2 Preparation of large animal / human-sized fibrin gel sheets (Manufacturing method 1—method using a cooled metal mold) <Method> Evaluation of gelation time Fibrinogen solution (40 mg / mL) and thrombin solutions (31 U / mL, 15.6 U / mL, 10.4 U / mL, 5.2 U / mL, 3.1 U / mL) were prepared, and 440 μL of each solution was combined in a 1.5 mL tube and mixed by pipetting once or twice. The state of gelation was then evaluated according to the following criteria, and the relationship between thrombin concentration and gelation time was analyzed.

[0154] Evaluation criteria for gelation state: ◯: Pipetting is possible in the tube △: Pipetting is possible, but only partial gelation occurs ×: Overall gelation prevents pipetting

[0155] ・Preparation of large animal / human size fibrin gel sheets iPIC 45x10 6The cells were separated into 15 mL tubes and centrifuged, and the medium supernatant was removed. 360 μL of fibrinogen solution (40 mg / mL) was added and suspended. 360 μL of thrombin solution (15.6 U / mL) was then added and suspended. The cells were quickly spread on a metal mold (40 × 60 × 0.4 mm) pre-cooled to 4°C, and a polyester mesh (opening ratio 69%, size 40 × 10 mm) was placed on top. The mixture was allowed to stand at room temperature for 6 minutes to gel.

[0156] <Results> Analysis of the relationship between thrombin concentration and gelation time showed that the gelation time was delayed as the thrombin concentration decreased, and that at a concentration of 15.6 U / mL or less, the time required to mold a fibrin gel sheet measuring 40 x 60 x 0.4 mm could be secured (Figure 4).

[0157] By rapidly spreading a fibrinogen solution (40 mg / mL) and a thrombin solution (15.6 U / mL) onto a cooled metal mold, we were able to fabricate a fibrin gel sheet measuring 40 x 60 x 0.4 mm suitable for use in large animals and humans (Figure 5). Furthermore, when the resulting fibrin gel sheet was examined under a phase-contrast microscope, it was confirmed that the encapsulated iPIC was uniformly dispersed throughout the sheet (iPIC is observed as black granules in Figure 5(B)).

[0158] Example 3: Preparation of Large Animal / Human-Sized Fibrin Gel Sheets (Production Method 2-1—Fibrin + Gelatin Microsphere Gel Sheet) <Method> - Confirmation of Cell Dispersion Maintenance Gelatin was dissolved in physiological saline to a concentration of 2 w / v%, cooled in a refrigerator at 4°C to gel, and then passed through a 200 μm mesh to prepare gelatin gel microspheres (hereinafter referred to as "gelatin microspheres") of approximately 200 μm. The gelatin microspheres were suspended in a fibrinogen solution (40 mg / mL) to a concentration of 20 w / v%, to prepare a fibrinogen + gelatin microsphere solution. iPIC was suspended in the fibrinogen solution (40 mg / mL) and the fibrinogen + gelatin microsphere solution and allowed to stand. The maintenance of cell dispersion was evaluated based on the presence or absence of precipitate formation.

[0159] In vivo test iPIC 3x10 6 The cells were placed in a 1.5 mL tube and centrifuged. The supernatant was removed, and 25 μL of the fibrinogen + gelatin microsphere solution prepared as described above was added and suspended. The fibrinogen + gelatin microsphere solution containing iPIC was thinly applied to a polyester mesh (opening ratio 69%, size 10 × 10 mm) impregnated with 12.5 μL of thrombin solution (62.5 U / mL). 25 μL of thrombin solution was then applied to the entire surface and allowed to stand for 5 minutes to gel, producing a thin fibrin + gelatin microsphere gel sheet containing iPIC.

[0160] The fibrin gel sheet containing iPIC described in Example 1 and the fibrin + gelatin microsphere gel sheet containing iPIC were subcutaneously transplanted into nude rats under anesthesia. After transplantation, blood was collected from the tail vein, and plasma was separated and measured for human c-peptide concentration by ELISA.

[0161] ・Large animal / human size iPIC 75x10 6 The cells were separated into 15 mL tubes and centrifuged, and the medium supernatant was removed. 600 μL of fibrinogen + gelatin microsphere solution was added and suspended. A thin layer of the fibrinogen + gelatin microsphere solution containing iPIC was applied to a polyester mesh (opening ratio 69%, size 40 x 60 mm) and a glycolic acid / lactic acid polyester (90 / 10, poly(glycolide-co-L-lactide), size 40 x 60 mm) mesh impregnated with 300 μL of thrombin solution (62.5 U / mL) using a pipette or a 3D printer. 600 μL of thrombin solution was then applied to the entire surface and allowed to stand for 5 minutes to gel, creating a thin layer of fibrin + gelatin microsphere gel sheet containing iPIC.

[0162] <Results> Figure 6 shows the state of iPIC suspended in a fibrinogen solution and a fibrinogen + gelatin microsphere solution and allowed to stand. In the fibrinogen solution (Figure 6(A)), precipitation of iPIC was observed after 10 minutes of standing (arrowhead). On the other hand, in the fibrinogen + gelatin microsphere solution (Figure 6(B)), the iPIC remained dispersed even after 30 minutes of standing.

[0163] Furthermore, in the thin fibrin + gelatin microsphere gel sheet containing iPIC that was produced, the iPIC was retained within the gel sheet, and no leakage of cells was observed.

[0164] 7 shows the results of measuring the blood human c-peptide concentration over time in nude rats subcutaneously implanted with an iPIC-encapsulated fibrin gel sheet or an iPIC-encapsulated fibrin + gelatin microsphere gel sheet. No significant difference in the blood human c-peptide level was observed between the fibrin gel sheet and the fibrin + gelatin microsphere gel sheet, demonstrating that the use of gelatin microspheres in the sheet does not negatively affect human c-peptide secretion from iPIC.

[0165] Figure 8 shows fibrin + gelatin microsphere gel sheets containing large animal / human-sized iPICs fabricated manually by pipette injection and using a 3D printer. Both methods enabled fabrication of large animal / human-sized fibrin + gelatin microsphere gel sheets, and the iPICs were retained within the gel, with no cell leakage observed (Figure 8(A)).

[0166] Image analysis of the brightness distribution of fibrin + gelatin microsphere gel sheets encapsulating large animal- and human-sized iPICs fabricated by manual pipetting and using a 3D printer yielded standard deviations of 19.437 and 15.630, respectively (Figure 8(B)). These results demonstrate that the 3D printer fabrication method allows for more uniform application of the fibrin + gelatin microsphere gel. Furthermore, four 12 mm diameter circular cutouts were randomly placed on the fibrin + gelatin microsphere gel sheets fabricated using a 3D printer, and the number of cells encapsulated in these cutouts was determined. This confirmed that the number of cells was approximately the same (Figure 8(C)). These results demonstrate that the use of a 3D printer makes it possible to fabricate fibrin + gelatin microsphere gel sheets encapsulating uniformly distributed cells.

[0167] Example 4: Preparation of Large Animal / Human-Sized Fibrin Gel Sheets (Manufacturing Method 2-2: Fibrin + Collagen Gel Sheet) <Method> Confirmation of Cell Dispersibility Maintenance A fibrinogen + collagen solution was prepared by mixing a fibrinogen solution (80 mg / mL) and a collagen solution (3 w / v%) at a 3:1 ratio. iPIC was suspended in the fibrinogen + collagen solution and then loaded into a syringe. The syringe was held vertically with the tip facing downwards, and after leaving the solution for 0, 10, 20, and 30 minutes, a fixed amount of the discharged solution was taken and the cell count (concentration) was measured to evaluate the cell dispersion maintenance function of the fibrinogen + collagen solution.

[0168] In vivo test: Fibrinogen solution (80 mg / mL) and collagen solution (3 w / v%) were mixed in a 3:1 ratio to prepare a fibrinogen + collagen solution. iPIC 3x10 6The cells were placed in a 1.5 mL tube and centrifuged. The culture supernatant was removed, and 25 μL of fibrinogen + collagen solution was added and suspended. A thin layer of the fibrinogen + collagen solution containing iPIC was applied to a glycolic acid / lactic acid polyester (90 / 10, poly(glycolide-co-L-lactide)) mesh impregnated with 12.5 μL of thrombin solution (125 U / mL). 25 μL of thrombin solution was then applied to the entire surface and allowed to stand for 5 minutes to gel, producing a thin fibrin + collagen gel sheet containing iPIC.

[0169] The fibrin+gelatin microsphere gel sheet encapsulating iPIC and the fibrin+collagen gel sheet encapsulating iPIC described in "In vivo test" in Example 3 were implanted subcutaneously into nude rats under anesthesia. After implantation, blood was collected from the tail vein, and plasma was separated and measured for human c-peptide concentration by ELISA.

[0170] ・Large animal / human size iPIC 75x10 6 The cells were separated into 15 mL tubes and centrifuged, after which the culture supernatant was removed and 600 μL of fibrinogen + collagen solution was added and suspended. A thin layer of the fibrinogen + collagen solution containing iPIC was applied to a poly(glycolide-co-L-lactide) mesh (40 x 60 mm) impregnated with 300 μL of thrombin solution (125 U / mL) using a 3D printer, and then 600 μL of thrombin solution was applied to the entire surface and left to gel for 5 minutes to produce a thin fibrin + collagen gel sheet containing iPIC.

[0171] <Results> Figure 9 shows the results of measuring the number (concentration) of cells in the fibrinogen + collagen solution discharged from the syringe. The cell concentration in the discharged solution was almost constant regardless of the standing time, and it was shown that even though the syringe was held vertically, iPIC did not precipitate and an almost constant number of cells was discharged. Therefore, it was shown that the use of the fibrinogen + collagen solution makes it possible to maintain a dispersed state of cells.

[0172] Furthermore, in the thin fibrinogen+collagen sheet containing iPIC that was produced, the iPIC was retained within the gel sheet, and no leakage of cells was observed.

[0173] 10 shows the results of measuring the blood human c-peptide concentration over time in nude rats subcutaneously implanted with a fibrin + gelatin microsphere gel sheet containing iPIC or a fibrin + collagen gel sheet containing iPIC. No significant difference in the blood human c-peptide level was observed between the fibrin + gelatin microsphere gel sheet and the fibrin + collagen gel sheet, demonstrating that both have equivalent performance.

[0174] It was possible to fabricate large animal and human-sized fibrin and collagen sheets using a 3D printer, and the iPIC was retained within the gel, with no cell leakage observed. Six 12 mm diameter circular cutouts were randomly placed in the fabricated large animal and human-sized fibrin and collagen gel sheets, and the number of cells encapsulated within each cutout was measured. The results confirmed that the number of cells was approximately the same (Figure 11). These results demonstrate that by suspending iPIC in a fibrinogen and collagen solution and applying it using a 3D printer, it is possible to fabricate fibrin and collagen gel sheets encapsulating cells in a uniform distribution.

Claims

1. The cells are uniformly dispersed and encapsulated within the fibrin gel sheet. The fibrin gel sheet further comprises 0.2 to 2 w / v% of collagen and / or 10 to 30 w / v% of gelatin gel particles that can pass through 100 μm to 1000 μm. Fibrin gel sheets for cell transplantation.

2. The surface area of ​​one side is 2.25 cm². ２ A fibrin gel sheet according to claim 1, having the above dimensions and a thickness of 1 mm or less.

3. The aforementioned cells are 1.5 × 10 ６ pieces / cm ２ A fibrin gel sheet according to claim 1, which is contained in the amount of [amount].

4. The fibrin gel sheet according to claim 1, wherein the cells are in the form of spheroids.

5. The fibrin gel sheet according to claim 1, wherein the cells are iPS cell-derived pancreatic islet cells.

6. The fibrin gel sheet according to claim 1, further comprising a support.

7. A method for producing a fibrin gel sheet for cell transplantation, wherein cells are uniformly dispersed and encapsulated within the fibrin gel sheet, The process includes reacting a fibrinogen solution containing the suspended cells with thrombin to form a sheet and gel it, A method for producing a fibrinogen solution in which the cells are suspended further comprises 0.2 to 2 w / v% of collagen and / or 10 to 30 w / v% of gelatin gel particles that can pass through 100 μm to 1000 μm.

8. The aforementioned gelling step involves reacting the fibrinogen solution in which the cells are suspended with thrombin, resulting in a surface area of ​​2.25 cm². ２ The manufacturing method according to claim 7, wherein the material is formed into a sheet having the above dimensions and a thickness of 1 mm or less, and then gelled.

9. The aforementioned cells were 1.5 × 10 in a fibrin gel sheet. ６ pieces / cm ２ The manufacturing method according to claim 7, wherein the amount is suspended in a fibrinogen solution.

10. The method for producing the cell according to claim 7, wherein the cell is in the form of a spheroid.

11. The method for producing the pancreatic islet cells derived from iPS cells, according to claim 7.

12. The manufacturing method according to claim 7, wherein the material is molded into a sheet by being applied to a support.

13. The manufacturing method according to claim 12, wherein the coating is applied onto a support by bioprinting.

14. The manufacturing method according to claim 12, wherein the support comprises thrombin.

15. A laminate of fibrin gel sheets for cell transplantation, comprising a plurality of fibrin gel sheets as described in claim 1.