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 uniformly dispersed and encapsulated while having an unprecedented larger size, and a method for producing the same, and to provide a fibrin gel sheet for cell transplantation in which cells are uniformly dispersed and encapsulated in the fibrin gel sheet, the fibrin gel sheet having a size in which the surface area of one surface is 2.25 cm2 or more, and the fibrin gel sheet having a size in which cells are uniformly dispersed and encapsulated in the fibrin gel sheet. The fibrin gel sheet for cell transplantation has a thickness of 1 mm or less; and a method for producing the fibrin gel sheet for cell transplantation.

Description

Technical Field

[0001] 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 and which has an unprecedented large size, and a method for producing the same. Background of the Invention Functional differentiated cells induced from pluripotent stem cells such as induced pluripotent stem cells and embryonic stem cells (ES cells) are expected to be a cell supply source for transplantation and regenerative medicine. Currently, various structures have been developed in which such functional differentiated cells are encapsulated in biodegradable gels, and research on cell transplantation therapy using them is progressing.

[0003] For example, non-patent document 1 discloses a sheet-like structure in which pancreatic islet cells encapsulated in a fibrin gel are sandwiched between 7 mm×7 mm Vicryl (registered trademark) meshes, and records that the structure is transplanted into a site of a diabetic model rat that has been pre-treated for angiogenesis as a recipient.

[0004] Non-patent document 2 discloses a sheet-like structure in which islet cells encapsulated in a hydrogel containing polyvinyl alcohol (PVA) are sandwiched between polyethylene terephthalate (PET) meshes of 20 mm×15 mm, and records that the structure is transplanted into diabetic model rats as recipients.

[0005] Patent Document 1 discloses a sheet-like structure in which pancreatic islet cells encapsulated in a hydrogel containing PVA are sandwiched between circular PET meshes having a diameter of 15 mm, and describes transplanting the structure into diabetic model mice as recipients. Prior art literature Patent Literature

[0006] Patent Document 1: WO2018 / 155622 Non-patent literature

[0007] Non-patent literature 1: Hirotake Komatsu et al., Transplant International 2020; 33: 806-818. Non-patent document 2: Meirigeng Qi et al., Biomaterials. 2004 Dec; 25(27): 5885-92. Summary of the invention Problems to be solved by the invention

[0008] On the other hand, the sheet-like structures in which functional differentiated cells are encapsulated in biodegradable gels reported in the past are intended for transplantation into diabetic model animals (rats and mice), but their sizes are not large enough for large mammals such as humans, and the number of cells carried is also insufficient. Therefore, the art is eager to develop the above-mentioned structures with a larger size that can be used for humans and the like and contain more cells.

[0009] To solve this problem, the inventors of the present invention, when trying to prepare the above-mentioned structure with a larger size using fibrin gel, found that when trying to spread a large amount of fibrin gel prepared for making a larger size to the desired size and forming it into a sheet, gelation (solidification) occurred before fully spreading, making it difficult to form it into the desired size. In addition, when trying to continuously inject this large amount of fibrin gel to form a sheet with the desired size, it was found that the cells in the solution precipitated, and the cell content gradually decreased from the beginning to the end of the continuous injection, and as a result, only the above-mentioned structure with uneven cell density could be produced. The uneven cell density may reduce the treatment efficiency and affect the long-term implantation of the transplanted cells, so it is not preferred.

[0010] Therefore, the present invention aims to solve the above-mentioned problems found by the present inventors, and has an object to provide a sheet-like fibrin gel structure (i.e., a fibrin gel sheet) having an unprecedentedly large size and containing more cells dispersed uniformly. Solution to the problem

[0011] The inventors of the present invention have conducted in-depth research to solve the above-mentioned problems, and found that in the formation of fibrin gel, by adjusting the mixing amount of fibrinogen and thrombin and / or the temperature during gelation (solidification), the gelation (solidification) speed of the formed fibrin gel can be adjusted, and a fibrin gel sheet of the desired large size can be spread and formed as needed.

[0012] In addition, in the formation of fibrin gel, by adding biodegradable particles to a fibrinogen solution in which cells are suspended and suspending the solution, or by adding a biodegradable gelling agent and dissolving the solution, cell precipitation can be inhibited in the fibrinogen solution, so that the cells are maintained in a uniformly dispersed suspended state, and by gelling (solidifying) the cells, a fibrin gel sheet with uniform cell density can be obtained even when molding is performed by continuous injection.

[0013] The present invention is based on these new findings and includes the following inventions. [1] A fibrin gel sheet for cell transplantation, wherein cells are uniformly dispersed and encapsulated in the fibrin gel sheet, and the sheet has a surface area of ​​2.25 cm 2The size is above and the thickness is below 1 mm. [2] The fibrin gel sheet according to [1], wherein the number of cells is greater than 1.5×10 6 Pieces / cm 2 The amount is insourced. [3] The fibrin gel sheet according to [1] or [2], wherein the cells are in the form of spheroids. [4] The fibrin gel sheet according to any one of [1] to [3], wherein the cells are pancreatic islet cells derived from iPS cells. [5] The fibrin gel sheet according to any one of [1] to [4], further comprising a biodegradable gelling agent. [6] The fibrin gel sheet according to [5], comprising the biodegradable gelling agent in an amount of 0.2 to 2 w / v%. [7] The fibrin gel sheet according to [5] or [6], wherein the biodegradable gelling agent is collagen. [8] The fibrin gel sheet according to any one of [1] to [4], further comprising biodegradable particles. [9] The fibrin gel sheet according to [8], wherein the size of the biodegradable particles that can pass through the openings is 100 μm to 1000 μm.

[10] The fibrin gel sheet according to [8] or [9], comprising 10 to 30 w / v% of biodegradable particles.

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

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

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

[11] , further comprising a carrier.

[13] A method for producing a cell-free gel sheet having a surface area of ​​2.25 cm on one side, wherein cells are uniformly dispersed and encapsulated in a fibrin gel sheet. 2 A method for producing a fibrin gel sheet for cell transplantation having a size of 1 mm or more and a thickness of 1 mm or less, The method comprises allowing a fibrinogen solution containing cells suspended therein to act with thrombin, and forming a film having a surface area of ​​2.25 cm on one side. 2 The process of forming the sheet into a size of 1 mm or more and a thickness of 1 mm or less and gelling it.

[14] The method according to

[13] , wherein the cells are present in a fibrin gel sheet in an amount of more than 1.5×10 6 Pieces / cm 2 The amount was suspended in the fibrinogen solution.

[15] The production method according to

[13] or

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

[16] The production method according to any one of

[13] to

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

[17] The production method according to any one of

[13] to

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

[18] The production method according to

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

[19] The production method according to

[17] or

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

[20] The production method according to any one of

[17] to

[19] , wherein the sheet is formed by coating on a support.

[21] The manufacturing method according to

[20] , wherein coating on the carrier is performed by bioprinting.

[22] The production method according to

[20] or

[21] , wherein the carrier contains thrombin.

[23] The production method according to any one of

[13] to

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

[24] The production method according to

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

[25] The production method according to

[23] or

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

[26] The production method according to any one of

[23] to

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

[27] The production method according to any one of

[23] to

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

[28] The manufacturing method according to

[27] , wherein coating on the carrier is performed by bioprinting.

[29] The production method according to

[27] or

[28] , wherein the carrier contains thrombin.

[30] The production method according to any one of

[13] to

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

[31] The production method according to

[30] , wherein the product is molded into a sheet while cooling at 2 to 8°C.

[32] The production method according to

[30] or

[31] , further comprising the step of integrating the carrier and the fibrin gel sheet.

[33] A laminate of fibrin gel sheets for cell transplantation, comprising a plurality of fibrin gel sheets according to any one of [1] to

[12] laminated together. [1a] A fibrin gel sheet for use in a cell transplantation therapy method, wherein cells are uniformly dispersed and encapsulated in the fibrin gel sheet, wherein the fibrin gel sheet has a surface area of ​​2.25 cm 2 The size is above and the thickness is below 1 mm. [2a] The fibrin gel sheet according to [1a], wherein the number of cells is greater than 1.5×10 6 Pieces / cm 2 The amount is insourced. [3a] The fibrin gel sheet according to [1a] or [2a], wherein the cells are in the form of spheroids. [4a] The fibrin gel sheet according to any one of [1a] to [3a], wherein the cells are pancreatic islet cells derived from iPS cells. [5a] The fibrin gel sheet according to any one of [1a] to [4a], further comprising a biodegradable gelling agent. [6a] The fibrin gel sheet according to [5a], comprising a biodegradable gelling agent in an amount of 0.2 to 2 w / v%. [7a] The fibrin gel sheet according to [5a] or [6a], wherein the biodegradable gelling agent is collagen. [8a] The fibrin gel sheet according to any one of [1a] to [4a], further comprising biodegradable particles. [9a] The fibrin gel sheet according to [8a], wherein the size of the biodegradable particles that can pass through the openings is 100 μm to 1000 μm. [10a] The fibrin gel sheet according to [8a] or [9a], wherein the biodegradable particles are contained in an amount of 10 to 30 w / v%. [11a] The fibrin gel sheet according to any one of [8a] to [10a], wherein the biodegradable particles are gelatin gel particles. [12a] The fibrin gel sheet according to any one of [1a] to [10a], further comprising a carrier. [33a] A stack of fibrin gel sheets for use in a cell transplantation therapy method, comprising a plurality of stacked fibrin gel sheets according to any one of [1a] to [12a]. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2022-113546 filed on Jul. 14, 2022, which is the basis of priority of this application. All publications, patents and patent applications cited in this specification are directly incorporated into this specification as reference. Effects of the Invention

[0014] According to the present invention, it is possible to provide a fibrin gel sheet having an unprecedentedly large size and containing a greater number of uniformly dispersed cells, and a method for producing the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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[0016] 1. Terminology The following is a description of the terms used in this specification.

[0017] In this specification, "about" means a value fluctuating within plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference value. Preferably, the term "about" means a range of plus or minus 15%, 10%, 5% or 1% relative to a reference value.

[0018] In this specification, "comprise(s) or comprising~" means including / containing the elements following the sentence, but is not limited thereto. Therefore, although it implies including / containing the elements following the sentence, it does not imply excluding any other elements.

[0019] In this specification, "consist(s) of or consisting of" means including and limited to all the elements following the sentence. Therefore, the sentence "consist(s) of" means that the listed elements are essential or necessary, and there are substantially no other elements.

[0020] In this specification, "not using feeder cells" means that feeder cells are substantially not contained, and a medium pretreated by culturing feeder cells is not used, etc. Therefore, the medium does not contain substances such as growth factors and cytokines secreted by feeder cells.

[0021] In addition, "feeder cells" or "feeders" refer to cells that are co-cultured with other types of cells and provide an environment that can support the cells and make them grow. Feeder cells can be of the same source as the cells they support, or they can be of different sources. For example, as feeders for human cells, human skin fibroblasts or human embryonic stem cells can be used, and primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts can also be used. Feeder cells can be inactivated by radiation irradiation or mitomycin C treatment.

[0022] In this specification, "adhesion" refers to the attachment of cells to a container, for example, cells adhere to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate culture medium. There are cells that cannot be maintained or grown in culture without adhering to the cell culture container. Non-adherent cells can still be maintained and proliferated in culture without adhering to the container.

[0023] In this specification, "culture" means maintaining cells in an in vitro environment and causing them to grow and / or differentiate. "Cultivating" means causing cells to continue, proliferate and / or differentiate in a tissue or in vitro, such as in a cell culture dish or flask. Cultivation includes two-dimensional culture (planar culture) and three-dimensional culture (suspension culture).

[0024] In this specification, "purify" and "purification" refer to removing impurities from a composition such as a composition of cells to make a specific constituent pure; "purified", when used to describe a composition of cells, refers to a composition of cells in which the amount of impurities is reduced compared to the proportion of the constituent in the composition of cells before purification, and the purity of the specific constituent is improved. For example, a composition such as a composition of cells related to a target cell type can be purified, so that the proportion of the target cell type is increased compared to the proportion of target cells present in the composition of cells before purification. The composition of cells related to the target cell type can also be purified by cell selection and screening methods known in the art. The composition of cells can also be purified by a specific screening or selection process described in this specification. In a specific embodiment of the present invention, the purity of the composition of the target cell is made to be at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99%, or it can be made to a degree where impurities (including mixed cells) cannot be detected.

[0025] In the present specification, "growth factor" refers to an endogenous protein that promotes differentiation and / or proliferation of specific cells. Examples of "growth factor" 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 effective for stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0026] In the present specification, "ROCK inhibitor" means 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-pyridyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (sometimes referred to as Y27632 in the present specification), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolyl)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]pyrrolidone-1α-carboxamide (Wf-547), and N-(1H-pyrrolo[2,3-b]pyrrolidone-1α-carboxamide (Wf-548). [4-(trifluoromethyl)phenyl]-1-(6-fluoro-1H-indazol-5-yl)-4-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A). The ROCK inhibitor is not limited to the above, and antisense oligonucleotides or siRNA to ROCK mRNA, antibodies binding to ROCK, dominant negative ROCK mutants, etc. can also be used as ROCK inhibitors, which are commercially available or can be synthesized according to known methods.

[0027] In this specification, "GSK3β inhibitor" refers to a substance that has inhibitory activity on GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a type of serine / threonine protein kinase, which is involved in many signal transduction pathways such as glycogen production or apoptosis, maintenance of stem cells, etc. GSK3 has 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 can also be a substance that is combined with GSK3β inhibitory activity and has GSK3α inhibitory activity.

[0028] Examples of 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)-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), and TWS-119 (3-[ 6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy]phenol), Kenpaullone, 1-azakenpaullone, 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) and AR-AO144-18, CT99021, CT20026, BIO, BIO-acetone oxime, pyridocarbazole-cyclopentadienylruthenium complex, OTDZT, α-4-dibromoacetophenone, lithium, etc. GSK3β is not limited to the above, and antisense oligonucleotides or siRNA to GSK3β mRNA, antibodies binding to GSK3β, dominant negative GSK3β mutants, etc. can also be used as GSK3β inhibitors, which are commercially available or can be synthesized according to known methods.

[0029] The "serum replacement" in this specification includes, for example, KnockOut TMSerum Replacement (KSR: Thermo Fisher Scientific), StemSure (registered trademark) 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'-thioglycerol or a mixture thereof (e.g., ITS-G). As serum substitutes, B-27 supplement, KSR, StemSure (registered trademark) Serum Replacement, ITS-G are preferred. When the serum substitute is added to the culture medium, its concentration in the culture medium is 0.01%-10% by weight, preferably 0.1%-2% by weight. In the present invention, it is preferred to use a "serum substitute" instead of serum.

[0030] In this specification, "factors having CDK8 / 19 inhibitory activity" or "CDK8 / 19 inhibitors" means all substances having CDK8 / 19 inhibitory activity. Compared with other proteins in the CDK family, CDK8 is not essential for cell proliferation, and the inhibition of CDK8 does not have much effect under normal conditions. CDK19 is similar to CDK8, and the inhibition of CDK8 is usually accompanied by the inhibition of CDK19. "Factors having CDK8 / 19 inhibitory activity" can use previously known factors, and such CDK8 / 19 inhibitors can be found in patent literature or non-patent literature. For example, US 2012 / 0071477, WO 2015 / 159937, WO2015 / 159938, WO 2013 / 116786, WO 2014 / 0038958, WO 2014 / 134169, JP 2015 / 506376, US2015 / 0274726, US2016 / 0000787, WO 2016 / 009076, WO 2016 / 0016951, WO 2016 / 018511, WO 2016 / 100782 and WO Among the compounds described in 2016 / 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, (E)-(4-(3-(5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamide)benzyl)phosphonic acid diethyl ester, 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]dioxin-6-yl)-1H-pyrazol-3-yl)benzene-1 ...benzene Alcohol, 3-(2-(imidazo[1,2-b]pyridazin-6-ylthio)ethyl)-4-(naphthalene-1-ylsulfonyl)-3,4-dihydroquinoxaline-2(1H)-one, and (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinylmethyl)phenyl)acrylamide or its salt can be used as "factors having CDK8 / 19 inhibitory activity". Factors having CDK8 / 19 inhibitory activity are not limited to the above, and antisense oligonucleotides or siRNAs to 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, which can be commercially available or synthesized according to a known method.

[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 in that it has a size that has not been achieved in conventional fibrin gel sheets for cell transplantation, and the surface area of ​​one side of the sheet (i.e., any one of the main surfaces of the sheet, also referred to as a single side of the sheet) is 2.25 cm 2 More than 4cm, preferably 2 More preferably 9 cm 2 More preferably, 16 cm 2 More preferably, 25 cm 2 More than, especially preferably 36cm 2 More preferably, 49 cm 2 More preferably, 64 cm 2 More preferably, 81 cm 2 More than, particularly preferably 100cm 2 The upper limit is not particularly limited and can be appropriately selected according to the size and shape of the transplant site, but for example, 400 cm 2 The following dimensions.

[0033] The range of the surface area of ​​the fibrin gel sheet of the present invention can be represented by two values ​​selected from the lower limit and the upper limit, for example, the surface area of ​​the fibrin gel sheet of the present invention can be from 2.25 cm 2 Above~400cm 2 Below, preferably 4cm 2 Above~400cm 2 Less than, more preferably 9cm 2 Above~400cm 2 Below, more preferably 16cm 2 Above~400cm 2 Below, more preferably 25cm 2 Above~400cm 2 Below, especially preferably 36cm 2 Above~400cm 2 Below, especially more preferably 49cm 2 Above~400cm 2 Below, especially more preferably 64cm 2 Above~400cm 2 Below, especially more preferably 81cm 2 Above~400cm 2 Less than, particularly preferably 100cm2 Above~400cm 2 Select the appropriate size from the following range.

[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 according to the size and shape of the transplant site. For example, polygons (for example, triangles, quadrilaterals, pentagons, hexagons, octagons, etc.), rounded polygons, circles, ellipses, etc. can be listed, but are not limited to these.

[0035] For example, when the fibrin gel sheet of the present invention has a quadrangular shape, its size is a longitudinal length of 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, further preferably 40 mm or more, further 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 more preferably 90 mm or more, particularly preferably 100 mm or more, and its upper limit is not particularly limited, for example, it can be 200 mm or less. In addition, the transverse length of the fibrin gel sheet of the present invention is 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, further preferably 40 mm or more, further 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 more preferably 90 mm or more, particularly preferably 100 mm or more, and its upper limit is not particularly limited, for example, it can be 200 mm or less.

[0036] The range of the longitudinal length and the transverse length can be expressed by two numerical values ​​selected from the above lower limit and upper limit, for example, the size of the fibrin gel sheet of the present invention can be (longitudinal length × transverse length (or transverse length × longitudinal length)), 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, further preferably 40 mm to 200 mm × 40 mm to 200 mm, further preferably 50 mm to 200 mm × 50 mm to 200 mm, and further preferably 60 mm to 200 mm × 60 mm to 200 mm. The size is preferably selected appropriately from the range of 50 mm to 200 mm × 50 mm to 200 mm, especially preferably 60 mm to 200 mm × 60 mm to 200 mm, even more preferably 70 mm to 200 mm × 70 mm to 200 mm, even more preferably 80 mm to 200 mm × 80 mm to 200 mm, even more preferably 90 mm to 200 mm × 90 mm to 200 mm, and particularly preferably 100 mm to 200 mm × 100 mm to 200 mm. For example, the size of the fibrin gel sheet of the present invention can be listed as (longitudinal length × transverse length (or transverse length × longitudinal length)), 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, further preferably 70 mm × 70 mm, 80 mm × 80 mm, 90 mm × 90 mm, 100 mm × 100 mm, or 200 mm × 200 mm, etc., but are not limited to these.

[0037] The thickness of the fibrin gel sheet of the present invention can be appropriately set according to factors such as the size and amount of the cells contained therein, the size of the transplant site, and is preferably less than 1 mm. By making the thickness of the fibrin gel sheet of the present invention less than 1 mm, it is possible to efficiently supply nutrients, oxygen, etc. to the cells contained in the fibrin gel sheet, and after transplantation, the rapid decomposition of the fibrin gel can be achieved, so it is preferred. The thickness of the fibrin gel sheet of the present invention can be, for example, less than 1 mm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, or less than 400 μm, and the lower limit is not particularly limited, for example, it can be more than 100 μm, more than 200 μm, or more than 300 μm. In addition, the range of the thickness of the fibrin gel sheet of the present invention can be represented by two values ​​selected from the above upper limit and the upper limit value, 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 the thickness can be appropriately selected from these ranges. For example, the thickness of the fibrin gel sheet of the present invention is about 400 μm. It should be noted that the thickness of the fibrin gel sheet of the present invention does not need to be uniform throughout the sheet, as long as the thickest part is within the above range.

[0038] In the present specification, "uniformly dispersed and contained" of cells means that the seeded cells are not limited to any part of the fibrin gel sheet of the present invention, but are widely distributed and contained in the fibrin gel sheet. The cells do not have to be completely embedded in the fibrin gel sheet, but for the cell culture seeded on the surface of the fibrin gel and cultured by adhesion, since the cells are only confined to the surface of the fibrin gel sheet, it is different from the scheme of "dispersed and contained" of cells. More specifically, in the present specification, "uniformly dispersed and contained" of cells means that the deviation of the number of each cell (cell density) contained in a given range of any multiple places (for example, 2 places, 4 places, or 6 places, etc.) of the fibrin gel sheet of the present invention is small, and preferably the value of each cell number is within ±20% of their average value, preferably within ±15%, and more preferably within ±10%. In addition, in this specification, "uniform dispersion and inclusion" of cells refers to the positional relationship between the seeded cells, for example, the cell culture and proliferation of the seeded cells on the surface or inside of the fibrin gel, the contact / combination of cell groups of different origins in the seeded cells, and since the cells are not dispersed between the seeded cells, this does not conform to the state of "uniform dispersion and inclusion" of the cells. As such a state of contact / combination between seeded cells, for example, a cell culture (for example, a cell sheet, etc.) formed by seeding and culturing on the surface / inside of a fibrin gel can be cited, and such a scheme is not intended to be included in the present invention. On the other hand, as long as it is not such a state of contact / combination between seeded cells, it is not intended to exclude the morphology of the seeded cells, for example, a cell mass or cell aggregate state formed by the assembly and aggregation of cells, that is, a spheroid morphology, or the proliferation of seeded cells.

[0039] In the present invention, "cells" refer to cells used for transplantation into patients in need for cell therapy, which can be appropriately selected according to the patient's symptoms. For example, cells with the same or similar functions as cells and progenitor cells constituting organs and tissues such as the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lungs, thyroid, pancreas, liver, muscles, bones, heart, blood vessels, spleen, kidneys, blood, etc. (but not limited to these) can be listed. For "cells", any one can be used alone, or different cells can be used in combination. The morphology of cells in the fibrin gel sheet of the present invention is not particularly limited, and can be cells separated from each other (single cell state), can be in the form of cell blocks or cell aggregates formed by assembling and agglutinating each other, that is, spheroids, or can be suspended cultured cells. Cells in spheroidal form can be listed as pancreatic islet cells, hepatocytes and cardiomyocytes, but are not limited to these.

[0040] The "cells" in the present invention may be cells collected from an organism, cultured cells, or frozen-thawed cells. Preferably, the "cells" in the present invention are cells derived from pluripotent stem cells obtained by differentiation induction from pluripotent stem cells.

[0041] In this specification, "pluripotency" means the ability to differentiate into various tissues or cells with different morphologies or functions, and the ability to differentiate into cells of any of the three germ layers. "Pluripotency" cannot differentiate into a blastoderm, and therefore, from the perspective of not having the ability to form an individual, it is distinguished from "totipotency" which can differentiate into all tissues of an organism including a blastoderm.

[0042] In this specification, "multipotency" means the ability to differentiate into multiple cells of a limited number of layers. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent, not pluripotent.

[0043] In this specification, "pluripotent stem cells" refer to embryonic stem cells (ES cells) and cells that potentially have the same differentiation pluripotency as ES cells, that is, the ability to differentiate into various tissues of an organism (entoderm, mesoderm, ectoderm). "Induced pluripotent stem cells" (also referred to as "iPS cells" in this specification) can be cited as cells that have the same differentiation pluripotency as ES cells. Preferably, in the present invention, pluripotent stem cells are human pluripotent stem cells.

[0044] If mouse ES cells are used as "ES cells", various mouse ES cell lines established by inGenious, RIKEN (RIKEN), etc. can be used; if human ES cells are used as "ES cells", various human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis can be used. For example, CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc. of NIH; H1 strain and H9 strain of WiCell Research Institute; KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. of RIKEN can be used as ES cell lines.

[0045] “Induced pluripotent stem cells” refer to cells obtained by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells and reprogramming them. Currently, there are various types of "induced pluripotent stem cells". In addition to the iPS cells established by Yamanaka et al. by introducing the four factors Oct3 / 4, Sox2, Klf4 and c-Myc into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), there are also iPS cells derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872.), Nanog-iPS cells established by screening with Nanog expression as an indicator after the introduction of 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 include c-Myc (Nakagawa M, Yamanaka S., et al., Nature Biotechnology, (2008) 26, 101-106), iPS cells established by introducing six factors by a virus-free method (Okita K et al., Nat. Methods 2011 May; 8(5): 409-12, Okita K et al., Stem Cells. 31(3): 458-66.). In addition, the following can also be used: 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. (Japanese Patent Application Laid-Open No. 2008-307007), etc.

[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., Scholer 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., Japanese Patent Application Laid-Open No. 2008-307007, Japanese Patent Application Laid-Open No. 2008-283972, US2008 / 2336610, US2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO Any of the induced pluripotent stem cells known in the art described in WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852).

[0047] As the induced pluripotent cell line, various iPS cell lines established by NIH, RIKEN (RIKEN), Kyoto University, etc. can be used. For example, in the case of human iPS cell lines, examples include: HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 of RIKEN; Ff-WJ-18, Ff-I01s01, Ff-I01s02, Ff-I01s04, Ff-I01s06, Ff-I14s03, Ff-I14s04, QHJI01s01, QHJI01s04, QHJI14s03, QHJI14s04, 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, and 648A1 of Kyoto University; and MyCell iPS Cells (21525.102.10A) strain, MyCell iPS Cells (21526.101.10A) strain, etc.

[0048] As cells derived from pluripotent stem cells that can be used in the present invention, pancreatic islet cells derived from iPS cells (hereinafter sometimes also written as "iPIC") can be listed, but are not limited to this. iPIC includes insulin-producing cells and / or pancreatic β cells. Insulin-producing cells are cells characterized by the expression of at least one marker in insulin and NK6 homeobox 1 (NKX6.1). Pancreatic β cells are cells that are more mature than insulin-producing cells and can be characterized by the expression of at least one marker in MAFA, UCN3 and IAPP. "Marker" means a cell antigen or its gene that is specifically expressed by a specified cell type, such as a "marker protein" or a "marker gene". Preferably, the marker is a cell surface marker, in which case concentration, separation and / or detection of surviving cells can be implemented. The marker can be a positive selection marker or a negative selection marker.

[0049] The detection of the marker protein can be performed by immunological analysis using antibodies specific for the marker protein, such as ELISA, immunostaining, and flow cytometry. The detection of the marker gene can be performed using nucleic acid amplification methods and / or nucleic acid detection methods known in the art, such as RT-PCR, microarrays, biochips, etc. In this specification, the marker protein is "positive" means that it is detected as positive by flow cytometry, and "negative" means that it is below the detection limit by flow cytometry. In addition, in this specification, the marker gene is "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 specific nucleotide sequence driven by a promoter in a cell.

[0051] iPICs can be obtained by inducing differentiation from pluripotent stem cells according to known methods (WO2009 / 012428, WO2016 / 021734, Stem Cell Research (2015) 14, 185-197). That is, iPICs can be obtained by the following differentiation induction steps: Step 1) inducing differentiation of pluripotent stem cells into definitive endoderm cells; Step 2) inducing the definitive endoderm cells to differentiate into gastrointestinal cells; Step 3) inducing differentiation of gastrointestinal cells into posterior foregut cells; Step 4) inducing differentiation of posterior foregut cells into pancreatic progenitor cells; Step 5) inducing differentiation of pancreatic precursor cells into endocrine precursor cells; and Step 6) Inducing differentiation of endocrine precursor cells into iPICs. Hereinafter, each step will be described, but the differentiation induction of each cell is not limited to these methods.

[0052] Step 1) Differentiation into definitive endoderm cells First, pluripotent stem cells are differentiated into definitive endoderm cells. Methods for inducing definitive endoderm from pluripotent stem cells are well 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, so as to differentiate into definitive endoderm cells. The number of cells at the start of culture is not particularly limited, but is 22,000 cells / cm 2 ~150,000 cells / cm 2 , preferably 22000 cells / cm 2 ~100,000 cells / cm 2 , more preferably 22000 cells / cm 2 ~80,000 cells / cm 2 The culture time is 1 to 4 days, preferably 1 to 3 days, and particularly preferably 3 days.

[0053] The culture temperature is not particularly limited, and is performed at 30° C. to 40° C. (for example, 37° C.). The carbon dioxide concentration in the culture container is, for example, about 5%. The culture can be performed in two-dimensional culture or three-dimensional culture.

[0054] The culture medium used in this step can be: RPMI 1640 medium, MEM medium, iMEM medium, DMEM / F12 medium, modified MEM Zinc Option medium, modified MEM / 1% B-27 / PenisilinStreptomycin medium, MCDB131 / 20mM Glucose / NaHCO3 / FAF-BSA / ITS-X / GlutaMAX TM / Ascorbic acid / Penisilin Streptomycin medium and other basic culture media used to culture mammalian cells.

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

[0056] In another embodiment, activin A may be included in the culture medium at a low dosage, for example, 5 ng / mL to 100 ng / mL, preferably 5 ng / mL to 50 ng / mL, more preferably 5 ng / mL to 10 ng / mL.

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

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

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

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

[0061] In a specific embodiment, after culturing for 1 day in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, the culture is further cultured for 2 days while the medium is exchanged every day with a medium containing only activin A. Alternatively, the pluripotent stem cells can be produced by first culturing in a medium containing 0.01 μM to 20 μM insulin in the presence of a low dose of activin A and then second culturing in a medium without insulin.

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

[0063] The culture temperature is not particularly limited, and is performed at 30° C. to 40° C. (for example, 37° C.). The carbon dioxide concentration in the culture container is, for example, about 5%. The culture can be performed in two-dimensional culture or three-dimensional culture.

[0064] The medium may be a basal medium for culturing mammalian cells in the same manner as in step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. may be appropriately added to the medium.

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

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

[0067] Step 3) Differentiation into posterior foregut cells The gastrointestinal cells obtained in step 2) are further cultured in a culture medium containing growth factors, cyclopamine, noggin, etc., and differentiated into posterior foregut cells. The culture time is 1 to 5 days, preferably about 2 days. The culture can be carried out by two-dimensional culture or three-dimensional culture.

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

[0069] The medium may be a basal medium for culturing mammalian cells in the same manner as in step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. may be appropriately added to the medium.

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

[0071] The concentration of the growth factor in the culture medium is appropriately set according to the type of growth factor used, but is generally about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. When EGF is used, its concentration is about 5 ng / mL to 2000 ng / mL (i.e., about 0.8 nM to 320 nM), preferably about 5 ng / mL to 1000 ng / mL (i.e., about 0.8 nM to 160 nM), and more preferably about 10 ng / mL to 1000 ng / mL (i.e., about 1.6 nM to 160 nM). When FGF10 is used, its concentration is about 5 ng / mL to 2000 ng / mL (i.e., about 0.3 nM to 116 nM), preferably about 10 ng / mL to 1000 ng / mL (i.e., about 0.6 nM to 58 nM). For example, when KGF is used as a growth factor, the concentration is usually 5 ng / mL to 150 ng / mL, preferably 30 ng / mL 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 μM to 1.5 μM, preferably 0.3 μM to 1.0 μM, and particularly preferably about 0.5 μM.

[0073] The concentration of Noggin in the culture medium is not particularly limited, but is usually 10 ng / mL to 200 ng / mL, preferably 50 ng / mL 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 further cultured in a culture medium containing a factor having CDK8 / 19 inhibitory activity, preferably in a culture medium containing a factor having CDK8 / 19 inhibitory activity and a proliferation factor, and differentiated into pancreatic progenitor cells. The culture time is 2 to 10 days, preferably about 5 days. The culture can be performed by two-dimensional culture or three-dimensional culture.

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

[0076] The medium may be a basal medium for culturing mammalian cells in the same manner as in step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0077] The aforementioned various compounds or salts thereof can be used as factors having CDK8 / 19 inhibitory activity, and the amount added to the culture medium is appropriately determined according to the compound or salt used, 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 culture medium is preferably a concentration that achieves 50% or more of the inhibitory activity on 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 further preferred.

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

[0080] The first day of culturing in step 4) may be performed in the presence of a ROCK inhibitor, and thereafter culturing may be performed in a medium not containing a ROCK inhibitor.

[0081] In addition, the culture medium may also contain a protein kinase C (PKC) activator. PDBu (PKC activator II), TPB (PKC activator V), etc. can be used as the PKC activator, but are not limited thereto. The PKC activator is added at a concentration of about 0.1 ng / mL to 100 ng / mL, preferably about 1 ng / mL to 50 ng / mL, and more preferably about 3 ng / mL to 10 ng / mL.

[0082] Furthermore, dimethyl sulfoxide and / or activin (1 ng / mL to 50 ng / mL) may be added to the culture medium.

[0083] In any step, in addition to the above-mentioned ingredients, serum substitutes (e.g., B-27 supplement, ITS-G) may be added to the culture medium. In addition, amino acids, L-glutamine, GlutaMAX (product name), non-essential amino acids, vitamins, nicotine amide, antibiotics (e.g., antibiotic-antifungal, penicillin, streptomycin or a mixture thereof), antibacterial agents (e.g., amphotericin B), antioxidants, pyruvic acid, buffers, inorganic salts, etc. may also be added as needed. When antibiotics are added to the culture medium, their concentration in the culture medium is generally 0.01% to 20% by weight, preferably 0.1% to 10% by weight. Cultivation may be performed by two-dimensional culture or three-dimensional culture.

[0084] In addition, when cell culture is two-dimensional culture, feeder cells are not used, and culture is carried out by adhesion. During cultivation, culture vessels such as cell culture sheets such as dish, flask, microplate, OptiCell (product name) (Nunc company) are used. Preferably, culture vessel is treated to improve the surface of the adhesiveness (hydrophilicity) with cell, or collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, matrigel (for example, BD matrigel (Beckton Dickinson, Japan), vitronectin etc. are coated thereon with the matrix for cell adhesion. Preferably, the culture vessel coated with type I collagen, matrigel, fibronectin, vitronectin or poly-D-lysine etc. is used as culture vessel, more preferably, the culture vessel coated with matrigel or poly-D-lysine is used as culture vessel.

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

[0086] The pancreatic progenitor cells obtained in step 4) can be further purified using glycoprotein 2 (GP2), a known surface marker, etc. The purification can be performed by a known method, for example, using beads to which an anti-GP2 antibody is immobilized.

[0087] Step 5) Differentiation into endocrine precursor cells The pancreatic precursor cells obtained in step 4) are further cultured in a culture medium containing proliferation factors to induce differentiation into endocrine precursor cells. The culture can be performed by two-dimensional culture 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, dispersed in the solution by pipetting to obtain a cell dispersion, the obtained dispersion is centrifuged, the recovered cells are resuspended in a small amount of fresh culture medium, and the cell suspension is re-inoculated into the new culture medium of step 5). The culture time is 2 to 3 days, preferably about 2 days.

[0088] The culture medium can be a basal medium for culturing mammalian cells in the same manner as in step 1). According to previous reports (Nature Biotechnology 2014; 32: 1121-1133), SANT1, retinoic acid, ALK5 inhibitor II, T3, and LDN are added to the culture medium. Furthermore, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum replacements, vitamins, antibiotics, etc. can be added to the culture medium in appropriate amounts.

[0089] The culture is performed by non-adhesive culture without using feeder cells. During the culture, a dish, flask, microplate, multi-well plate (Nunc) or a bioreactor is used. The culture container is preferably surface treated to reduce adhesion to the cells.

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

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

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

[0093] The culture medium can be a basal medium for culturing mammalian cells in the same manner as in step 1). According to a previous report (Nature Biotechnology 2014; 32: 1121-1133), ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor XX, γ-secretase inhibitor RO, N-cysteine, AXL inhibitor, and ascorbic acid are added to the culture medium. Furthermore, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum replacements, vitamins, antibiotics, etc. can be added to the culture medium in appropriate amounts. For example, ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, and ascorbic acid can be added to the culture medium, or T3, ALK5 inhibitor II, ZnSO4, heparin, N-acetylcysteine, Trolox, and R428 can be added to the culture medium.

[0094] Cultivation can be carried out by two-dimensional culture or three-dimensional culture. Cultivation does not use feeder cells. In the case of three-dimensional culture, it is carried out by non-adhesive culture. During cultivation, a dish, flask, microplate, multi-well plate (Nunc company) or a bioreactor is used. Preferably, the culture container is surface treated to reduce adhesion to the cells.

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

[0096] The fibrin gel sheet of the present invention is characterized in that it has a large number of cells (high-density cells) that cannot be achieved by conventional fibrin gel sheets for cell transplantation. 2 More than 1.5×10 6 , preferably 2×10 6 More than 2.5×10 6 More than 3×10 6 More than 4.5×10 6 More than 5×10 6 The amount includes cells, and the upper limit is not particularly limited. For example, it can be 10×10 6 Less than 7×10 6 The range of the number of cells contained in the fibrin gel sheet of the present invention can be expressed by two numerical values ​​selected from the above lower limit and upper limit, for example, the fibrin gel sheet of the present invention can contain more than 1.5×10 6 ~10×10 6 Less than 1 / cm 2 , preferably 2×10 6More than 10×10 6 Less than 1 / cm 2 , more preferably 2.5×10 6 More than 10×10 6 Less than 1 / cm 2 , and more preferably 3×10 6 More than 10×10 6 Less than 1 / cm 2 , and further preferably 4.5×10 6 More than 10×10 6 Less than 1 / cm 2 , especially preferably 5×10 6 More than 10×10 6 Less than 1 / cm 2 The cells are contained in an amount appropriately selected within the range.

[0097] The iPIC is usually present in the form of cell clusters with a size of about 100 μm to 200 μm. The number of cell clusters in the fibrin gel sheet of the present invention is about 100 μm to 200 μm per cm. 2 The number of iPICs contained in the fibrin gel sheet of the present invention is more than 3,000, preferably more than 4,000, more preferably more than 5,000, further preferably more than 6,000, further preferably more than 9,000, and particularly preferably more than 10,000. The upper limit is not particularly limited, for example, it can be 20,000 or less, preferably 14,000 or less. The range of the number of iPICs (cell blocks) contained in the fibrin gel sheet of the present invention can be represented by two numerical values ​​selected from the above lower limit and upper limit, for example, the fibrin gel sheet of the present invention can be more than 3,000 to less than 20,000 / cm 2 , preferably 4000 to 20000 / cm 2 , more preferably 5000 or more and 20000 or less / cm 2 , more preferably 6000 to 20000 / cm 2 , more preferably 9000 to 20000 / cm 2 , particularly preferably 10,000 to 20,000 / cm 2 An amount appropriately selected within the range of comprises iPIC (cell block).

[0098] The fibrin gel sheet and fibrin gel of the present invention can be formed by reacting fibrinogen and thrombin in a suitable solvent (e.g., water, saline, etc.). The amount of fibrinogen and thrombin used in the formation of the fibrin gel and the mixing method of the two can be appropriately determined based on the method for producing the fibrin gel sheet of the present invention described in detail below.

[0099] In one embodiment, the fibrin gel sheet of the present invention further contains a biodegradable material having a particle-like morphology (hereinafter sometimes referred to as "biodegradable particles").

[0100] The "biodegradable particles" in the present invention are added to a fibrinogen solution together with cells in the method for producing a fibrin gel sheet of the present invention described in detail below to be suspended, thereby inhibiting the precipitation of cells in the solution and maintaining the dispersion of cells, thereby allowing the cells to be dispersed and encapsulated in the final fibrin gel sheet.

[0101] The biodegradable material constituting the "biodegradable particles" is not particularly limited as long as it is a substance that can be decomposed by hydrolysis or the like in a living body. For example, it can be selected from polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polyethylene glycol (PEG), polyhydroxyethyl methacrylate, polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), poly(3-hydroxybutyrate-hydroxyvalerate copolymer) (PHBV), poly(ethylene-vinyl acetate copolymer) (PEVA), polyacrylamide, polyethylene oxide, polyethyleneimine, polyhydroxybutyric acid, poly(N-vinyl pyrrolidone), polyvinyl alcohol, polypropylene fumarate, polyacrylic acid, polye- Caprolactone, polymethacrylic acid, polyvinylidene fluoride (PVDF), pectic acid, hyaluronic acid, heparan sulfate, chondroitin sulfate, heparan sulfate proteoglycan, heparin, chitin, chitosan, xanthan gum, carboxymethyl cellulose, carboxymethyl chitosan, alginic acid, alginate, collagen, cellulose, fibroin, keratin, gelatin, fibrin, pullulan, laminin, gellan gum, silica gel, ethyl formate, elastin, etc. and their deformations 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, carboxymethyl cellulose, PLA, PGA, PLGA, etc. and their deformations and combinations thereof.

[0102] The form of the "biodegradable particles" may be any form such as blocks, beads, particles, sheets, gels, etc., as long as they can suppress the precipitation of cells and maintain the dispersion of cells in the above-mentioned solution, and may be solid or porous. The form of the "biodegradable particles" is preferably a three-dimensional shape, for example, a sphere, a tetrahedron, a hexahedron or other polyhedron, a cylinder, a prism, a cone, a truncated cone, a pyramid, a truncated pyramid, a torus, a disk, an ellipsoid, and a deformed three-dimensional shape thereof, but not limited thereto.

[0103] The size of the "biodegradable particles" can be any size as long as it can inhibit cell precipitation and maintain cell dispersion in the above-mentioned solution and does not hinder the formation of the fibrin gel sheet of the present invention. It can be a size that can pass through an opening of 100μm to 1000μm, preferably 100μm to 800μm, more preferably 150μm to 400μm, and further preferably about 200μm.

[0104] In one embodiment of the present invention, the biodegradable particles are gelatin gel and have a spherical shape capable of passing through an opening of 200 μm.

[0105] The amount of biodegradable particles contained in the fibrin gel sheet of the present invention is any amount that can inhibit cell precipitation and maintain cell dispersion in the above-mentioned fibrinogen solution and does not hinder the formation of the fibrin gel sheet of the present invention. For example, the biodegradable particles can be contained in the above-mentioned 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 further preferably 20 w / v%.

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

[0107] In 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 the fibrinogen solution together with the cells in the method for producing the fibrin gel sheet of the present invention described in detail below, and the solution is given viscosity, thereby being able to inhibit the precipitation of cells in the solution and maintain the dispersion of cells, thereby allowing the cells to be dispersed and encapsulated in the fibrin gel sheet finally obtained.

[0109] The "biodegradable gelling agent" is not particularly limited as long as it is a substance that can be decomposed by hydrolysis or the like in a living body, and gives the above solution a viscosity that can inhibit cell precipitation and maintain cell dispersion, and does not hinder the formation of the fibrin gel sheet of the present invention. For example, the aforementioned biodegradable materials (but excluding fibrin) can be used. Preferably, the biodegradable gelling agent can use a biodegradable material selected from collagen, hyaluronic acid, heparin sulfate, chondroitin sulfate, heparin sulfate proteoglycan, carboxymethyl cellulose, alginic acid, cellulose, silk protein, keratin, gelatin, dextran, PEG, etc., and their deformations and combinations thereof.

[0110] The amount of the biodegradable gelling agent contained in the fibrin gel sheet of the present invention is any amount that can inhibit the precipitation of cells and maintain the dispersion of cells in the above-mentioned fibrinogen solution and does not hinder the formation of the fibrin gel sheet of the present invention. For example, the biodegradable gelling agent can be contained in the above-mentioned 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 further preferably 0.75 w / v%.

[0111] In addition, the fibrin gel sheet of the present invention may further have a carrier. The carrier supports the fibrin gel sheet during the manufacture and / or use of the fibrin gel sheet of the present invention, and can contribute to improving the operability of the fibrin gel sheet. The carrier is composed of the aforementioned biodegradable material or non-biodegradable material, for example, polyester (PEs), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), nylon, silk (not limited to these), and has an arbitrary shape (for example, sheet, mesh, disc, woven fabric, non-woven fabric, etc.) that can support the fibrin gel sheet of the present invention. The carrier can be bonded to the surface of the fibrin gel sheet of the present invention, or a part or all of it can be fixed in the fibrin gel sheet of the present invention.

[0112] In one embodiment of the present invention, the fibrin gel sheet of the present invention has a mesh-shaped carrier composed of polyester or poly(lactic acid-glycolic acid copolymer).

[0113] The fibrin gel sheet of the present invention may be provided in the form of a stack of multiple fibrin gel sheets, which may be a stack of fibrin gel sheets containing all the same cells or a stack of fibrin gel sheets partially or entirely containing different cells.

[0114] 3. Method for producing the 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 sometimes referred to as "the method of the present invention"), which comprises allowing a fibrinogen solution containing cells suspended therein to act with thrombin to form and gel (solidify) a fibrin gel sheet having a surface area of ​​2.25 cm on one side. 2 The fibrin gel can be obtained by mixing fibrinogen and thrombin in an appropriate solvent (eg, water, physiological saline, etc.) and gelling (solidifying).

[0115] The cells are evenly dispersed and suspended in the fibrinogen solution, and the amount of cells can be determined based on the fibrin gel sheet after forming, that is, the fibrin gel sheet is formed into a 1 cm 2More than 1.5×10 6 , preferably 2×10 6 More than 2.5×10 6 More than 3×10 6 More than 4.5×10 6 More than 5×10 6 The upper limit is not particularly limited, for example, 10×10 6 Less than 7×10 6 The range of the number of cells suspended in the fibrinogen solution can be represented by two numerical values ​​selected from the above lower limit and upper limit, for example, the number of cells in the fibrinogen solution can be more than 1.5×10 6 ~10×10 6 Less than 1 / cm 2 , preferably 2×10 6 More than 10×10 6 Less than 1 / cm 2 , more preferably 2.5×10 6 More than 10×10 6 Less than 1 / cm 2 , and more preferably 3×10 6 More than 10×10 6 Less than 1 / cm 2 , and further preferably 4.5×10 6 More than 10×10 6 Less than 1 / cm 2 , especially preferably 5×10 6 More than 10×10 6 Less than 1 / cm 2 The amount of suspension is appropriately selected within the range.

[0116] In one embodiment of the method of the present invention, when the cells are iPICs, the number of iPICs in the fibrinogen solution is calculated as the number of cell blocks so that the fibrin gel sheet has a density of 1.5-1.5 W per 1 cm 2 The number of iPICs (cell masses) suspended in the fibrinogen solution is more than 3,000, preferably more than 4,000, more preferably more than 5,000, further preferably more than 6,000, further preferably more than 9,000, and particularly preferably more than 10,000. The upper limit is not particularly limited, for example, it is suspended in an amount of 20,000 or less, preferably 14,000 or less. The range of the number of iPICs (cell masses) suspended in the fibrinogen solution can be represented by two values ​​selected from the above lower limit and upper limit, for example, the fibrinogen solution can be suspended in an amount of more than 3,000 to less than 20,000 / cm in the fibrin gel sheet.2 , preferably 4000 to 20000 / cm 2 , more preferably 5000 or more and 20000 or less / cm 2 More preferably, it is 6000 or more and 20000 or less / cm 2 More preferably, it is 9000 to 20000 / cm 2 , particularly preferably 10,000 to 20,000 / cm 2 The iPIC (cell mass) is suspended in an amount appropriately selected within the range of .

[0117] When formed into a fibrin gel sheet, the surface area of ​​one side of the sheet (i.e., any one of the main surfaces of the sheet, or also referred to as a single side of the sheet) is 2.25 cm 2 More than 4cm, preferably 2 More preferably 9 cm 2 More preferably, 16 cm 2 More preferably, 25 cm 2 More than, especially preferably 36cm 2 More preferably, 49 cm 2 More preferably, 64 cm 2 More preferably, 81 cm 2 More than, particularly preferably 100cm 2 The upper limit is not particularly limited and can be appropriately selected according to the size and shape of the transplant site, for example, 400 cm 2 The following dimensions.

[0118] The range of the surface area of ​​the fibrin gel sheet obtained by molding can be represented by two values ​​selected from the lower limit and the upper limit, respectively. For example, the surface area of ​​the fibrin gel sheet obtained by molding can be 2.25 cm 2 Above~400cm 2 Below, preferably 4cm 2 Above~400cm 2 Less than, more preferably 9cm 2 Above~400cm 2 Below, more preferably 16cm 2 Above~400cm 2 Below, more preferably 25cm 2 Above~400cm 2 Below, especially preferably 36cm 2 Above~400cm 2 Below, especially more preferably 49cm 2Above~400cm 2 Below, especially more preferably 64cm 2 Above~400cm 2 Below, especially more preferably 81cm 2 Above~400cm 2 Less than, particularly preferably 100cm 2 Above~400cm 2 Select the appropriate size from the following range.

[0119] The shape of the above-mentioned one surface of the fibrin gel sheet obtained by molding is not particularly limited and can be appropriately selected according to the size and shape of the transplantation site. For example, polygons (for example, triangles, quadrilaterals, pentagons, hexagons, octagons, etc.), rounded polygons, circles, ellipses, etc. can be listed, but are not limited to these.

[0120] When the fibrin gel sheet obtained by molding has a quadrilateral shape, the longitudinal length is 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, further preferably 40 mm or more, further 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 more preferably 90 mm or more, and especially preferably 100 mm or more, and the upper limit is not particularly limited, for example, it can be 200 mm or less. In addition, the transverse length is 15 mm or more, preferably 20 mm or more, more preferably 30 mm or more, further preferably 40 mm or more, further 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 more preferably 90 mm or more, and especially preferably 100 mm or more, and the upper limit is not particularly limited, for example, it can be 200 mm or less.

[0121] The range of the longitudinal length and the transverse length can be represented by two numerical values ​​selected from the above lower limit and upper limit, respectively. For example, the size of the fibrin gel sheet obtained by molding (longitudinal length × transverse length (or transverse length × longitudinal length)) can be 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, further preferably 40 mm to 200 mm × 40 mm to 200 mm, and further preferably 50 mm to 200 mm × 50 mm to 200 mm. The size selected is appropriately selected from the range of 50 mm to 200 mm × 50 mm to 200 mm, especially preferably 60 mm to 200 mm × 60 mm to 200 mm, especially more preferably 70 mm to 200 mm × 70 mm to 200 mm, especially further preferably 80 mm to 200 mm × 80 mm to 200 mm, especially further preferably 90 mm to 200 mm × 90 mm to 200 mm, and particularly preferably 100 mm to 200 mm × 100 mm to 200 mm. For example, the size of the fibrin gel sheet obtained by molding (longitudinal length × transverse length (or transverse length × longitudinal length)) can be listed as 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, further preferably 70 mm × 70 mm, 80 mm × 80 mm, 90 mm × 90 mm, 100 mm × 100 mm, or 200 mm × 200 mm, etc., but are not limited to these.

[0122] The thickness of the fibrin gel sheet obtained by molding can be appropriately set according to the size and amount of the cells contained therein, the size of the transplant site and other factors, preferably less than 1 mm. By making the thickness of the fibrin gel sheet obtained by molding less than 1 mm, it is possible to efficiently supply nutrients, oxygen, etc. to the cells contained in the fibrin gel sheet, and after transplantation, it is possible to achieve rapid decomposition of the fibrin gel, so it is preferred. The thickness of the fibrin gel sheet obtained by molding can be, for example, less than 1 mm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm or less than 400 μm, and its lower limit is not particularly limited, for example, it can be more than 100 μm, more than 200 μm, or more than 300 μm. In addition, the range of the thickness of the fibrin gel sheet obtained by molding can be represented by two values ​​selected from the above upper limit and the upper limit value, 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, which can be a thickness appropriately selected from these ranges. For example, the thickness of the fibrin gel sheet obtained by molding can be about 400 μm. It should be noted that the thickness of the fibrin gel sheet obtained by molding does not need to be uniform throughout the sheet, as long as the thickest part is within the above range.

[0123] In one embodiment of the method of the present invention, the gelation (solidification) speed of the formed fibrin gel can be adjusted by adjusting the mixing amounts of fibrinogen and thrombin and / or the temperature during molding, thereby allowing a fibrin gel sheet of a desired size to be spread and molded.

[0124] The fibrinogen solution in this scheme contains fibrinogen in an amount of 5 to 150 mg / mL, preferably 10 to 80 mg / mL, and more preferably 20 mg / mL. For every 1 mg of fibrinogen, thrombin is 0.4U or less, preferably 0.3U or less or 0.25U or less, more preferably 0.2U or less or 0.15U or less, and further preferably 0.1U or less or 0.08U or less, and its lower limit is not particularly limited, and it is allowed to act in an amount that becomes a mixing ratio of 0.04U or more. When the mixing amount of fibrinogen and / or thrombin is more than the above range, gelation (solidification) proceeds rapidly, and there is a situation where it is difficult or impossible to spread and form into a sheet with a desired size. On the other hand, when the mixing amount of fibrinogen and / or thrombin is less than the above range, it cannot be fully gelled (solidified), and there is a situation where it is difficult or impossible to form into a sheet with a desired size. Thrombin can act on fibrinogen in any form, and can be directly added to the fibrinogen solution, or can be dissolved in a suitable solvent (such as water, physiological saline, etc.) to prepare a thrombin solution and then mixed with the fibrinogen solution.

[0125] The method of molding the fibrinogen solution that acts on thrombin into a sheet with a desired size and thickness can be performed by any method, for example, it can be performed by using a combination of one or more of any molding methods (e.g., mold molding, extrusion molding, calendering molding, blow molding, etc.) currently known in the field of manufacturing polymer sheets, preferably by mold molding. In this embodiment, the fibrinogen solution that acts on thrombin can be pressed and spread into a sheet with a desired size and thickness, and mold molding can be easily implemented, that is, the fibrinogen solution is injected into a sheet mold with a desired size and thickness, and is pressed and spread using a scraper, trowel, etc. as needed.

[0126] The shaping of the fibrinogen solution reacted with thrombin is carried out under cooling. By placing the fibrin gel solution under cooling, the gelation (solidification) rate can be suppressed and it can be shaped into a sheet having a desired size and thickness. More specifically, it can be spread and shaped into a sheet. The cooling temperature is not particularly limited as long as it can shape the fibrin gel solution into a sheet having a desired size and thickness. For example, it is 2 to 8°C, preferably 2 to 6°C, and more preferably 4°C. Cooling can be any temperature that can cool the fibrin gel solution. The fibrin gel solution can be cooled, or a molding means (for example, a mold, etc.) in contact with the fibrin gel solution can be cooled.

[0127] As required, before the shaped fibrin gel solution is gelled (solidified), the above-mentioned carrier and the fibrin gel sheet can be integrated. The carrier can be bonded to the surface of the fibrin gel solution, or a part or all of it can be immersed in the fibrin gel solution. By gelling (solidifying), a fibrin gel sheet with the carrier integrated can be obtained.

[0128] In another embodiment of the method of the present invention, by further adding or suspending "biodegradable particles" in the fibrinogen solution in which cells are suspended, or by adding or dissolving a "biodegradable gelling agent", the cell precipitation in the fibrinogen solution can be inhibited, and the cells in the fibrinogen solution can be maintained in a uniformly dispersed and suspended state, thereby producing a fibrin gel sheet with dispersed and encapsulated cells.

[0129] "Biodegradable particles" can be used as defined above, and can be added or suspended in a fibrinogen solution in which cells are suspended in an amount that can inhibit cell precipitation and maintain cell dispersion, and does not hinder the formation of the fibrin gel sheet of the present invention. For example, biodegradable particles are added or suspended in an amount of 10 to 30 w / v%, preferably 15 to 25 w / v%, more preferably 18 to 22 w / v%, and further preferably 20 w / v% in a fibrinogen solution in which cells are suspended. In one embodiment of the present scheme, the biodegradable particles are gelatin gel particles having a spherical shape that can pass through an opening of 200 μm, and are added or suspended in an amount of 20 w / v% in a fibrinogen solution in which cells are suspended.

[0130] The "biodegradable gelling agent" can be used as defined above, and is added or suspended in a fibrinogen solution in which cells are suspended in an amount that can inhibit cell precipitation and maintain cell dispersion, and does not hinder the formation of the fibrin gel sheet of the present invention. For example, the biodegradable gelling agent is added or dissolved in a fibrinogen solution in which cells are suspended 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 further preferably 0.75 w / v%. In one embodiment of the present scheme, the biodegradable gelling agent is collagen, and is added or dissolved in a fibrinogen solution in which cells are suspended in an amount of 0.75 w / v%.

[0131] The fibrinogen solution in this embodiment can contain fibrinogen in an amount of 5 to 150 mg / mL, preferably 20 to 100 mg / mL, and more preferably 80 mg / mL. Thrombin can act in an amount sufficient to gel (solidify) the fibrinogen solution, for example, in an amount of 0.5 to 5 U, preferably 1 to 4 U, and more preferably 1.5 to 3.5 U relative to 1 mg of fibrinogen. Thrombin can act on fibrinogen in any form, and can be added directly to the fibrinogen solution, added to the fibrinogen solution after being dissolved in a suitable solvent (such as water, saline, etc.) in advance to make a thrombin solution, by including thrombin in the "substrate" described in detail below, or a combination of multiple thereof. In the case where thrombin is included in the substrate, fibrinogen and thrombin can be mixed by applying the fibrinogen solution on the substrate.

[0132] The method of molding the fibrinogen solution in which cells are suspended into a sheet having a desired size and thickness can be performed by any method, for example, the fibrin gel solution can be applied to an appropriate substrate, and one or more combinations of any molding methods known in the field of polymer sheet production (for example, mold molding, extrusion molding, calendar molding, blow molding, etc.) can be used, preferably by applying to an appropriate substrate. In this embodiment, the suspended cells can be maintained uniformly dispersed, so that not only when the fibrinogen solution in which cells are suspended is injected once, but also when the fibrinogen solution in which cells are suspended is continuously injected (taking time from the start of injection to the end), it can be easily molded into a sheet in which cells are uniformly dispersed.

[0133] The "substrate" is not particularly limited as long as it has a flat surface capable of forming a sheet, and the above-mentioned carrier can be preferably used as the substrate. The fibrinogen solution in which cells are suspended can be applied to the substrate by any method, such as one or more of injection, scraper, trowel, spraying, bioprinter (2D or 3D printer), etc., and can be performed manually and / or mechanically, preferably mechanically using a bioprinter.

[0134] The fibrin gel sheet of the present invention manufactured by the method of the present invention can be stored in a cell culture medium, such as Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, MEM medium, CMRL medium, etc. (not limited to these), a buffer solution, such as phosphate-buffered saline (PBS), Hanker's balanced salt solution (HBSS), etc. (not limited to these), an isotonic solution, such as physiological saline, etc. (not limited to these) until it is used.

[0135] 4. Use of the fibrin gel sheet of the present invention The fibrin gel sheet of the present invention has functions equivalent to or similar to those of cells or progenitor cells constituting organs or tissues such as the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lungs, thyroid, pancreas, liver, muscles, bones, heart, blood vessels, spleen, kidneys, blood, etc. (not limited to these) based on the above-mentioned cells dispersed therein, and can be used as a cell therapy for enhancing or maintaining the functions of these organs or tissues or for assisting or supplementing the functions of these organs or tissues that have been reduced or disappeared due to diseases or disorders, and can be transplanted into patients in need.

[0136] As one embodiment of the present invention, when the fibrin gel sheet of the present invention includes iPICs derived from pluripotent stem cells, the fibrin gel sheet of the present invention can improve and / or maintain the blood sugar (glucose) level of the transplanted patient through the action of insulin or glucagon secreted by iPIC, thereby controlling the blood sugar level to a normal value. Therefore, the fibrin gel sheet of the present invention including iPIC can be used for the treatment or prevention of diseases, disorders, or symptoms that need to improve and / or maintain blood sugar levels. As such diseases, disorders, or symptoms, diabetes (type 1 diabetes, type 2 diabetes), changes in fasting and postprandial glucose concentrations, hypoglycemia (for example, hypoglycemia in diabetic patients through insulin administration), etc. can be cited, but are not limited to these. It should be noted that "treatment" refers to the improvement of the treatment, cure, prevention or relief of a disease, disorder, or symptom, or the reduction of the rate of progression of a disease, disorder, or symptom. In addition, "prevention" refers to reducing the possibility and risk of the onset of a disease, disorder, or symptom, or delaying the onset of a disease, disorder, or symptom.

[0137] The fibrin gel sheet of the present invention is transplanted to patients who need cell therapy, for example, mammals such as mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, and humans (not limited to these), preferably large mammals, and particularly preferably humans.

[0138] The fibrin gel of the fibrin gel sheet of the present invention is rapidly decomposed after transplantation, and the cells contained therein can rapidly contact the host's tissues and receive oxygen, nutrition, etc. Therefore, the fibrin gel sheet of the present invention can be transplanted into tissues lacking blood vessels (e.g., subcutaneous tissue, etc.), and in addition, angiogenesis treatment is not required in advance during transplantation, which is not essential.

[0139] The fibrin gel sheet of the present invention may be transplanted in one or more sheets depending on the patient's symptoms and the size of the transplant site. In addition, since the fibrin gel sheet of the present invention is thin, a plurality of sheets may be stacked and transplanted as needed.

[0140] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples. [Example]

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

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

[0143] Next, the cells were cultured in a differentiation induction medium (modified MEM / 1% B-27 / PenisilinStreptomycin medium) containing ALK5iII (10 μM) together with differentiation factors (T3, LDN, γ-secretase inhibitor RO, and FGF receptor 1 inhibitor PD-166866) for 7 days, and then cultured in a differentiation induction medium (MCDB131 / 20mM Glucose / NaHCO3 / FAF-BSA / ITS-X / Glutamax / PenisilinStreptomycin 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 for 4 days to induce differentiation into iPICs.

[0144] The obtained iPIC had the morphology of an aggregate (spheroid) (about 150 μm in diameter) formed by aggregation of about 500 cells.

[0145] Example 1: Evaluation of transplant shape <Method> Preparation of fibrin gel blocks iPIC3x10 6After centrifugation, the cells (about 6000 cells in aggregate) were divided into 1.5 mL test tubes, the medium was removed, and 25 μL of fibrinogen solution (80 mg / mL) was added to suspend the cells. Then, 25 μL of thrombin solution (125 U / mL) was added and the cells were allowed to stand for 5 minutes to gel, thereby producing a fibrin gel block (about 5 mm in diameter) containing iPIC.

[0146] Preparation of fibrin gel sheets iPIC3x10 6 After centrifugation, the cells were divided into 1.5 mL test tubes, the culture supernatant was removed, and 25 μL of fibrinogen solution (80 mg / mL) was added to suspend the cells. The iPIC-suspended fibrinogen solution was thinly coated on a polyester mesh (opening rate 69%, 10x10 mm) impregnated with 12.5 μL of thrombin solution (125 U / mL), and then 25 μL of thrombin solution was coated on the entire mesh, and the mesh was allowed to stand for 5 minutes to gel, thereby preparing a thin layer of fibrin gel sheet containing iPIC.

[0147] In vivo evaluation The iPIC-encapsulated fibrin gel block and iPIC-encapsulated fibrin gel sheet were transplanted subcutaneously into anesthetized nude rats. After transplantation, blood was collected from the tail vein, plasma was separated, and human c-peptide concentration was measured by ELISA.

[0148] <Results> Figure 1 The appearance of the fibrin gel block and fibrin gel sheet are shown in . In both cases, the iPICs were retained in the gel, and no leakage of cells was observed.

[0149] Figure 2 , shows the results of time-dependent measurement of the human c-peptide concentration in the blood of nude rats to which a fibrin gel block or a fibrin gel sheet containing iPIC was subcutaneously transplanted. Figure 3 The results of histological analysis of each transplantation site 10 days and 6 weeks after transplantation are shown in FIG.

[0150] Figure 2 The results showed that compared with block transplantation, the fibrin gel containing iPIC transplanted in sheet form showed higher blood human c-peptide concentration values.

[0151] In addition, from Figure 3 The histological analysis results 10 days after transplantation show that in the case of block-shaped iPICs, only the peripheral part of the fibrin gel was decomposed, and the iPIC in this part was in contact with the host tissue, but most of the fibrin gel remained, and iPICs that were not in contact with the host tissue were observed. On the other hand, in the case of sheet-shaped iPICs, almost all of the fibrin gel was rapidly decomposed, and the transplanted iPIC was in contact with the host tissue.

[0152] Six weeks after transplantation, although the gel transplanted in block form was completely decomposed, the one transplanted in sheet form showed a higher blood human c-peptide concentration. Therefore, even in the case of using a gel made of biodegradable material, the gel decomposes quickly in the early stage of transplantation, and the transplanted cells come into contact with the host, and the supply of oxygen, nutrients, etc. is important for the survival of the transplanted cells. This indicates that it may have an impact on the long-term efficacy of the drug, showing the usefulness of transplantation in thin sheet form.

[0153] Example 2: Preparation of large animal and human-sized fibrin gel sheets (Production method 1: Method using a cooled metal mold) <Method> Evaluation of gelation time Fibrinogen solution (40 mg / mL) and thrombin solution (31 U / mL, 15.6 U / mL, 10.4 U / mL, 5.2 U / mL, 3.1 U / mL) were prepared, 440 μL of each was added to a 1.5 mL test tube, and after pipetting 1 to 2 times to mix, the gelation state was evaluated according to the following standards, and the relationship between thrombin concentration and gelation time was analyzed.

[0154] Evaluation criteria for gelation state: 0:Can perform pipetting operations in test tubes △: Although the pipetting operation can be performed, some of the samples have gelled. ×: All gelled and liquid transfer is not possible

[0155] Preparation of large animal and human-sized fibrin gel sheets iPIC45x10 6 After centrifugation, the cells were divided into 15 mL test tubes, the culture supernatant was removed, 360 μL of fibrinogen solution (40 mg / mL) was added, and the mixture was suspended. Then, 360 μL of thrombin solution (15.6 U / mL) was added, and the mixture was quickly spread on a metal mold (40x60x0.4 mm) cooled to 4°C in advance, and a polyester mesh (opening rate 69%, size 40x10 mm) was placed on the mold, and the mixture was allowed to stand at room temperature for 6 minutes to gel.

[0156] <Results> The relationship between thrombin concentration and gelation time was analyzed. The results showed that as the thrombin concentration decreased, the gelation time was delayed. It was shown that if the concentration was below 15.6 U / mL, the time required for forming a fibrin gel sheet of 40x60x0.4 mm could be ensured ( Figure 4 ).

[0157] By rapidly spreading a fibrinogen solution (40 mg / mL) and a thrombin solution (15.6 U / mL) on a metal mold, a 40x60x0.4 mm fibrin gel sheet (100 μm) for large animals and humans can be prepared. Figure 5 ). In addition, the obtained fibrin gel sheet was confirmed using a phase contrast microscope, and the results showed that the iPIC contained therein was evenly dispersed at any position ( Figure 5 (B) iPIC is observed as black granules).

[0158] Example 3 Preparation of large animal and human-sized fibrin gel sheets (Manufacturing method 2-1_fibrin + gelatin microsphere gel sheets) <Method> Confirmation of cell dispersion Gelatin was dissolved to 2 w / v% using physiological saline, cooled in a refrigerator at 4°C to gel, and then gelatin gel microspheres (hereinafter referred to as "gelatin microspheres") of about 200 μm were made by passing the gelatin gel through a mesh with an opening of 200 μm. Gelatin microspheres were suspended in a fibrinogen solution (40 mg / mL) at 20 w / v% to prepare a fibrinogen + gelatin microsphere solution. iPIC was suspended in a fibrinogen solution (40 mg / mL) and a fibrinogen + gelatin microsphere solution and allowed to stand, and the maintenance of the cell dispersion state was evaluated by the presence or absence of precipitation.

[0159] In vivo testing iPIC3x10 6 After the cells were divided into 1.5 mL test tubes and centrifuged, the culture medium supernatant was removed, and 25 μL of the fibrinogen + gelatin microsphere solution prepared as described above was added and suspended. After the fibrinogen + gelatin microsphere solution was thinly applied on a polyester mesh (opening rate 69%, size 10x10 mm) impregnated with 12.5 μL of thrombin solution (62.5 U / mL), 25 μL of thrombin solution was applied to the entire mesh and allowed to stand for 5 minutes to gel, a thin layer of fibrin + gelatin microsphere gel sheet containing iPIC was prepared.

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

[0161] Large Animal and Human-Sized Production iPIC75x10 6After centrifugation, the cells were divided into 15 mL test tubes, the culture medium supernatant was removed, and 600 μL of fibrinogen + gelatin microsphere solution was added to suspend. The fibrinogen + gelatin microsphere solution with iPIC suspended was thinly applied to a polyester mesh (opening rate 69%, size 40x60 mm) and a glycolic acid / lactic acid polyester (90 / 10, poly (glycolide-co-L-lactide), size 40x60 mm) mesh impregnated with 300 μL of thrombin solution (62.5 U / mL) using a pipette or a 3D printer, and 600 μL of thrombin solution was applied to the entire mesh, and it was allowed to stand for 5 minutes to gel, thereby producing a thin layer of fibrin + gelatin microsphere gel sheet containing iPIC.

[0162] <Results> Figure 6 The state of iPIC suspended and allowed to stand in fibrinogen solution and fibrinogen + gelatin microsphere solution is shown in FIG. Figure 6 (A)) was allowed to stand for 10 minutes, and iPIC precipitation (arrow) was observed. On the other hand, the fibrinogen + gelatin microsphere solution ( Figure 6 (B)) The iPIC dispersion was maintained even after standing for 30 minutes.

[0163] In addition, in the thin layer of fibrin + gelatin microsphere gel sheet containing iPIC, the iPIC was retained in the gel sheet and no leakage of cells was observed.

[0164] Figure 7 The results of time-dependent measurement of the concentration of human c-peptide in the blood of nude rats implanted subcutaneously with a fibrin gel sheet containing iPIC or a fibrin + gelatin microsphere gel sheet containing iPIC are shown. No significant difference in the amount of human c-peptide in the blood was observed between the fibrin gel sheet and the fibrin + gelatin microsphere gel sheet, indicating that the use of gelatin microspheres in the sheet does not have a negative impact on the secretion of human c-peptide from iPIC.

[0165] Figure 8 The fibrin + gelatin microsphere gel sheet containing iPICs in large animal and human sizes was produced manually by pipette operation and by 3D printer. The fibrin + gelatin microsphere gel sheet in large animal and human sizes can be produced by either method, and the iPICs are retained in the gel, and no cell leakage is observed ( Figure 8 (A)).

[0166] The standard deviation of the brightness distribution of the large animal / human-sized iPIC-encapsulated fibrin + gelatin microsphere gel sheets produced by pipette manual manipulation and 3D printer was calculated by image analysis, and the results were 19.437 and 15.630 ( Figure 8(B)). This result shows that the fibrin + gelatin microsphere gel produced by 3D printer can be coated more evenly. In addition, a circle with a diameter of 12 mm was dug out at any four locations in the fibrin + gelatin microsphere gel sheet produced by 3D printer, and the number of cells contained therein was measured. The result confirmed that the number of cells was almost the same level ( Figure 8 (C)). This result shows that it is possible to produce a fibrin + gelatin microsphere gel sheet with cells evenly distributed inside by using a 3D printer.

[0167] Example 4: Preparation of large animal and human-sized fibrin gel sheets (manufacturing method 2-2_fibrin + collagen gel sheets) <Method> Confirmation of cell dispersion A fibrinogen solution (80 mg / mL) and a collagen solution (3 w / v%) were mixed at a ratio of 3:1 to prepare a fibrinogen + collagen solution. iPIC was suspended in the fibrinogen + collagen solution and then filled into a syringe. The syringe was held vertically with the tip of the syringe barrel facing downward, and a certain amount of the effluent was dispensed after standing for 0, 10, 20, and 30 minutes. The cell number (concentration) was measured to evaluate the cell dispersion retention performance of the fibrinogen + collagen solution.

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

[0169] The iPIC-encapsulated fibrin + gelatin microsphere gel sheet described in the "In vivo test" of Example 3 and the iPIC-encapsulated fibrin + collagen gel sheet described above were transplanted subcutaneously into anesthetized nude rats. After transplantation, blood was collected from the tail vein to separate plasma, and the human c-peptide concentration was measured by ELISA.

[0170] Large Animal and Human-Sized Production iPIC75x10 6After centrifugation, the cells were divided into 15 mL test tubes, the culture medium supernatant was removed, and 600 μL of fibrinogen + collagen solution was added to suspend. The fibrinogen + collagen solution with iPIC suspended was thinly coated on a poly (glycolide-co-L-lactide) (size 40x60mm) mesh 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 mesh and allowed to stand for 5 minutes to gel, thus producing a thin layer of fibrin + collagen gel sheet containing iPIC.

[0171] <Results> Fig. 9 The results of measuring the number (concentration) of cells in the fibrinogen + collagen solution discharged from the syringe are shown in . It is shown that the cell concentration in the discharged liquid remains almost constant regardless of the length of the standing time, and even if the syringe is held vertically, iPIC does not precipitate and discharges an almost constant number of cells. Therefore, it is shown that the dispersed state of cells can be maintained by using a fibrinogen + collagen solution.

[0172] In addition, in the manufactured thin layer of fibrinogen+collagen sheet containing iPIC, the iPIC was retained inside the gel sheet, and no leakage of cells was observed.

[0173] Fig.10 The results of time-dependent measurement of the concentration of human c-peptide in the blood of nude rats implanted subcutaneously with a fibrin + gelatin microsphere gel sheet containing iPIC or a fibrin + collagen gel sheet containing iPIC are shown in the figure. No significant difference in the amount of human c-peptide in the blood was observed between the fibrin + gelatin microsphere gel sheet and the fibrin + collagen gel sheet, indicating that the two have equivalent performance.

[0174] The fibrin + collagen sheet of large animal and human size can be made using a 3D printer. The iPIC is kept in the gel and no cell leakage is observed. A circle with a diameter of 12 mm was dug out at any 6 locations in the fibrin + collagen gel sheet of large animal and human size, and the number of cells contained therein was measured. The result confirmed that the number of cells was almost the same level ( Fig.11 ). This result shows that by suspending iPIC in a fibrinogen + collagen solution and coating it with a 3D printer, it is possible to produce a fibrin + collagen gel sheet with cells evenly distributed inside.

Claims

1. A fibrin gel sheet for cell transplantation, which is formed by uniformly dispersing cells and encapsulating them in the fibrin gel sheet, wherein 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 capable of passing through 100 μm to 1000 μm.

2. The fibrin gel sheet according to claim 1, which has a surface area of ​​2.25 cm 2 The size is above 1 mm and the thickness is below 1 mm.

3. The fibrin gel sheet according to claim 1, wherein the number of cells is more than 1.5×10 6 Pieces / cm 2 The amount is insourced. The fibrin gel sheet according to claim 1 , wherein the cells are in the form of spheroids. The fibrin gel sheet according to claim 1 , wherein the cells are pancreatic islet cells derived from iPS cells. The fibrin gel sheet according to claim 1 , further comprising a carrier.

7. A method for producing a fibrin gel sheet for cell transplantation, wherein cells are uniformly dispersed and encapsulated in a fibrin gel sheet. The method comprises the steps of allowing a fibrinogen solution containing the cells suspended therein to act with thrombin to form a sheet and gel the sheet. in, The fibrinogen solution in which the cells are suspended further contains 0.2 to 2 w / v% of collagen and / or 10 to 30 w / v% of gelatin particles capable of passing through 100 μm to 1000 μm.

8. The manufacturing method according to claim 7, wherein: The gelling step is to allow the fibrinogen solution containing the suspended cells to react with thrombin to form a gel having a surface area of ​​2.25 cm 2 The sheet is gelled to have a size of at least 1 mm and a thickness of 1 mm or less.

9. The method according to claim 7, wherein the number of cells in the fibrin gel sheet exceeds 1.5×10 6 Pieces / cm 2 The amount was suspended in the fibrinogen solution. The production method according to claim 7 , wherein the cells are in the form of spheroids. The production method according to claim 7 , wherein the cells are pancreatic islet cells derived from iPS cells.

12. The production method according to claim 7, wherein the film is formed into a sheet shape by coating on a carrier.

13. The manufacturing method according to claim 12, wherein coating on the carrier is performed by bioprinting. The production method according to claim 12 , wherein the carrier comprises thrombin. 15 . A laminate of fibrin gel sheets for cell transplantation, which is formed by laminating a plurality of the fibrin gel sheets according to claim 1 .

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