Immune isolation device

Abstract

The problem addressed by the present invention is to provide: an immune isolation device which not only achieves a reduction in the diffusion distance to effectively improve the permeability of substances such as physiologically active substances and nutrients, but also achieves an improvement in durability to withstand long-term transplantation; and an immune control technique using the cell sheet. The present invention provides an immunoisolation device having a sheet-like cell mass containing cells and an extracellular matrix, and an immunoisolation layer covering the cell mass.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority based on Japanese Patent Application No. 2022-075135 filed on April 28, 2022 (the entire disclosure of which is incorporated herein by reference). Background Art

[0003] Immunoisolation devices have been developed as a means of cell transplantation therapy without the need for the administration of immunosuppressants. In particular, for example, in the transplantation of somatic cells derived from iPS cells where the risk of carcinogenesis is concerned or when the function of transplanted cells is reduced, macroencapsulation immunoisolation devices are considered to be an effective method because the transplantation site can be confirmed and the device can be replaced. The important functions required for macroencapsulation immunoisolation devices are as follows: cells or cell clusters can be uniformly dispersed and fixed without inducing the binding of cells or cell clusters; the device allows oxygen and / or nutrients to easily penetrate into transplanted cells; the device allows the expected physiologically active substances (cytokines, hormones, growth factors, etc.) required for the therapeutic effect to be easily released by cells according to cell response; prevent the penetration of immune response cells and immune response factors; the transplant device has excellent biocompatibility, is less likely to adhere to surrounding tissues, and is less likely to induce inflammatory reactions such as granulation.

[0004] Citation List

[0005] Patent Literature

[0006] PTL 1:JP2012-508584A

[0007] PTL 2: Japanese Patent No. 5717253 Summary of the invention

[0008] Technical issues

[0009] Many immunoisolation devices using porous membranes have been studied (Patent Document (PTL) 1), and it has been considered that there is a problem that the permeability is reduced due to protein adsorption to the porous membrane material or fibrosis, or due to adhesion to surrounding tissues. When the diameter exceeds 500 μm, it is believed that internal necrosis of cell clusters, etc. will be induced. In order to easily maintain the implantation of embedded cells into the recipient and release physiologically active substances, the total thickness of such an immunoisolation device must be properly controlled, and it is hoped that the immunoisolation layer constituting the device is as thin as possible. On the other hand, in order to be transplanted into the body, the strength and durability of the membrane must be improved to prevent the device from breaking or twisting. However, it is not easy to achieve a reduction in membrane thickness and an improvement in durability at the same time.

[0010] In addition, temperature-responsive culture dishes can be used to culture cells for transplantation, which enable cells to be produced in a monolithic form containing an extracellular matrix, adhesion factors, and the like. For example, myocardial sheets, which are used by applying skeletal muscle cell sheets as "cell sheets" to an affected area, have been put into practical use as regenerative medicine products. In addition, Patent Document (PTL) 2 discloses a method for producing a cell sheet of pancreatic islet cells and a method for using the same. One advantage of a cell sheet is that the cell sheet contains an extracellular matrix, cell adhesion factors, and the like, and achieves excellent cell implantation in transplantation without excessive progression of cell cluster formation caused by cell binding.

[0011] However, the cell sheet transplantation method is currently only put into practical use in autologous cells. When allogeneic cells are used, the cell sheet is not implanted into the host, but dies due to immune rejection. Therefore, immunosuppressants must be administered. However, since HLA homology between the donor and the host is required, there are many challenges to overcome before this method can be popularized and commercialized as a regenerative medicine technology. The embodiments of patent document (PTL) 2 also disclose transplantation into allogeneic rats, and PTL 2 further discloses an embodiment of transplantation into immunodeficient mice; however, no immune control technology is used therein.

[0012] One object of the present invention is to provide an immunoisolation device that achieves both a reduction in diffusion distance to effectively increase the permeability of substances such as physiologically active substances and nutrients and an improvement in durability to withstand long-term transplantation. Another object of the present invention is to provide an immunocontrol technology using a cell sheet.

[0013] Solutions to the Problem

[0014] The present invention provides the following immunoisolation device.

[0015] [1] An immunoisolation device comprising a sheet-like cell aggregate and an immunoisolation layer,

[0016] The sheet-like cell aggregates contain cells and an extracellular matrix, and

[0017] The immunoisolation layer covers the cell aggregates.

[0018] [2] The immunoisolation device according to [1], wherein the cell aggregate is a cell sheet comprising cells and an extracellular matrix.

[0019] [3] The immunoisolation device according to [1] or [2], wherein the cell aggregate has a thickness of 300 μm or less.

[0020] [4] The immunoisolation device according to any one of [1] to [3], wherein the immunoisolation layer comprises a porous membrane or a fiber structure,

[0021] The porous film or the fiber structure includes at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate.

[0022] [5] The immunoisolation device according to [4], wherein the immunoisolation layer is an immunoisolation multilayer comprising the porous membrane or the fiber structure and a hydrogel.

[0023] [6] The immunoisolation device according to [5], wherein the outermost layer of the immunoisolation multilayer is the porous membrane or the fiber structure, and the innermost layer is the hydrogel.

[0024] [7] The immunoisolation device according to [5], wherein

[0025] The outermost layer of the immunoisolation multilayer is the hydrogel, and

[0026] The innermost layer of the immunoisolation multilayer is the porous membrane or the fiber structure.

[0027] [8] The immunoisolation device according to any one of [1] to [7], wherein

[0028] The immunoisolation layer comprises a plurality of layers, and

[0029] The innermost layer of the immune isolation layer is a hydrogel or a porous membrane fixed with cell adhesion proteins and / or cell adhesion peptides.

[0030] [9] The immunoisolation device according to any one of [5] to [8], wherein the hydrogel comprises at least one member selected from polyvinyl alcohol and polyethylene glycol.

[0031] Advantageous Effects of the Invention

[0032] The present invention can provide an immunoisolation device that achieves both a reduction in diffusion distance to effectively increase the permeability of substances such as physiologically active substances and nutrients and an improvement in durability to withstand long-term transplantation. In addition, the present invention can provide an immunocontrol technology using a cell sheet. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a perspective view of a bag-shaped immunoisolation device.

[0034] Figure 2 is a cross-sectional view of a tubular immunoisolation device.

[0035] Figure 3 This is a conceptual diagram of a cell sheet.

[0036] Figure 4 is a conceptual diagram of a device having a cell sheet encapsulated therein.

[0037] Figure 5 is a cross-sectional view of an immunoisolation layer obtained by multilayer molding using a porous membrane and a hydrogel.

[0038] Figure 6 is a cross-sectional view of an immunoisolation layer obtained by multilayer molding using a porous membrane and a hydrogel.

[0039] Figure 7 is a SEM image of a cross section of the immunoisolation layer (B) obtained in Production Example 1.

[0040] Figure 8 Schematic diagram of an instrument used to measure the amount of glucose, insulin, immune system humoral factors, etc. that permeate through an immune barrier.

[0041] Fig. 9 This is a conceptual diagram of the device obtained in Example 1.

[0042] Fig.10 This is a graph showing the functional evaluation performed in Example 1.

[0043] Fig.11 The results of the histopathological evaluation performed in Example 1 are shown.

[0044] Fig.12 The results of the histopathological evaluation performed in Example 1 are shown. Fig.12 A is a histological staining image. Fig.12 B to 12D are fluorescent staining images. Fig.12 B is the original image. Fig.12 C is the original image ( Fig.12 B) The image in which the insulin-positive area (anti-insulin (pancreatic β cells)) appears in black. Fig.12 D is the original image ( Fig.12 B) The cell nucleus part (Hoechst dye stained part) is shown in black. Fig.12 As shown in B to 12D, cell nuclei were observed in the insulin-positive areas in the fluorescent staining images. DETAILED DESCRIPTION

[0045] As used in this specification, the singular forms ("a", "an" and "the") are intended to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0046] The immunoisolation device of the present invention comprises a sheet-like cell aggregate and an immunoisolation layer, wherein the sheet-like cell aggregate comprises cells and an extracellular matrix, and the cell aggregate is covered by the immunoisolation layer, thereby inhibiting immune cells and cytokines from invading the cell aggregate. The immunoisolation layer is preferably an immunoisolation multilayer comprising a porous membrane or a fiber structure and a hydrogel.

[0047] Cell aggregates

[0048] The cell aggregates used in the immunoisolation device of the present invention are sheet-like cell aggregates comprising cells and extracellular matrix. Examples of cell aggregates include complexes of cells and extracellular matrix; cells immobilized on sheets formed using collagen sheets or collagen sponges as scaffold materials; and cell sheets prepared using, for example, temperature-responsive culture dishes. Examples of available cell aggregates further include cell aggregates that do not contain scaffold materials such as collagen sheets. Cells can be in the form of cell clusters.

[0049] In a preferred embodiment, the cell aggregate is a cell sheet. As used herein, the term "cell sheet" refers to a cell aggregate in which cells are connected to each other through cell-cell bonding and in the form of a sheet comprising a single layer or multiple layers (preferably a single layer). The cell sheet is composed of cells and an extracellular matrix, and the adhesion between cells is maintained. In a preferred embodiment, as Figure 3 As shown in , the cell sheet has a structure in which sheet-like cells (11) are laminated on a sheet-like extracellular matrix (10). In this embodiment, the cells are preferably arranged as a single layer on the extracellular matrix.

[0050] For example, a cell sheet can be obtained by culturing cells on a stimulus-responsive culture substrate coated with a polymer whose molecular structure changes in response to a stimulus, such as temperature, pH or light, and by changing the conditions of the stimulus (such as temperature, pH or light) to change the surface of the stimulus-responsive culture substrate. An example of a stimulus-responsive culture substrate is UpCell (registered trademark), which is a commercially available temperature-responsive culture dish produced by CellSeed Inc. In a preferred embodiment, an extracellular matrix is ​​formed during the cell culture step, and the cells are kept in an adherent state.

[0051] A single cell sheet may be used as a cell aggregate, or two or three cell sheets may be laminated and used as a cell aggregate in a laminated state.

[0052] When using a cell sheet as a cell aggregate, the cell sheet can be laminated on a scaffold material such as a collagen sponge to improve the ease of operation and used in this state; however, it is not always necessary to use a scaffold material. When using a cell sheet as a cell aggregate, no scaffold material is required to fix the cells, and an immunoisolation device can be produced without intentionally adding a scaffold material. Therefore, in one embodiment, the present invention provides an immunoisolation device as described above, wherein the cell aggregate is a cell sheet comprising cells and an extracellular matrix without any intentionally added scaffold material.

[0053] The thickness of the cell aggregate is not particularly limited, but is preferably 10 μm or more, more preferably 20 μm or more. In addition, the thickness of the cell aggregate is preferably 300 μm or less, more preferably 290 μm or less, and even more preferably 150 μm or less. The thickness within the above range is preferred because it is less likely to hinder the supply of oxygen to cells, etc.

[0054] Animal species as the source of cells used in the cell aggregates can include, for example, mammals, such as humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees and immunodeficient animals thereof, birds, reptiles, amphibians, fish, insects, etc. When the immunoisolation device of the present invention is used to treat humans, it is preferred to use human cells. When the immunoisolation device is used for human treatment, the cells may be taken from the patient himself or others, or from commercially available cell lines.

[0055] Examples of cells used in cell aggregates include somatic cells that constitute an organism (cardiomyocytes, hepatocytes, kidney cells, adrenal cortical cells, epidermal cells, vascular endothelial cells, mucosal cells, pancreatic islet cells, etc.), germ cells (sperm, eggs, etc.), stem cells (mesenchymal stem cells, ES cells, iPS cells, etc.), precursor cells, cells that are isolated from an organism and have acquired immortalization ability and are stably maintained in vitro, cells that are isolated from an organism and have undergone artificial genetic modification, and cells that are isolated from an organism and whose cell nuclei have been artificially replaced.

[0056] In a preferred embodiment, the cells used in the cell aggregates include those that release bioactive substances from the immunoisolation device. Examples include mesenchymal stem cells, pancreatic islet cells, islet-like insulin-producing cells, pituitary hormone-producing cells, and lysosomal enzyme-producing cells.

[0057] Cell aggregates can be composed of the various types of cells mentioned above.

[0058] Immune barrier

[0059] The immunoisolation device of the present invention comprises an immunoisolation layer covering the cell aggregates. In a typical embodiment, from the perspective of obtaining a sufficient immunoisolation effect, it is preferred that the immunoisolation layer in the device of the present invention covers the entire surface of the cell aggregates. For the same reason, the immunoisolation layer preferably does not have any pores, such as pinholes, that penetrate the membrane.

[0060] The immunoisolation layer preferably comprises a porous membrane or a fiber structure. More preferably, the immunoisolation layer is preferably an immunoisolation multilayer comprising a porous membrane or a fiber structure and a hydrogel. The immunoisolation layer can be an immunoisolation multilayer comprising a porous membrane and a fiber structure, or can be an immunoisolation multilayer comprising a porous membrane, a fiber structure and a hydrogel.

[0061] porous membrane

[0062] The porous membrane of the immunoisolation layer is a membrane having a plurality of pores. The membrane can be confirmed to be a porous membrane by a scanning electron microscope (SEM) image or a transmission electron microscope (TEM) image of a membrane cross section. The porous membrane is preferably a semipermeable membrane.

[0063] The thickness of the porous membrane is not particularly limited, but is preferably 300 μm or less, more preferably 15 μm to 290 μm, and even more preferably 30 μm to 150 μm. A thickness within the above range is preferred because the strength of the immunoisolation layer is maintained while being less likely to hinder the supply of oxygen and other substances to cells.

[0064] The average pore size of the porous film is not particularly limited, but is preferably 0.01 μm to 10 μm, more preferably 0.01 μm to 5 μm, and even more preferably 0.01 to 3 μm. The average pore size can be determined from a SEM image or a TEM image. For example, the surface of the porous film is observed using SEM, and 50 pores are randomly selected from the pores formed on the surface. The major diameter of each pore is measured, and the average value of the major diameters of the 50 pores is calculated to obtain the average pore size.

[0065] The maximum pore size of the porous membrane is not particularly limited, but preferably 0.01 μm to 10 μm, more preferably 0.01 μm to 5 μm, and more preferably 0.01 μm to 4 μm. When the maximum pore size is within the above range, immune response cells can be inhibited from invading the device while allowing nutrients such as carbon sources such as amino acids, vitamins, inorganic salts, glucose, and physiologically active substances such as oxygen, carbon dioxide, cytokines, hormones, and insulin to fully penetrate. The maximum pore size can be determined by SEM images or TEM images. For example, the surface of the porous membrane is observed using SEM, and 50 pores are randomly selected from the pores formed on the surface. The major diameter of each pore is measured, and the maximum value among the 50 major diameters is defined as the maximum pore size.

[0066] From the viewpoint of having to have the function of inhibiting cell infiltration from the recipient and also preventing leakage of cells as a material to be transplanted, the average pore size or maximum pore size of the porous membrane is preferably 5 μm or less to be smaller than the cell size.

[0067] The porous membrane comprises polymer. Preferably, the porous membrane is substantially composed of polymer. The polymer can be, for example, a thermoplastic or thermosetting polymer. The polymer can be biocompatible. The specific example of the polymer includes ethylene-vinyl alcohol copolymer, polysulfone, cellulose acetate, nitrocellulose, sulfonated polysulfone, polyethersulfone, polyacrylonitrile, styrene-acrylonitrile copolymer, styrene-butadiene copolymer, polyvinyl alcohol, polycarbonate, organosiloxane-polycarbonate copolymer, polyester carbonate, organopolysiloxane, polyphenylene oxide, polyamide, polyimide, polyamide-imide, polybenzimidazole and polytetrafluoroethylene (PTFE). Considering solubility, optical properties, electrical properties, strength, elasticity, etc., the polymer can be a homopolymer or can be a copolymer, a polymer blend, a polymer alloy, etc. The polymer constituting the porous membrane can contain a hydrophilic polymer, such as polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose or polyethylene glycol. Combining hydrophilic polymers and hydrophobic polymers can improve biocompatibility.

[0068] Ethylene-vinyl alcohol copolymers can usually be obtained by saponifying ethylene-vinyl ester copolymers. The production and saponification of ethylene-vinyl ester copolymers can be carried out by known methods. A representative example of vinyl ester is vinyl acetate, but other fatty acid vinyl esters such as vinyl formate, vinyl propionate, vinyl valerate, vinyl caprate, vinyl laurate, vinyl stearate, vinyl pivalate and vinyl versatate are also available.

[0069] The ethylene unit content of the ethylene-vinyl alcohol copolymer is preferably 20 mol% or more, more preferably 25 mol% or more. In addition, the ethylene unit content in the ethylene-vinyl alcohol copolymer is preferably 60 mol% or less, more preferably 55 mol% or less, and even more preferably 50 mol% or less.

[0070] The saponification degree of the ethylene-vinyl alcohol copolymer is preferably 80 mol% or more, more preferably 90 mol% or more, and even more preferably 95 mol% or more. In addition, the saponification degree of the ethylene-vinyl alcohol copolymer may be 100 mol% or less, or may be 99.99 mol% or less. The saponification degree of the ethylene-vinyl alcohol copolymer can be determined by 1 H-NMR measurement and calculation are performed by measuring the peak area of ​​hydrogen atoms contained in the vinyl ester structure and the peak area of ​​hydrogen atoms contained in the vinyl alcohol structure.

[0071] As long as the purpose of the present invention is not impaired, the ethylene-vinyl alcohol copolymer may further contain units derived from monomers other than ethylene, vinyl esters and saponification products thereof. When the ethylene-vinyl alcohol copolymer has such other monomer units, the content of such other monomer units is preferably 30 mol% or less, more preferably 20 mol% or less, even more preferably 10 mol% or less, and particularly preferably 5 mol% or less, based on the total monomer units of the ethylene-vinyl alcohol copolymer. When the ethylene-vinyl alcohol copolymer contains units derived from such other monomers, the lower limit of the content may be 0.05 mol% or 0.10 mol%. Examples of other monomers include olefins such as propylene, butene, pentene and hexene; olefins containing an ester group or saponification products thereof, such as 3-acyloxy-1-propylene, 3-acyloxy-1-butene, 4-acyloxy-1-butene, 3,4-diacyloxy-1-butene, 3-acyloxy-4-methyl-1-butene, 4-acyloxy-2-methyl-1-butene, 4-acyloxy-3-methyl-1-butene, 3,4-diacyloxy-2-methyl-1-butene, 4-acyloxy-1-pentene, 5-acyloxy-1-pentene, 4,5-diacyloxy-1-pentene, 4-acyloxy-1-hexene, 5-acyloxy-1-hexene, 6-acyloxy-1-hexene, 5,6-diacyloxy-1-hexene and 1,3-diethylhexene; Acyloxy-2-methyleneprylene (1,3-diacetoxy-2-methyleneprylene); unsaturated acids such as acrylic acid, methacrylic acid, crotonic acid and itaconic acid, or anhydrides, salts or monoalkyl esters or dialkyl esters thereof; nitriles such as acrylonitrile and methacrylonitrile; amides such as acrylamide and methacrylamide; olefin sulfonic acids such as vinyl sulfonic acid, allyl sulfonic acid and methyl allyl sulfonic acid, or salts thereof; vinyl silane compounds such as vinyl trimethoxysilane, vinyl triethoxysilane, vinyl tri(β-methoxy-ethoxy) silane and γ-methacryloxypropyl methoxysilane; alkyl vinyl ethers, vinyl ketones, N-vinyl pyrrolidone, vinyl chloride, vinylidene chloride, etc.

[0072] The ethylene-vinyl alcohol copolymer can be post-modified by amination, acetalization, cyanoethylation, oxyalkyleneization, or the like.

[0073] Such ethylene-vinyl alcohol copolymers may be used alone or in combination of two or more.

[0074] The polymer used to form the porous film is preferably a highly biocompatible material that is unlikely to cause adhesion to recipient tissues around the transplant site or induce inflammation, etc. The porous film preferably contains at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate.

[0075] The porous membrane may be composed of one type of porous membrane, or may be composed of two or more types of porous membranes laminated to each other. When the porous membrane comprises two or more porous membranes, these porous membranes may be directly laminated, or the hydrogel or fiber structure may be sandwiched between the two porous membranes.

[0076] In a preferred embodiment, the porous membrane is a single layer formed from one composition. In a preferred embodiment, the porous membrane does not have a multi-layer laminate structure.

[0077] Fiber structure

[0078] The fiber structure of the immunoisolation layer is, for example, a nonwoven, a woven fabric or a knitted fabric. The fiber structure is preferably a nonwoven. The fibers are bonded or entangled together to form the fiber structure by heat, mechanical or chemical action. The basis weight (weight per unit area) can be adjusted by adjusting the fiber diameter and / or the fiber amount so that in addition to strength, permeability and / or filtration can also be controlled. The fiber structure preferably has a density of 10 to 100 g / m 2 Considering the diffusion efficiency of the physiologically active substance released from the material to be implanted, the thickness of the fiber structure is preferably 300 μm or less, more preferably 200 μm or less and as thin as possible.

[0079] The example of the fiber material of fiber structure includes gelatin, collagen, chitin, chitosan, fibronectin, dextran, cellulose, polyethylene (PE), polypropylene (PP), polyurethane, polyamide, polyester, polyvinyl alcohol (PVA), ethylene-vinyl alcohol copolymer, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, PVA (such as methacrylic acid modified PVA and acrylic acid modified PVA) modified with monomer, polycaprolactone, polyglycerol sebacate, polyhydroxyalkanoic acid, polybutylene succinate, polymethylene carbonate, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, cellulose acetate such as carboxymethylcellulose, silk core protein and silk. As with porous membrane, the fiber material of the fiber structure is preferably biocompatible. The fiber structure preferably contains at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate. Preferably, the surface smoothing of the fiber structure is made by heat, mechanical or chemical treatment. When using ethylene-vinyl alcohol copolymer, the ethylene-vinyl alcohol copolymer described in the explanation of porous membrane can be preferably used.

[0080] Hydrogel

[0081] Examples of hydrosols for producing hydrogels of immunoisolation layers include sols that gel in the presence of metal ions to form hydrogels; sols that gel in response to temperature to form hydrogels; sols that gel in response to pH to form hydrogels; and sols that gel in response to light to form hydrogels. Metal ions and pH are examples of chemical effects. In order to gel these hydrosols, operations can be performed according to the characteristics of the gel used, such as contacting metal ions with the hydrosol, adjusting the temperature to gelling conditions, adjusting the pH to gelling conditions, applying light under gelling conditions, or providing a magnetic field under gelling conditions.

[0082] Examples of hydrogels obtainable by gelation in the presence of metal ions include alginate gels obtainable by gelation in the presence of divalent or trivalent metal ions, preferably alkaline earth metal ions, such as calcium ions or magnesium ions; carrageenan gels obtainable by gelation in the presence of calcium ions and / or potassium ions; and acrylic acid-based synthetic gels obtainable by gelation in the presence of sodium ions.

[0083] Examples of temperature-responsive hydrogels include those obtainable by cross-linking poly(N-isopropylacrylamide) and polyethylene glycol (trade name: Mebiol Gel), methylcellulose, hydroxypropylcellulose, copolymers of lactic acid and ethylene glycol, triblock copolymers of polyethylene glycol and polypropylene oxide (trade name: Pluronic (registered trademark), poloxamer), agarose, polyvinyl alcohol, and the like.

[0084] Examples of pH-responsive hydrogels include alginate gel, chitosan gel, carboxymethyl cellulose gel, and acrylic acid-based synthetic gel.

[0085] Examples of photoresponsive hydrogels include synthetic gels in which azobenzene and cyclodextrin are incorporated in the backbone, gels composed of supramolecules having fumaric acid amide as spacers, gels obtainable by crosslinking or bonding through nitrobenzyl groups, and gels composed of modified polyvinyl alcohol.

[0086] The modified polyvinyl alcohol may be, for example, (meth)acryloyl-modified polyvinyl alcohol. The (meth)acryloyl group may be introduced by subjecting the hydroxyl group on the side chain of the polyvinyl alcohol to an esterification reaction or an ester exchange reaction with a compound containing an ethylenically unsaturated group in the presence of a base. Examples of compounds containing an ethylenically unsaturated group include (meth)acrylic acid and derivatives thereof, such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acryloyl halide, and (meth)acrylate.

[0087] Preferred examples of hydrogels include polyvinyl alcohol, polyethylene glycol, chitosan, alginate, etc. The hydrogel preferably contains at least one member selected from polyvinyl alcohol and polyethylene glycol, more preferably polyvinyl alcohol. Polyvinyl alcohol-based polymers can be produced, for example, by saponification of polyvinyl esters, which can be obtained by polymerization of vinyl ester-based monomers, and by converting ester groups in the polyvinyl esters into hydroxyl groups.

[0088] Examples of vinyl ester monomers include aliphatic vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate. These vinyl ester monomers can be used alone or in combination of two or more.

[0089] Among the vinyl ester monomers, aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of production cost. In other words, the polyvinyl ester is preferably polyvinyl acetate obtainable by polymerization of vinyl acetate.

[0090] Polyvinyl ester may optionally contain structural units derived from monomers other than vinyl ester monomers as long as the effects of the present invention are not impaired. Examples of such other monomers include α-olefins such as ethylene, propylene, n-butene and isobutylene; acrylic acid or its salts; acrylic acid alkyl esters such as methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate and octadecyl acrylate; methacrylic acid or its salts; methacrylic acid alkyl esters such as methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate and octadecyl methacrylate; acrylamide derivatives such as acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetoneacrylamide, acrylamidopropanesulfonic acid or its salts, acrylamidopropyldimethylamine or its salts or quaternary salts, and N-hydroxymethyl acrylamide or its derivatives; methacrylamide derivatives such as methacrylamide, N-methyl methacrylamide, N-ethyl methacrylamide, methacrylamidopropanesulfonic acid or its salts, methacrylamidopropyl dimethylamine or its salts or quaternary salts, N-hydroxymethyl methacrylamide or its derivatives; N-vinylamide derivatives such as N-vinyl formamide and N-vinylacetamide; vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, isopropyl vinyl ether, n-butyl vinyl ether, isobutyl vinyl ether, tert-butyl vinyl ether, dodecyl vinyl ether and stearyl vinyl ether; nitriles such as acrylonitrile and methacrylonitrile; vinyl halides such as vinyl chloride and vinyl fluoride; vinylidene halides such as vinylidene chloride and vinylidene fluoride; allyl compounds such as allyl acetate and allyl chloride; maleic acid or its salts, esters or anhydrides; vinyl silyl compounds such as vinyltrimethoxysilane; and isopropenyl acetate. These may be used alone or in combination of two or more.

[0091] The average degree of polymerization of polyvinyl alcohol is preferably 300 to 10000, more preferably 500 to 5000, still more preferably 1000 to 5000, particularly preferably 2000 to 5000. The thickness within the above range is preferred in view of the permeability of substances and ease of handling in multilayer formation.

[0092] The average degree of polymerization of polyvinyl alcohol in this specification refers to the average degree of polymerization measured in accordance with JIS K 6726: 1994. Specifically, the average degree of polymerization can be determined from the intrinsic viscosity measured in 30° C. water after the raw material PVA has been saponified and purified.

[0093] From the viewpoint of improving the water solubility of polyvinyl alcohol, the saponification degree of polyvinyl alcohol is preferably 50 mol % or more, more preferably 60 mol % or more, and still more preferably 65 mol % or more.

[0094] The saponification degree of the polyvinyl alcohol is preferably 99 mol% or less from the viewpoint of suppressing the hydrosol solution from having high viscosity and improving the storage stability of the hydrosol solution.

[0095] In this specification, the saponification degree of polyvinyl alcohol refers to the ratio (mol %) of the number of moles of vinyl alcohol units relative to the total number of moles of structural units (e.g., vinyl acetate units) and vinyl alcohol units in the raw material PVA that can be converted into vinyl alcohol units by saponification. The saponification degree can be measured according to JIS K 6726:1994.

[0096] The thickness of the hydrogel is not particularly limited, but is preferably 1 to 300 μm, more preferably 5 to 200 μm, and still more preferably 10 to 100 μm.

[0097] The hydrogel may consist of one hydrogel, or a laminate of two or more hydrogels. When the immunoisolation device of the present invention comprises two or more hydrogels, the hydrogel layers may be directly laminated, or a porous membrane or fiber structure may be sandwiched between the two hydrogels.

[0098] Cross-linking of hydrogels enables the regulation of permeability, strength, etc. of nutrients such as glucose, physiologically active substances such as insulin, and humoral factors of the immune system.

[0099] The gel strength of the hydrogel is preferably 20 to 300 kPa, more preferably 50 to 200 kPa. The gel strength can be measured by the following procedure using a tensile tester.

[0100] First, the hydrosol solution was poured between two glass plates separated from each other by inserting a 1 mm thick spacer between them, and processed under predetermined gelling conditions to obtain a gel sheet with a thickness of 1 mm. A test piece was cut from the gel sheet using a dumbbell cutter specified in JIS K 6251-3. The test piece was set on a tensile tester (model 5566) produced by Instron Co., Ltd., and the fracture stress and fracture strain were measured while collecting image data. The stress when the test piece fractured was defined as the gel strength.

[0101] The structure of immune isolation layer, etc.

[0102] The immunoisolation layer of the present invention comprises a porous membrane or a fiber structure, wherein the porous membrane or the fiber structure preferably comprises at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate. In addition, the immunoisolation layer of the present invention is preferably an immunoisolation multilayer comprising a porous membrane or a fiber structure and a hydrogel.

[0103] Forming multiple layers with hydrogels increases the strength of the immunoisolation layer and improves immunoisolation. The permeability and immunoisolation can be adjusted by adjusting the pore size of the porous membrane and the gel strength and / or crosslinking degree of the hydrogel.

[0104] Cell infiltration and cell leakage can be suppressed by porous membranes. However, it is not easy to suppress the infiltration of immune system humoral factors such as IgG antibodies without reducing the permeability of essential physiologically active substances. Therefore, by adjusting the gel strength or cross-linking density of the hydrogel formed with the porous membrane, the infiltration of immune system humoral factors such as IgG antibodies can be suppressed without reducing the permeability of physiologically active substances.

[0105] When the fiber structure layer used is a single layer, it is not easy to suppress the infiltration of immune system humoral factors such as IgG antibodies and cell infiltration and cell leakage without reducing the permeability of the necessary physiologically active substances. Therefore, by adjusting the gel strength or cross-linking density of the hydrogel formed with the porous membrane in multiple layers, in addition to cell infiltration and cell leakage, the infiltration of immune system humoral factors such as IgG antibodies can also be suppressed without reducing the permeability of physiologically active substances.

[0106] Porous membrane (b1), fibrous structure (b2) and hydrogel (b3) form layers separately, and these layers can be clearly separated at their boundaries, or the boundary between two adjacent layers may not be clearly distinct, or two or three different types of layers can be integrated to form one layer. For example, when the immunoisolation layer comprises two types of layers, i.e. fibrous structure and hydrogel, the immunoisolation layer can be an immunoisolation multilayer in which the fibrous structure and the hydrogel are clearly separated, or a mixed layer of the fibrous structure and the hydrogel can be present between the fibrous structure and the hydrogel, or the fibrous structure and the hydrogel can be fully integrated into one layer. For example, when the immunoisolation layer comprises two types of layers, i.e. fibrous structure and porous membrane, the immunoisolation layer can be an immunoisolation multilayer in which the fibrous structure and the porous membrane are clearly separated, or a mixed layer of the fibrous structure and the porous membrane can be present between the fibrous structure and the hydrogel, or the fibrous structure and the porous membrane can be fully integrated into one layer. For example, when the immunoisolation layer comprises two types of layers, i.e., a hydrogel and a porous membrane, the immunoisolation layer may be an immunoisolation multilayer in which the hydrogel and the porous membrane are clearly separated, or a mixed layer of the hydrogel and the porous membrane may exist between the hydrogel and the porous membrane, or the hydrogel and the porous membrane may be completely integrated into one layer.

[0107] The immunoisolation device according to a particularly preferred embodiment of the present invention comprises any one of the following three multilayer structures (i) to (iii):

[0108] (i) A porous film is used as a substrate, and a hydrogel is applied to the porous film or the porous film is impregnated with the hydrogel to form a multilayer structure.

[0109] (ii) A fiber structure is used as a substrate, and a hydrogel is applied to the fiber structure or the fiber structure is impregnated with the hydrogel to form a multilayer structure.

[0110] (iii) Using the fiber structure as a substrate and forming a porous film thereon, and further applying the hydrogel to the porous film or impregnating the porous film with the hydrogel to form a multilayer structure.

[0111] When the immunoisolation membrane (B) comprises a plurality of layers, the layers may be bonded by adhesives, heat, pressure, etc. When adjacent layers are formed of highly compatible materials, the layers may be bonded by sequentially forming the layers.

[0112] Specifically, porous membrane is used as substrate, and hydrosol solution is directly applied to the porous membrane and formed into hydrogel by heat, temperature, light or chemical action to form multilayer. Alternatively, fiber structure is used as substrate, and hydrosol solution is directly applied to the fiber structure and formed into hydrogel by heat, temperature, light or chemical action to form multilayer structure. When using fiber structure coated with hydrogel such as nonwoven to replace porous membrane, an immunoisolation layer with higher material permeability and higher strength than porous membrane can be formed, and hydrogel ensures immunoisolation at the same time.

[0113] The hydrosol solution preferably has a solid concentration of 3 to 15% by mass, more preferably 3 to 10% by mass, still more preferably 3 to 8% by mass, and particularly preferably 3 to 5% by mass. A solid concentration within the above range is preferred because the permeability of immune system fluid factors such as IgG can be suppressed while maintaining the permeability of substances such as glucose and insulin.

[0114] Alternatively, a hydrogel is used as a substrate, and a polymer solution as a raw material of the porous membrane is directly applied to the substrate. The raw material of the porous membrane solidifies by phase separation (which is a phase transition phenomenon), thereby forming a porous membrane. Some raw materials of the porous membrane may need pretreatment to reduce water content by pre-drying the hydrogel.

[0115] In a preferred embodiment of the present invention, a fiber structure with excellent durability is used as a substrate; and a porous polymer film or a hydrogel or both are formed on the fiber structure, thereby achieving both a reduction in the thickness of the device to improve the diffusion efficiency of physiologically active substances and durability due to increased strength while maintaining an immunoisolation effect.

[0116] The thickness of the immunoisolation layer is not particularly limited, but is preferably 10 μm or more and 500 μm or less, more preferably 300 μm or less, even more preferably 200 μm or less, even more preferably 170 μm or less, and particularly preferably 150 μm or less. Even when the immunoisolation layer is an immunoisolation multilayer, the preferred thickness of the immunoisolation layer is within the above range. Considering the mass diffusion efficiency of the physiologically active substance released from the material to be transplanted, the film thickness of the multilayer film is preferably 100 μm or less and as thin as possible.

[0117] The outermost layer of the immunoisolation layer is preferably biocompatible to prevent the immunoisolation layer from being recognized as a foreign body. The immunoisolation layer needs to have a permeability to allow oxygen and nutrients to fully penetrate into the material to be transplanted present inside the device. Since it is necessary to block the invasion of immune active cells into the device, on the outer surface, inner surface or inside of the immunoisolation layer, there is preferably an area with a pore size that blocks the penetration of immune active cells in the entire immunoisolation layer.

[0118] The outermost layer of the immunoisolation layer can be any of a porous membrane, a fiber structure or a hydrogel, or a mixture of two or three thereof. The innermost layer of the immunoisolation layer can be any of a porous membrane, a fiber structure or a hydrogel, or a mixture of two or three thereof. In this specification, the outermost layer of the immunoisolation layer refers to the layer constituting the outside of the immunoisolation device of the present invention in the immunoisolation layer, i.e., the layer contacting the tissue around the transplantation site (host), and the innermost layer of the immunoisolation layer refers to the layer constituting the part (inner part) of the immunoisolation layer contacting the cell aggregates in the immunoisolation device of the present invention.

[0119] When the outermost layer of the immunoisolation layer is a porous membrane, the porous membrane is ideally made of a material that is more biocompatible than the hydrogel, and also serves to prevent the hydrogel from adhering to the recipient's tissue at the transplant site and inducing inflammation.

[0120] When the outermost layer of the immunoisolation layer is a fiber structure, the fiber structure is preferably a material with higher biocompatibility than the hydrogel. Since the outer layer is also used to prevent the hydrogel from adhering to the tissue of the recipient at the transplant site and causing inflammation, etc., it is ideal to smooth the surface of the fiber structure as the outermost layer by heat, mechanical or chemical treatment.

[0121] By modifying the hydrogel to suppress the induction of inflammatory response, it is also possible to use the hydrogel as the outermost layer and the porous membrane as the innermost layer. In this case, it is also possible to load physiologically active substances on the hydrogel and exert functions such as inducing angiogenesis.

[0122] In one embodiment, the innermost layer of the immunoisolation layer is preferably composed of a hydrogel or porous membrane on which a cell adhesion protein and / or cell adhesion peptide is fixed. This configuration enables cell aggregates to adhere to the innermost layer of the immunoisolation layer so that the cell aggregates do not require a scaffold material, thereby reducing the overall thickness of the immunoisolation device.

[0123] When the inner layer is composed of multiple layers, these layers can be made of the same material or different materials. In the present invention, all layers (single layer or multiple layers) including the innermost layer but excluding the outermost layer are collectively referred to as the inner layer. Therefore, in the present invention, a single innermost layer and a layer composed of multiple layers including the innermost layer can be included in the concept of "inner layer". For example, when the multilayer has a four-layer structure, the innermost layer of the multilayer can be interpreted as the "inner layer", or the innermost layer and the second inner layer can be interpreted as the "inner layer", or the three consecutive layers including the innermost layer can be interpreted as the "inner layer". The inner layer can be any one of a porous membrane, a fiber structure and a hydrogel, or can be a mixture of two or three thereof.

[0124] The cell adhesion protein can be, for example, one or more proteins such as gelatin, fibrin, fibronectin, laminin, collagen, retronectin, vitronectin and elastin. The cell adhesion peptide can be, for example, one or more peptides such as RGD peptide, RGDS peptide, GRGD peptide and GRGDS peptide.

[0125] There is no limitation on the method of fixing the cell adhesion protein and / or cell adhesion peptide on the hydrogel or porous membrane. Immobilization can be achieved, for example, by known common methods, such as physical adsorption by applying an aqueous solution of a cell adhesion protein or a cell adhesion peptide. Alternatively, immobilization can also be achieved by using a condensing agent such as a water-soluble carbodiimide to perform active esterification on the functional groups on the surface of the hydrogel, and then covalently bonding the esterified functional groups to the amino groups of the cell adhesion protein and / or cell adhesion peptide.

[0126] The amount of cell adhesion protein and / or cell adhesion peptide immobilized is not particularly limited, but is preferably 0.05 μg / cm 2 More preferably 0.1 μg / cm 2 The immobilized amount of the cell adhesion protein and / or cell adhesion peptide can be measured by immersing a portion of the hydrogel or porous membrane in an excess of PBS (an aqueous solution of a PBS tablet (produced by Takara Bio Inc.) in a predetermined amount of ion-exchanged water) overnight and measuring the immobilized amount using a bicinchoninic acid (BCA) method (BCA Protein Assay Kit (produced by Takara Bio Inc.)).

[0127] Since the immune isolation layer of the present invention has sufficient strength and is stably present in the recipient's body and can inhibit the invasion of immune-competent cells into the device, it can also inhibit the invasion of the transplant material present in the device into the recipient's body. Therefore, even if the transplant material comes from iPS cells that are worried about becoming cancerous, the material is safe to use.

[0128] Material permeability of the immunoisolation layer

[0129] The amount of glucose, insulin, immune system humoral factors, etc. that permeate through the immunoisolation layer can be measured by inserting the immunoisolation layer into the connection point between two glass chambers of the same volume, pouring a sample solution of insulin, etc. of known concentration into chamber a, pouring the same amount of water into chamber b, and quantifying the amount of insulin, etc. contained in the liquid sampled from chamber b by ELISA, etc. at 37° C. under stirring after a period of time ( Figure 8 ). It is important to adjust the liquid amounts in chambers a and b to be equal when pouring the sample solution into chamber a.

[0130] The permeability of glucose, insulin, immune system humoral factors, etc. through the immune isolation layer is expressed as a percentage, which represents the amount of each substance that has penetrated into chamber b as measured by the above method after 20 hours relative to the concentration at equilibrium, that is, half of the concentration of the substance poured into chamber a. More specifically, for example, the permeability can be obtained according to the following formula:

[0131] Permeability of each substance (%) = concentration of each substance in chamber b after 20 hours of measurement / (concentration of each substance in chamber a at the beginning of measurement / 2) × 100

[0132] The insulin and glucose permeability of the immune isolation layer of the present invention is preferably above 50%, more preferably above 90%, and even more preferably above 95%.

[0133] The immune system humoral factor permeability of the immunoisolation layer (B) of the present invention is preferably 30% or less, more preferably 10% or less.

[0134] The material permeability can be controlled by the pore size of the porous membrane or the gel strength and cross-linking degree of the hydrogel. In order to exhibit the immunoisolation function, it is desirable that the pore size of the porous membrane is equal to or smaller than a size that does not allow cell penetration. It is desirable that the hydrogel does not inhibit the penetration of physiologically active substances, but can inhibit the penetration of immune response factors such as cells and antibodies.

[0135] Examples of immune response cells include macrophages, cytotoxic T cells, natural killer cells, dendritic cells, and helper T cells.Examples of humoral factors of the immune system include antibodies, complement, and cytokines.

[0136] Immunoisolation device

[0137] The immunoisolation device according to a preferred embodiment of the present invention is in the form of a bag, a tube, a cylinder, a rectangular tube, a sphere, a cube, a cuboid, a sheet or a hollow fiber. The cell aggregates are encapsulated in the immunoisolation device. In addition to the cell aggregates, physiologically active substances such as enzymes, hormones, cytokines and drugs can also be encapsulated.

[0138] The immunoisolation device can be produced in the following manner. After the immunoisolation layer is arranged around the cell aggregate so that the cell aggregate is covered by the immunoisolation layer, the peripheral portion is sealed by heat fusion to form the device into a pouch-like shape. Alternatively, an opening is provided in a device prepared in advance using the immunoisolation layer and after the cell aggregate is inserted from the opening, the opening is closed to thereby block the invasion of immune response cells and immune system humoral factors from the opening. Since oxygen, nutrients, etc. can penetrate the immunoisolation layer in the portion outside the opening, the opening can be closed to suppress the penetration of substances including nutrients.

[0139] Alternatively, the immunoisolation device can be produced by allowing the cell aggregate to adhere to the innermost side of the immunoisolation layer, then arranging the immunoisolation layer so that the cell aggregate is present inside the device, and then sealing by heat fusion. In heat fusion, the resin can be sandwiched between the immunoisolation layers, and then heat fusion can be performed. By performing heat fusion in a state where the resin is sandwiched, heat fusion can be easily performed even when the innermost layers of the upper and lower immunoisolation layers in the immunoisolation device are both hydrogels (when the hydrogels of the upper and lower immunoisolation layers face each other).

[0140] After the immunoisolation device of the present invention is implanted in a living body, the transplant material whose function has been reduced can be removed, and new functional material to be transplanted can be introduced. This operation can be repeated. The immunoisolation device can be repeatedly used to introduce the material to be transplanted. The immunoisolation device can be removed together with the transplant material.

[0141] The immunoisolation device according to a preferred embodiment of the present invention preferably has shape retention to provide sufficient strength in vivo.

[0142] In order to maintain functionality as the device, the fiber structure, porous membrane, and hydrogel are preferably materials excellent in safety and biocompatibility.

[0143] In order to prevent adhesion with surrounding tissues and prevent fibrosis, the immunoisolation layer is ideally composed of materials with excellent biocompatibility. When the immunoisolation membrane is composed of various materials, it is desirable that the material with excellent biocompatibility is arranged on the transplant side, that is, on the contact surface of the outermost layer in contact with the transplant site in the recipient. The example of the material with excellent biocompatibility is ethylene-vinyl alcohol copolymer. As ethylene-vinyl alcohol copolymer, those described in the explanation of porous membrane can be preferably used.

[0144] Preferred embodiments of the present invention will be described below with reference to the accompanying drawings.

[0145] Figure 1 is a schematic diagram of a device made by molding an immunoisolation layer into a bag shape. Figure 2 Schematic diagram of a device made by molding an immunoisolation layer into a tubular shape. A bag-shaped device ( Figure 1 ). A spacer may be provided to ensure a certain distance (a4).

[0146] Tubular device ( Figure 2 ) is composed of immunoisolation layers (b1, b2 and b3) formed into a tubular shape. The cell aggregate is encapsulated inside the tubular portion (b4) and heat, ultrasound, high frequency, electron beam, etc. are applied to fuse and seal both ends of the tubular layer to form a tubular device.

[0147] like Figure 1 As shown in or 2, in the conceptual diagram, the device is in the shape of a bag or a tube, and the material to be transplanted, such as cells or cell clusters, is encapsulated in the device as a cell aggregate.

[0148] Figure 3 This is a conceptual diagram of a cell sheet. The cell sheet is composed of cells (11) as the material to be transplanted and an extracellular matrix (10).

[0149] Figure 4 is a conceptual diagram of an immunoisolation device including a cell sheet encapsulated therein. is a conceptual diagram showing that a cell sheet (14) prepared using cells or cell clusters to be transplanted is inserted into the device. The immunoisolation device is formed into a bag shape by using an immunoisolation multilayer in which the outermost layer is a porous membrane (12) and the innermost layer is a hydrogel (13). The porous membrane is preferably formed of ethylene-vinyl alcohol copolymer or the like. The innermost layer of the hydrogel is preferably polyvinyl alcohol or the like.

[0150] Figure 5 and 6The immunoisolation layer shown in the figure is composed of a combination of the following materials. The outermost layer is to be in contact with the recipient's tissue at the transplantation site, and the innermost layer is to be in contact with the cell aggregate. Such an immunoisolation layer is formed into a bag shape ( Figure 1 ) or tubular ( Figure 2 ), and encapsulate a cell aggregate having cells or cell clusters fixed therein as materials to be transplanted, thereby making an immunoisolation device.

[0151] Figure 5 An immunoisolation multilayer consisting of a porous membrane (15) and a hydrogel (17) is shown. The hydrogel (17) is applied to the porous membrane (15), or the porous membrane (15) is impregnated with the hydrogel (17) to form a multilayer (16) wherein the outermost surface (18) consists of the porous membrane and the innermost surface (19) consists of the hydrogel.

[0152] Figure 6 An immunoisolation multilayer consisting of a fiber structure (20) and a hydrogel (21) is shown. The hydrogel (21) is applied to the fiber structure (20), or the fiber structure (20) is impregnated with the hydrogel (21) to form a multilayer, wherein the outermost surface (22) consists of the fiber structure and the innermost surface (23) consists of the hydrogel.

[0153] Example

[0154] The present invention is described in more detail below based on examples.

[0155] Production of immunoisolated multilayers

[0156] Production Example 1

[0157] A porous film formed using an ethylene-vinyl alcohol copolymer (hereinafter referred to as EVOH) and a hydrogel containing a methacryloyl-modified polyvinyl alcohol (hereinafter referred to as MA-PVA) as a main component were laminated to form a multilayer by the following procedure.

[0158] (1) A porous film (average pore diameter: 1.8 μm, maximum pore diameter: 3.4 μm, thickness: 100 μm) was produced by subjecting EVOH (EVAL (registered trademark), F101A, produced by Kuraray Co., Ltd.) to a polymer phase separation reaction.

[0159] (2) Phenyl (2,4,6-trimethylbenzoyl) lithium phosphinate was added as a water-soluble photoradical polymerization initiator to a 10 mass % aqueous solution of MA-PVA (average degree of polymerization: 1700, degree of saponification: 98.0 to 99.0 mol %, methacryloyl modification rate: 1.2 mol %) to achieve a concentration of 0.1 mass %, thereby preparing a sol.

[0160] (3) This sol was applied to a PET film to a thickness of 50 μm using a bar coater.

[0161] (4) A porous film is placed on the sol, and the sol and the porous film are closely adhered to each other using a laminator.

[0162] (5) Then put the sol surface on top and use 365nm light at 15mW / cm 2 Irradiation was performed at an intensity of 1000 nm for 3 minutes to form a hydrogel on the porous membrane, thereby producing an immunoisolation multilayer. Figure 7 SEM images of a cross section of the resulting immunoisolation multilayer are shown. The hydrogel was found to have penetrated the porous membrane to a depth of approximately 3 to 6 μm.

[0163] Production Example 2

[0164] Using 4 mass % of MA-PVA (average degree of polymerization: 3500, degree of saponification: 87.0 to 89.0 mol %, methacryloyl modification rate: 1.2 mol %) aqueous solution, a hydrogel was formed on a porous membrane in the same manner as in Production Example 1 to prepare an immunoisolation multilayer.

[0165] Material permeability test

[0166] The permeabilities of glucose, insulin and IgG of the porous membrane prepared in Production Example 1 and the immunoisolation multilayer obtained in Production Example 1 or 2 were measured in the following manner.

[0167] (1) Place a porous membrane or an immunoisolation multilayer between chambers a and b respectively ( Figure 8 ). An aqueous solution (65 mL) containing insulin (30 U / L), glucose (5 mg / mL) and IgG (0.5 μg / mL) was prepared in chamber a, while water (65 mL) was placed in chamber b.

[0168] (2) Stirring was performed with a stirrer at a constant temperature of 37° C. After 20 hours, the liquid was sampled from chamber b, and changes in the concentrations of insulin, glucose, and IgG were measured by ELISA.

[0169] (3) The permeability of each substance was calculated from the results of (2) (Table 1).

[0170] Permeability of each substance (%) = concentration of each substance in chamber b after 20 hours of measurement / (concentration of each substance in chamber a at the beginning of measurement / 2) × 100

[0171] Table 1

[0172]

[0173] Immobilization of cell adhesion proteins to hydrogels

[0174] Production Example 3

[0175] Collagen as a cell adhesion protein was immobilized (covalently bonded) on the hydrogel surface of the immunoisolation multilayer by the following procedure.

[0176] (1) Twenty immunoisolation multilayers (size: 24 mm×32 mm) having a MA-PVA hydrogel formed on one side of an EVOH porous membrane were prepared by the method described in Production Example 2.

[0177] (2) 20 mL of MES (2-morpholineethanesulfonic acid) buffer was placed in a petri dish, and the immunoisolation multilayer prepared in (1) was immersed in the buffer and shaken at 100 rpm for 30 minutes.

[0178] (3) 10 mL of MES buffer was placed in a separately prepared beaker, and 100 mg of N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Corporation, hereinafter referred to as NHS) and 170 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (manufactured by Dojindo Laboratories, Ltd., hereinafter referred to as WSC) were dissolved in the buffer to prepare an NHS / WSC solution.

[0179] (4) After removing the MES buffer from the Petri dish used in (2), 5 mL of the NHS / WSC solution was added to the Petri dish and shaken at 100 rpm for 60 minutes at room temperature.

[0180] (5) Remove the NHS / WSC solution from the Petri dish and immerse the immunoisolated multilayer in 20 mL of MES buffer and wash with shaking at room temperature.

[0181] (6) Next, a collagen solution (Cellmatrix (registered trademark) IC type, Nitta Gelatin Inc.) was diluted with MES buffer to a concentration of 0.2 mg / mL. The immunoisolated multilayer was immersed in 20 mL of this solution and shaken at 100 rpm for 20 hours at room temperature.

[0182] (7) Then, the immunoisolation multilayer was taken out, immersed in 20 mL of distilled water, and washed by shaking at 100 rpm for 5 minutes at room temperature.

[0183] (8) A release PET film (Cosmopeel (registered trademark) Model: E7004, manufactured by Toyobo Co., Ltd.) was placed on a glass plate. An immunoisolation multilayer was placed on the film, and a release PET film and a glass plate were further placed thereon. The resultant was dried at room temperature under reduced pressure for 20 hours to obtain an immunoisolation multilayer having collagen immobilized on the surface of the hydrogel.

[0184] (9) A portion of the hydrogel was immersed overnight in an excess of PBS (an aqueous solution of a PBS tablet (produced by Takara Bio Inc.) in a predetermined amount of ion-exchanged water). The amount of immobilized collagen was measured by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (produced by Takara Bio Inc.)) and found to be 0.5 μg / cm 2 .

[0185] Cell sheet production

[0186] Production Example 4

[0187] A co-culture cell sheet (circular, diameter: 8 mm, thickness: approximately 100 μm) of a rat pancreatic β cell line capable of secreting insulin-Gaussia luciferase (iGL cells, produced by Cosmo Bio Co., Ltd.) and human adipose-derived mesenchymal stem cells (hASC, produced by Lonza KK) was produced by the following procedure.

[0188] (1) 45×10 4 iGL cells and 40×10 4 hASCs were seeded on a temperature-responsive culture dish (12-well multiwell "UpCell" (registered trademark), manufactured by CellSeed Co., Ltd.) and cultured in a 37°C incubator for 24 hours.

[0189] (2) The temperature-responsive culture dish was then taken out of the incubator and allowed to stand at room temperature (approximately 20° C.) for 10 minutes to peel off the cells attached to and spread on the bottom of the culture dish, thereby obtaining a co-cultured cell sheet.

[0190] iGL cells are cells whose parent cell line is the rat pancreatic β cell line INS-1E and into which the gene of the fusion protein of insulin and secretory Gaussia luciferase is inserted. iGL cells react with the luminescent substrate coelenterazine (CTZ) and emit light. Using this luminescence intensity, the insulin release capacity can be measured.

[0191] Production of immunoisolation devices: Cell sheets encapsulated in immunoisolation layers

[0192] Example 1

[0193] A collagen sponge (Pelanc (registered trademark), product number: PN-S82060, manufactured by Gunze Limited) was cut into 1 cm squares. The cell sheet prepared in Production Example 4 was placed in the center of the cut collagen sponge. The collagen sponge was covered in such a manner that the sponge was sandwiched between the immunoisolation multilayers prepared in Production Example 2. The peripheral portion of the immunoisolation multilayer was processed into a bag shape by heat sealing, thereby manufacturing a strip-shaped cell sheet encapsulation type device with a size of 2 cm x 3 cm.

[0194] In the above method, the cell sheet is sandwiched between the immunoisolation multilayers so that on the two sides of each immunoisolation multilayer, namely the MA-PVA hydrogel side and the EVOH porous membrane side, the MA-PVA hydrogel of each immunoisolation multilayer faces the cell sheet (so that the EVOH porous membrane is exposed to the surface of the device). Fig. 9 A conceptual diagram showing the device. Fig. 9 The dashed lines in FIG. 1 indicate that a portion of the device is omitted from the figure.

[0195] Immune isolation function test

[0196] The cell sheet encapsulated device prepared in Example 1 was transplanted into SD rats (wild rats with immune function), and the immunoisolation performance of the device was confirmed based on cell engraftment and viability.

[0197] 1) Transplantation of immunoisolation device

[0198] The rat was subjected to laparotomy, and the cell sheet encapsulating type device was inserted between the liver lobes and fixed with fibrin glue (product name: Bolheal Tissue Sealant, manufactured by KM Biologics).

[0199] 2) Functional assessment

[0200] 2-1) In vitro insulin release capacity

[0201] On the 10th day after transplantation, the device was removed, and then the cell sheet was removed. The cell sheet was immersed in a 20 mM glucose solution for 2 hours, and the supernatant was collected. The insulin release capacity of iGL cells was compared based on the fluorescence intensity of the cell sheet supernatant.

[0202] like Fig.10 As shown in , the results demonstrated that the cell sheet supernatant had a luminescence intensity of iGL cells that was significantly correlated with the insulin-releasing capacity.

[0203] 2-2) Histopathological evaluation

[0204] On day 10 after transplantation, tissue sections of each device were prepared and subjected to HE staining and fluorescent immunostaining using an insulin antibody. Fig.11 and 12 Results of histopathological evaluation are shown.

[0205] In the group implanted with the device prepared in Example 1, the survival of insulin-producing cells was confirmed, and the absence of inflammatory cell infiltration was confirmed.

[0206] The above results confirmed that the device has immune isolation effect.

[0207] Example 2

[0208] The cell sheet prepared in Production Example 4 is placed on the surface of the collagen-immobilized hydrogel of the immunoisolation multilayer prepared in Production Example 3 and placed in a 60 mm dish. 5 mL of culture medium ("DMEM / F12 GlutaMAX" (registered trademark), produced by Thermo Fisher Scientific, Inc.) is injected into the dish. The 60 mm dish is placed in an incubator at 37°C. After 24 hours, the dish is taken out from the incubator and visually observed. It is found that the cell sheet has adhered to the surface of the immunoisolation multilayer.

[0209] In addition, cells were labeled with a red fluorescent reagent (Cell Explorer (registered trademark), Live Cell Tracking Orange Fluorescence, produced by AAT Bioquest, Inc.) and observed under a stereofluorescence microscope. It was found that cells around the cell sheet had adhered to the surface of the immunoisolation multilayer and extended. The results clearly show that the use of the collagen-immobilized immunoisolation multilayer enables the cell sheet to adhere directly to the immunoisolation multilayer without using a collagen sponge, thereby making the layer of the device thinner.

[0210] Industrial Applicability

[0211] The present invention relates to a transplantation device for cell transplantation therapy, and more particularly to an immunoisolation device for protecting materials to be transplanted from immune rejection.

[0212] The immunoisolation device is considered to be mainly used as a regenerative medicine product for cell transplantation therapy. However, the immunoisolation device is also suitable for the transplantation of non-cellular physiologically active substances such as enzymes, hormones and drugs.

[0213] Explanation of Reference Symbols

[0214] a1 Immune isolation layer

[0215] a2 Immune isolation layer

[0216] a3 Fusion

[0217] A4 Certain distance

[0218] b1 Immune isolation layer

[0219] b2 Immune isolation layer

[0220] b3 Immune isolation layer

[0221] b4 Tubular interior

[0222] 10 Extracellular Matrix

[0223] 11 Cells

[0224] 12 porous membrane

[0225] 13 Hydrogel

[0226] 14 Cell Sheet

[0227] 15 porous membrane

[0228] 16 Impregnation and Fixation

[0229] 17 Hydrogel

[0230] 18 Outermost surface

[0231] 19 Innermost surface

[0232] 20 Fiber structure

[0233] 21 Hydrogel

[0234] 22 Outermost surface

[0235] 23 Innermost surface

Claims

1. An immunoisolation device comprising a sheet-like cell aggregate and an immunoisolation layer, The sheet-like cell aggregates contain cells and an extracellular matrix, and The immunoisolation layer covers the cell aggregates.

2. The immunoisolation device according to claim 1, wherein the cell aggregate is a cell sheet comprising cells and an extracellular matrix.

3. The immunoisolation device according to claim 1 or 2, wherein the cell aggregates have a thickness of 300 μm or less.

4. The immunoisolation device according to any one of claims 1 to 3, wherein The immunoisolation layer comprises a porous membrane or a fiber structure, and The porous film or the fiber structure includes at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate. 5 . The immunoisolation device according to claim 4 , wherein the immunoisolation layer is an immunoisolation multilayer comprising the porous membrane or the fiber structure and a hydrogel.

6. The immunoisolation device according to claim 5, wherein The outermost layer of the immunoisolation multilayer is the porous membrane or the fiber structure, and The innermost layer of the immunoisolation multilayer is the hydrogel.

7. The immunoisolation device according to claim 5, wherein The outermost layer of the immunoisolation multilayer is the hydrogel, and The innermost layer of the immunoisolation multilayer is the porous membrane or the fiber structure.

8. The immunoisolation device according to any one of claims 1 to 7, wherein The immunoisolation layer comprises a plurality of layers, and The innermost layer of the immune isolation layer is a hydrogel or a porous membrane fixed with cell adhesion proteins and / or cell adhesion peptides.

9. The immunoisolation device according to any one of claims 5 to 8, wherein the hydrogel comprises at least one member selected from polyvinyl alcohol and polyethylene glycol.

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