Immunoisolation device

Abstract

The problem to be solved by the present invention is to provide: an immunoisolation device which can achieve both of the reduction in diffusion distance effective for the improvement in permeability of a substance such as a physiologically active substance and a nutrient and the improvement in durability for withstanding transplantation for a long period of time; and an immunological control technology using a cell sheet. The present invention provides an immunoisolation device provided with a sheet-like cell mass containing cells and an extracellular matrix and an immunoisolation layer that covers the cell mass.

Description

Immunoisolation Device

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Japanese Patent Application No. 2022-075135, filed on April 28, 2022, the entire disclosure of which is incorporated herein by reference. The present invention relates to an immunoisolation device.

[0002] Immunoisolation devices have been developed as a means of cell transplantation therapy without the need for immunosuppressants. Macroencapsulated immunoisolation devices are considered effective in the case of iPS cell-derived somatic cell transplantation, which is particularly susceptible to cancer risk, or when the transplanted cells are functionally impaired, because they allow for specific transplantation sites and device replacement. Required functions of a macroencapsulated immunoisolation device include uniformly dispersing and immobilizing cells or cell clusters without aggregation, readily allowing oxygen and / or nutrients to be transmitted to the transplanted cells, readily releasing physiologically active substances (e.g., cytokines, hormones, growth factors) required for therapeutic effects in response to cellular responses, and being impermeable to immune response cells and immune response factors. Furthermore, the implanted device must be highly biocompatible and unlikely to induce adhesions or inflammatory reactions, such as granulation, with surrounding tissues.

[0003] Special Publication No. 2012-508584 Patent No. 5717253

[0004] Many immunoisolation devices using porous membranes have been investigated to date (Patent Document 1), but issues have been identified, such as protein adsorption to the porous membrane material, reduced permeability due to fibrosis, and reduced permeability due to adhesion to surrounding tissues. Internal necrosis of cell aggregates and other particles is believed to occur when the diameter exceeds 500 μm. To facilitate the maintenance of engraftment of embedded cells into the recipient and the release of physiologically active substances, the overall thickness of the device must be appropriately controlled, and it is desirable for the immunoisolation layer constituting the device to be as thin as possible. Meanwhile, when considering implantation into the body, improved membrane strength and durability are also required to prevent tearing, twisting, and other damage to the device. However, achieving both thinness and improved durability is not easy.

[0005] Furthermore, by culturing cells for transplantation using a temperature-responsive culture dish, it is possible to produce a sheet of cells containing extracellular matrix, adhesion factors, etc., and myocardial sheets, in which skeletal muscle cell sheets are attached to hospital beds as "cell sheets," have been put into practical use as regenerative medicine products. Patent Document 2 also discloses a method for producing and using cell sheets for pancreatic islet cells. Advantages of cell sheets include the fact that they contain extracellular matrix, cell adhesion factors, etc., and do not excessively form cell clusters due to cell aggregation, resulting in high cell engraftment upon transplantation.

[0006] However, at present, this cell sheet transplantation method is limited to practical use with autologous cells, and when allogeneic cells are used, the cell sheet dies without engrafting into the host due to an immune rejection reaction, making the administration of immunosuppressants essential. However, since HLA homology between the donor and host is essential, there are many challenges to overcome before it can be widely adopted and commercialized as a regenerative medicine technology, and as with other cell transplantation therapies, the development of immunoregulatory technologies is necessary. Patent Document 2 also discloses examples of transplantation into allogeneic rats and immunodeficient mice, but does not use immunoregulatory technologies.

[0007] One object of the present invention is to provide an immunoisolation device that achieves both a reduced diffusion distance, which is effective in improving the permeability of substances such as physiologically active substances and nutrients, and improved durability that allows it to withstand long-term implantation. Another object of the present invention is to provide an immunoregulation technology using cell sheets.

[0008] The present invention provides the following immunoisolation devices: [1] An immunoisolation device having a sheet-like cell aggregate containing cells and an extracellular matrix, and an immunoisolation layer covering the cell aggregate. [2] The immunoisolation device of [1], wherein the cell aggregate is a cell sheet composed of cells and an extracellular matrix. [3] The immunoisolation device of [1] or [2], wherein the cell aggregate has a thickness of 300 μm or less. [4] The immunoisolation device of any of [1] to [3], wherein the immunoisolation layer comprises a porous membrane or a fiber structure, and the porous membrane or the fiber structure comprises at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate. [5] The immunoisolation device of [4], wherein the immunoisolation layer is a multilayered immunoisolation layer comprising the porous membrane or fiber structure and a hydrogel. [6] The immunoisolation device of [5], wherein the outermost layer of the multilayered immunoisolation layer is the porous membrane or fiber structure, and the innermost layer is the hydrogel. [7] The immunoisolation device according to [5], wherein the outermost layer of the multilayered immunoisolation layer is the hydrogel and the innermost layer is the porous membrane or fiber structure. [8] The immunoisolation device according to any one of [1] to [7], wherein the immunoisolation layer is composed of multiple layers, and the innermost layer of the immunoisolation layer is a hydrogel or porous membrane to which a cell adhesion protein and / or a cell adhesion peptide is immobilized. [9] The immunoisolation device according to any one of [5] to [8], wherein the hydrogel comprises at least one selected from the group consisting of polyvinyl alcohol and polyethylene glycol.

[0009] According to the present invention, it is possible to provide an immunoisolation device that achieves both a reduction in diffusion distance, which is effective in improving the permeability of substances such as physiologically active substances and nutrients, and improved durability that can withstand long-term implantation. Furthermore, the present invention can provide an immune control technology using cell sheets.

[0010] Fig. 12A: Histological evaluation results in Example 1. Fig. 12B: Histological evaluation results in Example 1. Fig. 12C: Histological evaluation results in Example 1. Fig. 12D: Histological evaluation results in Example 1. Fig. 12E: Histological evaluation results in Example 1. Fig. 12F: Histological evaluation results in Example 1. Fig. 12F: Histological evaluation results in Example 1. Fig. 12G: Histological evaluation results in Example 1. Fig. 12H ... Figures 12B to 12D: Fluorescent staining images (Figure 12B: original drawing. Figure 12C: original drawing (Figure 12B) with insulin-positive areas (anti-insulin (pancreatic islet β cells) displayed in black. Figure 12D: original drawing (Figure 12B) with cell nuclei (Hoechest dye areas) displayed in black). As shown in Figures 12B to 12D, cell nuclei were observed in the insulin-positive areas in the fluorescent staining images.

[0011] As used herein, the singular forms "a," "an," "the," etc. are intended to include both the singular and the plural unless otherwise expressly stated herein or clearly contradicted by context.

[0012] The immunoisolation device of the present invention comprises a sheet-like cell aggregate containing cells and an extracellular matrix, and an immunoisolation layer, and the cell aggregate is covered with the immunoisolation layer, thereby suppressing the infiltration of immune cells and cytokines into the cell aggregate. Furthermore, the immunoisolation layer is preferably a multi-layered immunoisolation layer containing a porous membrane or a fibrous structure and a hydrogel.

[0013] <Cell aggregate> The cell aggregate used in the immunoisolation device of the present invention is a sheet-shaped cell aggregate containing cells and an extracellular matrix. Examples of the cell aggregate include a complex of cells and an extracellular matrix, a sheet in which cells are immobilized on a scaffold material such as a collagen sheet or collagen sponge, or a cell sheet produced using a temperature-responsive culture dish. Examples of the cell aggregate include those that do not contain a scaffold material such as a collagen sheet. The cells may be in the form of a cell mass.

[0014] 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 by intercellular junctions and form a single-layer or multi-layer (preferably single-layer) sheet. The cell sheet is composed of cells and an extracellular matrix, and maintains adhesion between the cells. In a preferred embodiment, as shown in FIG. 3, the cell sheet has a structure in which sheet-shaped cells (11) are layered on a sheet-shaped extracellular matrix (10). In this embodiment, the cells are preferably arranged in a single layer on the extracellular matrix.

[0015] A cell sheet can be obtained by culturing cells on a stimuli-responsive culture substrate coated with a polymer whose molecular structure changes in response to stimuli such as temperature, pH, or light, and then varying the surface of the stimuli-responsive culture substrate by changing the stimuli such as temperature, pH, or light. An example of a stimuli-responsive culture substrate is UpCell (registered trademark), a temperature-responsive culture dish commercially available from CellSeed Inc. In a preferred embodiment, an extracellular matrix is ​​formed during the cell culture process, and the cells are maintained in an adherent state.

[0016] A single cell sheet may be used as a cell aggregate, or two or three sheets may be stacked together to form a cell aggregate.

[0017] When a cell sheet is used as the cell aggregate, it may be layered on a scaffold material such as collagen sponge to improve operability, but a scaffold material need not be used. When a cell sheet is used as the cell aggregate, a scaffold material for holding the cells is not required, and an immunoisolation device can be produced without intentionally adding a scaffold material. Therefore, in one embodiment, the present invention provides the immunoisolation device, in which the cell aggregate is a cell sheet composed of cells and extracellular matrix, without intentionally adding a scaffold material.

[0018] The thickness of the cell aggregate is not particularly limited, but is preferably 10 μm or more, more preferably 20 μm or more. It is also preferably 300 μm or less, more preferably 290 μm or less, and even more preferably 150 μm or less. A thickness within the above range is preferable because the supply of oxygen and the like to the cells is less likely to be inhibited.

[0019] The animal species from which the cells used in the cell aggregates are derived may include, for example, mammals such as humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, or immunodeficient animals thereof, as well as birds, reptiles, amphibians, fish, and insects. When the immunoisolation device of the present invention is used for human treatment, it is preferable to use cells derived from humans. Furthermore, when the immunoisolation device is used for human treatment, the cells may be collected from the patient himself / herself, cells collected from other people, or commercially available cell lines.

[0020] Examples of cells used in cell aggregates include somatic cells that make up a living organism (cardiomyocytes, hepatic parenchymal cells, 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.), progenitor cells, cells that have been isolated from a living organism and acquired immortalization ability and are stably maintained outside the body, cells that have been isolated from a living organism and artificially genetically modified, and cells that have been isolated from a living organism and whose nuclei have been artificially exchanged.

[0021] In one preferred embodiment, the cells used in the cell aggregates include those that release physiologically active substances to the outside of the immunoisolation device, such as mesenchymal stem cells, pancreatic islet cells, pancreatic islet-like insulin-producing cells, pituitary hormone-producing cells, and lysosomal enzyme-producing cells.

[0022] The cell aggregate may also be composed of multiple types of cells listed above.

[0023] <Immunoisolation Layer> The immunoisolation device of the present invention includes an immunoisolation layer that covers the cell aggregates. In a typical embodiment, from the viewpoint of obtaining a sufficient immunoisolation effect, it is preferable that the immunoisolation layer in the device of the present invention covers the entire surface of the cell aggregates. For the same purpose, it is preferable that the immunoisolation layer does not have any holes such as pinholes that penetrate the membrane.

[0024] The immunoisolation layer preferably includes a porous membrane or a fiber structure, and more preferably is a multi-layer immunoisolation layer including a porous membrane or a fiber structure and a hydrogel. The immunoisolation layer may be a multi-layer immunoisolation layer including a porous membrane and a fiber structure, or may be a multi-layer immunoisolation layer including a porous membrane, a fiber structure, and a hydrogel.

[0025] (Porous membrane) The porous membrane constituting the immunoisolation layer is a membrane having a plurality of pores. The porous membrane can be confirmed by a scanning electron microscope (SEM) image or a transmission electron microscope (TEM) image of the membrane cross section. The porous membrane is preferably a semipermeable membrane.

[0026] 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 preferable because it maintains the strength of the immunoisolation layer while preventing the supply of oxygen and the like to cells from being inhibited.

[0027] The average pore size of the porous membrane 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 SEM images or TEM images. For example, the surface of the porous membrane is observed using an SEM, and 50 pores formed on the surface are arbitrarily selected. The major axis of each pore is measured, and the average value of the major axes of the 50 pores is calculated to determine the average pore size.

[0028] The maximum pore size of the porous membrane 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 μm to 4 μm. A maximum pore size within the above range can inhibit the intrusion of immune response cells into the device while allowing sufficient permeation of nutrients such as amino acids, vitamins, inorganic salts, and carbon sources such as glucose, as well as physiologically active substances such as oxygen, carbon dioxide, cytokines, hormones, and insulin. The maximum pore size can be determined from SEM or TEM images. For example, the surface of the porous membrane is observed using an SEM, and 50 pores formed on the surface are randomly selected. The major axis of each pore is measured, and the largest of the 50 major axes is taken as the maximum pore size.

[0029] The porous membrane must have the function of suppressing cell infiltration from the recipient and preventing leakage of transplanted cells, and therefore the average or maximum pore size is preferably 5 μm or less, which is smaller than the cell size.

[0030] The porous membrane preferably contains a polymer and is substantially composed of a polymer. Examples of polymers include thermoplastic or thermosetting polymers. The polymer may be biocompatible. Specific examples of polymers include ethylene-vinyl alcohol copolymer, polysulfone, cellulose acetate such as 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, polyamideimide, polybenzimidazole, polytetrafluoroethylene (PTFE), and the like. These may be homopolymers, copolymers, polymer blends, polymer alloys, and the like, from the viewpoints of solubility, optical properties, electrical properties, strength, elasticity, and the like. The polymer constituting the porous membrane may include a hydrophilic polymer such as polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, or polyethylene glycol. Combining hydrophilic and hydrophobic polymers can improve biocompatibility.

[0031] 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 typical 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 may also be used.

[0032] The ethylene unit content in the ethylene-vinyl alcohol copolymer is preferably 20 mol% or more, more preferably 25 mol% or more, and is preferably 60 mol% or less, more preferably 55 mol% or less, and even more preferably 50 mol% or less.

[0033] 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. 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 is １ It can be calculated by carrying out H-NMR measurement and measuring the peak area of ​​the hydrogen atoms contained in the vinyl ester structure and the peak area of ​​the hydrogen atoms contained in the vinyl alcohol structure.

[0034] Furthermore, the ethylene-vinyl alcohol copolymer may contain units derived from other monomers other than ethylene, vinyl esters, and saponified products thereof, as long as the object of the present invention is not impaired. When the ethylene-vinyl alcohol copolymer contains other monomer units, the content of the other monomer units relative to the total monomer units of the ethylene-vinyl alcohol copolymer 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. When the ethylene-vinyl alcohol copolymer contains units derived from the other monomers, the lower limit may be 0.05 mol% or 0.10 mol%. Examples of other monomers include alkenes such as propylene, butylene, pentene, and hexene; 3-acyloxy-1-propene, 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, and 4-acyloxy-3-methyl-1-butene. -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, 1,3-diacetoxy-2-methylenepentene unsaturated acids such as acrylic acid, methacrylic acid, crotonic acid, itaconic acid, or the like, or their anhydrides, salts, or mono- or dialkyl esters; nitriles such as acrylonitrile and methacrylonitrile; amides such as acrylamide and methacrylamide; olefin sulfonic acids such as vinyl sulfonic acid, allyl sulfonic acid, and methallylsulfonic acid, and salts thereof; vinyl silane compounds such as vinyltrimethoxysilane, vinyltriethoxysilane, vinyltri(β-methoxyethoxy)silane, and γ-methacryloxypropylmethoxysilane; alkyl vinyl ethers, vinyl ketone, N-vinylpyrrolidone, vinyl chloride, and vinylidene chloride.

[0035] The ethylene-vinyl alcohol copolymer may be post-modified by urethanization, acetalization, cyanoethylation, oxyalkylenation, or the like.

[0036] The ethylene vinyl alcohol copolymer may be used alone or in combination of two or more.

[0037] The polymer forming the porous membrane is preferably a material with excellent biocompatibility that is unlikely to cause adhesion to the recipient's surrounding tissues, inflammation, etc. The porous membrane preferably contains at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate.

[0038] The porous membrane may be composed of one type of porous membrane, or may be a laminate of two or more types of porous membranes. When the porous membrane includes two or more porous membranes, the porous membranes may be directly laminated, or a hydrogel or a fibrous structure may be interposed between the two porous membranes.

[0039] In one preferred embodiment, the porous film is a film formed from one composition as a single layer, and is not a laminated structure of multiple layers.

[0040] (Fiber Structure) Examples of the fiber structure constituting the immunoisolation layer include nonwoven fabrics, woven fabrics, and knitted fabrics, with nonwoven fabrics being preferred. The fiber structure is formed by bonding or entangling fibers by thermal, mechanical, or chemical action. The fiber diameter and / or amount can be adjusted to adjust the basis weight (weight per unit area), which in turn makes it possible to control the strength as well as the permeability and / or filtration properties. The basis weight of the fiber structure is 10 to 100 g / m 2 The thickness of the fiber structure is preferably 300 μm or less, and more preferably as thin as possible, such as 200 μm or less, in consideration of the diffusion efficiency of the physiologically active substance from the recipient.

[0041] Examples of fiber materials for the fiber structure include 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 modified with a monomer such as methacrylic-modified PVA or acrylic-modified PVA, polycaprolactone, polyglycerol sebacic acid, polyhydroxyalkanoic acid, polybutylene succinate, polymerylene carbonate, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzyl cellulose, and carboxymethylcellulose, as well as cellulose acetates such as fibroin and silk. As with the porous membrane, the fiber material for the fiber structure is preferably biocompatible. It is also preferable to include at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate. It is also preferable to smooth the surface of the fiber structure by thermal, mechanical, or chemical treatment. When an ethylene vinyl alcohol copolymer is used, the one described in the description of the porous membrane can be preferably used.

[0042] (Hydrogels) Examples of hydrosols for producing the hydrogel constituting the immunoisolation layer include sols that gel in the presence of metal ions to form a hydrogel, sols that gel in response to temperature to form a hydrogel, sols that gel in response to pH to form a hydrogel, and sols that form a hydrogel in response to light. Metal ions and pH are examples of chemical interactions. To gel these hydrosols, operations such as contacting them with metal ions, adjusting the temperature to gelation conditions, adjusting the pH to gelation conditions, irradiating them with light that satisfies gelation conditions, or applying a magnetic field that satisfies gelation conditions may be performed depending on the properties of the gel used.

[0043] Examples of hydrogels that gel in the presence of metal ions include alginate gels that gel in the presence of divalent or trivalent metal ions, preferably alkaline earth metal ions such as calcium ions and magnesium ions; carrageenan gels that gel in the presence of calcium ions and / or potassium ions; and acrylic acid-based synthetic gels that gel in the presence of sodium ions.

[0044] Examples of temperature-responsive hydrogels include temperature-responsive hydrogels in which poly(N-isopropylacrylamide) is crosslinked with 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, and polyvinyl alcohol.

[0045] Examples of pH-responsive hydrogels include alginate gel, chitosan gel, carboxymethyl cellulose gel, and acrylic acid-based synthetic gel.

[0046] Examples of photoresponsive hydrogels include synthetic gels that combine azobenzene and cyclodextrin in the skeleton, gels made of supramolecules with fumaric acid amide as a spacer, gels that are crosslinked or bonded via nitrobenzyl groups, and gels made of modified polyvinyl alcohol.

[0047] The modified polyvinyl alcohol may be, for example, a (meth)acryloyl group-modified polyvinyl alcohol. The (meth)acryloyl group can be introduced by esterifying or transesterifying an ethylenically unsaturated group-containing compound with a hydroxyl group in the side chain of the polyvinyl alcohol in the presence of a base. The ethylenically unsaturated group-containing compound may be, for example, (meth)acrylic acid or a derivative thereof, such as (meth)acrylic acid, (meth)acrylic acid anhydride, (meth)acrylic acid halide, or (meth)acrylic acid ester.

[0048] Preferred examples of hydrogels include polyvinyl alcohol, polyethylene glycol, chitosan, alginate, etc. The hydrogel preferably contains at least one selected from the group consisting of polyvinyl alcohol and polyethylene glycol, and more preferably contains polyvinyl alcohol. Polyvinyl alcohol can be produced, for example, by saponifying polyvinyl ester obtained by polymerizing vinyl ester monomers and converting the ester groups in the polyvinyl ester to hydroxyl groups.

[0049] Examples of the vinyl ester monomer 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; aromatic vinyl esters such as vinyl benzoate; etc. These may be used alone or in combination of two or more.

[0050] Among the vinyl ester monomers, aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of production costs. That is, the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.

[0051] Furthermore, the polyvinyl ester may optionally contain structural units derived from monomers other than vinyl ester-based monomers, as long as the effects of the present invention are not impaired.Examples of the other monomers include α-olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or its salts; alkyl acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, i-propyl acrylate, n-butyl acrylate, i-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate, and octadecyl acrylate; methacrylic acid or its salts; methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, and methacrylic acid. methacrylic acid alkyl esters such as i-propyl methacrylate, n-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, and octadecyl methacrylate; acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetone acrylamide, acrylamidopropanesulfonic acid or a salt thereof, acrylamidopropyldimethylamine or a salt or quaternary salt thereof, N-methylolacrylamide or a salt thereof methacrylamide derivatives such as methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, methacrylamidepropanesulfonic acid or a salt thereof, methacrylamidepropyldimethylamine or a salt or quaternary salt thereof, N-methylolmethacrylamide or a derivative thereof; N-vinylamide derivatives such as N-vinylformamide and N-vinylacetamide; vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, i-propyl vinyl ether, n-butyl vinyl ether, i-butyl vinyl ether, t-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 a salt, ester, or acid anhydride thereof; vinylsilyl compounds such as vinyltrimethoxysilane; and isopropenyl acetate. These may be used alone or in combination of two or more.

[0052] The average degree of polymerization of polyvinyl alcohol is preferably 300 to 10,000, more preferably 500 to 5,000, even more preferably 1,000 to 5,000, and particularly preferably 2,000 to 5,000. A degree of polymerization within the above range is preferable from the viewpoints of substance permeability and multilayer handling properties.

[0053] 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, it can be determined from the intrinsic viscosity measured in water at 30°C after saponifying and purifying the raw material PVA.

[0054] The degree of saponification of polyvinyl alcohol is preferably 50 mol % or more, more preferably 60 mol % or more, and even more preferably 65 mol % or more, from the viewpoint of improving the water solubility of polyvinyl alcohol.

[0055] In order to prevent the hydrosol solution from becoming too viscous and improve the storage stability of the hydrosol solution, the degree of saponification of the polyvinyl alcohol is preferably 99 mol % or less.

[0056] In this specification, the degree of saponification of polyvinyl alcohol means the ratio (mol %) of the number of moles of vinyl alcohol units to the total number of moles of structural units (e.g., vinyl acetate units) that can be converted into vinyl alcohol units by saponification in the raw material PVA and vinyl alcohol units, and can be measured in accordance with JIS K6726:1994.

[0057] The thickness of the hydrogel is not particularly limited, but is preferably 1 to 300 μm, more preferably 5 to 200 μm, and even more preferably 10 to 100 μm.

[0058] The hydrogel may be composed of one type of hydrogel or may be a laminate of two or more types of hydrogels. When the hydrogel contains two or more hydrogels, the hydrogels may be laminated directly, or a porous membrane or a fibrous structure may be interposed between the two hydrogels.

[0059] The crosslinking of the hydrogel makes it possible to adjust the permeability, strength, etc. of nutrients such as glucose, physiologically active substances such as insulin, and humoral factors of the immune system.

[0060] The gel strength of the hydrogel is preferably 20 to 300 kPa, more preferably 50 to 200 kPa. The gel strength can be measured using a tensile tester according to the following procedure.

[0061] First, the hydrosol solution is poured between glass plates sandwiching a 1 mm thick spacer and processed under specified gelation conditions to obtain a 1 mm thick gel sheet. Test specimens are then cut out using a dumbbell cutter conforming to JIS K-6251-3. The test specimens are placed in an Easton tensile tester (Model 5566), and the breaking stress and breaking strain are measured while image data is acquired. The stress at which the test specimen breaks is defined as the gel strength.

[0062] (Configuration of Immunoisolation Layer, etc.) The immunoisolation layer of the present invention preferably comprises a porous membrane or a fiber structure, and the porous membrane or fiber structure preferably comprises at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate. Furthermore, the immunoisolation layer of the present invention is more preferably a multi-layer immunoisolation layer comprising a porous membrane or a fiber structure and a hydrogel.

[0063] The combination of a porous membrane and a hydrogel layer enhances immunoisolation and improves its strength. The balance between permeability and immunoisolation can be adjusted by adjusting the pore size of the porous membrane and the gel strength and / or cross-linking degree of the hydrogel.

[0064] Although porous membranes can suppress cell infiltration and cell leakage, it is not easy to suppress the infiltration of humoral immune factors such as IgG antibodies without suppressing the permeability of necessary physiologically active substances. Therefore, by adjusting the gel strength or crosslink density of the hydrogel layered on the porous membrane, it becomes possible to suppress the infiltration of humoral immune factors such as IgG antibodies without suppressing the permeability of physiologically active substances.

[0065] Furthermore, with a single layer of fiber structure, it is not easy to suppress not only cell infiltration and cell leakage but also the infiltration of humoral immune factors such as IgG antibodies without suppressing the permeability of necessary physiologically active substances. Therefore, by adjusting the gel strength or crosslinking density of the hydrogel layered on the fiber structure, it becomes possible to suppress not only cell infiltration and cell leakage but also the infiltration of humoral immune factors such as IgG antibodies without suppressing the permeability of physiologically active substances.

[0066] The porous membrane, fiber structure, and hydrogel each form a layer, and the boundary between them may be clearly separated, the boundary between the two layers may not be clearly separated, or two or three types may be integrated to form a single layer. For example, an immunoisolation layer containing two types, a fiber structure and a hydrogel, may be a multilayer immunoisolation layer in which the fiber structure and the hydrogel are clearly separated, may have a layer in which the fiber structure and the hydrogel are mixed between the fiber structure and the hydrogel, or may be a single layer in which the fiber structure and the hydrogel are completely integrated. An immunoisolation layer containing two types, a fiber structure and a porous membrane, may be a multilayer immunoisolation layer in which the fiber structure and the porous membrane are clearly separated, may have a layer in which the fiber structure and the porous membrane are mixed between the fiber structure and the porous membrane, or may be a single layer in which the fiber structure and the porous membrane are completely integrated. The immunoisolation layer containing both hydrogel and porous membrane may be a multi-layer immunoisolation layer in which the hydrogel and porous membrane are clearly separated, may have a layer in which the hydrogel and porous membrane are mixed between the hydrogel and porous membrane, or may be a layer in which the hydrogel and porous membrane are completely integrated to form a single layer.

[0067] Particularly preferred immunoisolation devices of the present invention are composed of any of the following three types of multilayer structures (i) to (iii): (i) A porous membrane is used as a substrate, and a hydrogel is applied to or impregnated into the porous membrane; (ii) A fibrous structure is used as a substrate, and a hydrogel is applied to or impregnated into the fibrous structure; or (iii) A porous membrane is formed using a fibrous structure as a substrate, and a hydrogel is then applied to or impregnated into the fibrous structure.

[0068] When the immunoisolation layer has multiple layers, the layers may be bonded together using adhesive, heat, pressure, etc., and when adjacent layers are made of highly compatible materials, they can be bonded together by forming the layers sequentially.

[0069] Specifically, a porous membrane is used as a substrate, and a hydrosol solution is applied directly to the porous membrane, followed by hydrogelation through heat, temperature, light, or chemical action, resulting in a multilayer structure. Alternatively, a fiber structure is used as a substrate, and a hydrosol solution is applied directly to the fiber structure, followed by hydrogelation through heat, temperature, light, or chemical action, resulting in a multilayer structure. By using a fiber structure such as a hydrogel-coated nonwoven fabric instead of a porous membrane, it is possible to construct an immunoisolation layer that is stronger and more permeable to substances than a porous membrane, while still ensuring immunoisolation through the hydrogel.

[0070] The solids concentration of the hydrosol solution is preferably 3 to 15% by mass, more preferably 3 to 10% by mass, even more preferably 3 to 8% by mass, and particularly preferably 3 to 5% by mass. A solids concentration within the above range is preferable because it is possible to suppress the permeation of immune system humoral factors such as IgG while maintaining permeability to substances such as glucose and insulin.

[0071] Alternatively, a porous membrane can be formed by directly applying a polymer solution, which is the raw material for the porous membrane, to the substrate using a hydrogel as the base material, and solidifying the porous membrane raw material through phase separation, a phase transition phenomenon. Depending on the porous membrane raw material, a pretreatment may be required to dry the hydrogel in advance to reduce its water content.

[0072] In one preferred embodiment of the present invention, a highly durable fiber structure is used as a substrate, and a porous membrane or hydrogel, or both a porous membrane and a hydrogel, are formed on the fiber structure. This makes it possible to achieve both a thinner device that improves the diffusion efficiency of physiologically active substances while maintaining the immunoisolation effect, and durability due to improved strength.

[0073] 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. Similarly, when the immunoisolation layer is a multi-layered immunoisolation layer, the thickness is preferably within the above range. Considering the diffusion efficiency of physiologically active substances from the transplant recipient, the thickness of the immunoisolation layer is preferably as thin as possible, 100 μm or less.

[0074] The outermost layer of the immunoisolation layer is preferably biocompatible to prevent it from being recognized as a foreign body. The immunoisolation layer is required to have sufficient permeability to allow sufficient oxygen and nutrients to pass through to the recipient body. Furthermore, to prevent immunocompetent cells from entering the device, it is preferable that the immunoisolation layer has pores on its outer surface, inner surface, or interior throughout the entire layer that have a pore size that prevents the penetration of immunocompetent cells.

[0075] The outermost layer of the immunoisolation layer may be a porous membrane, a fibrous structure, or a hydrogel, or a mixture of two or three of these. The innermost layer of the immunoisolation layer may be a porous membrane, a fibrous structure, or a hydrogel, or a mixture of two or three of these. As used herein, the outermost layer of the immunoisolation layer refers to the layer that constitutes the outer portion of the immunoisolation device of the present invention, i.e., the layer that comes into contact with the tissue surrounding the (host) transplantation site. The innermost layer of the immunoisolation layer refers to the layer that constitutes the portion of the immunoisolation layer that comes into contact with the cell aggregates (inner portion) of the immunoisolation device of the present invention.

[0076] When the outermost layer of the immunoisolation layer is a porous membrane, the porous membrane is preferably made of a material that is more biocompatible than hydrogel, and also serves to prevent the hydrogel from adhering to the recipient's transplant site tissue and causing inflammation.

[0077] When the outermost layer of the immunoisolation layer is a fiber structure, it is desirable that the fiber structure be made of a material that is more biocompatible than hydrogel, and since it also plays a role in preventing adhesion of the hydrogel to the recipient's transplant site tissue and the induction of inflammation, it is desirable to smooth the surface of the fiber structure, which is the outermost layer, by thermal, mechanical, or chemical treatment.

[0078] By suppressing the induction of inflammatory responses through hydrogel modification, it is possible to make the outermost layer a hydrogel and the innermost layer a porous membrane. In this case, it is possible to impart functionality such as angiogenesis by loading a physiologically active substance onto the hydrogel.

[0079] In one embodiment, the innermost layer of the immunoisolation layer is preferably composed of a hydrogel or a porous membrane to which a cell adhesion protein and / or a cell adhesion peptide is immobilized, which allows cell aggregates to adhere to the innermost layer of the immunoisolation layer, eliminating the need for a scaffold material for the cell aggregates and allowing the overall thickness of the immunoisolation device to be reduced.

[0080] When the inner layer is composed of multiple layers, the layers may be made of the same material or different materials. In the present invention, layers (single layer or multiple layers) that include the innermost layer but not the outermost layer are collectively referred to as the "inner layer." Therefore, in the present invention, both the innermost layer and multiple layers including the innermost layer are included in the concept of the "inner layer." For example, in the case of a multiple-layer structure with four layers, the innermost layer can be interpreted as the "inner layer," or the two layers including the innermost layer and the next 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 may be a porous membrane, a fiber structure, or a hydrogel, or a mixture of two or three of these.

[0081] Examples of cell adhesive proteins include those composed of one or more of gelatin, fibrin, fibronectin, laminin, collagen, retronectin, vitronectin, elastin, etc. Examples of cell adhesive peptides include those composed of one or more of RGD peptide, RGDS peptide, GRGD peptide, GRGDS peptide, etc.

[0082] Immobilization of cell adhesive proteins and / or cell adhesive peptides on a hydrogel or porous membrane can be achieved by, but is not limited to, conventional physical adsorption methods such as applying an aqueous solution of the cell adhesive protein or cell adhesive peptide. Alternatively, immobilization can be achieved by using a condensing agent such as a water-soluble carbodiimide to convert functional groups on the hydrogel surface into active esters, which are then covalently bonded to amino groups on the cell adhesive protein and / or cell adhesive peptide.

[0083] The amount of cell adhesive protein and / or cell adhesive peptide immobilized is not particularly limited, but is preferably 0.05 μg / cm 2 Preferably 0.1 μg / cm or more 2 The amount of immobilized cell adhesive protein and / or cell adhesive peptide can be measured by immersing a portion of the hydrogel or porous membrane overnight in excess PBS (PBS tablets (manufactured by Takara Bio Inc.) dissolved in a specified amount of ion-exchanged water) and then measuring the amount by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (manufactured by Takara Bio Inc.)).

[0084] The immunoisolation layer of the present invention has sufficient strength, can remain stable in the recipient's body, and can inhibit the infiltration of immunocompetent cells into the device, thereby simultaneously inhibiting the infiltration of the recipient inside the device into the recipient's body. Therefore, it can be used safely even if the recipient is derived from iPS cells, which are at risk of becoming cancerous.

[0085] (Substance permeability of the immunoisolation layer) The permeation amounts of glucose, insulin, immune system humoral factors, etc. through the immunoisolation layer can be measured by sandwiching the immunoisolation layer between the connecting portions of two glass chambers of the same volume, placing a sample solution of insulin or other substances with a known concentration in chamber a, and placing the same amount of water in chamber b, and then quantitating the amount of insulin or other substances contained in the solution sampled from chamber b after a certain period of time at 37°C with stirring by ELISA or the like ( Figure 8 ). The volumes of the liquids in chambers a and b are adjusted to be equal at the time the sample solution is placed in chamber a.

[0086] The permeability of the immunoisolation layer for glucose, insulin, immune system humoral factors, etc. is expressed as a percentage of the amount of each substance that has permeated into chamber b after 20 hours, as measured by the above method, relative to the equilibrium concentration, i.e., half of the concentration in chamber a. More specifically, the permeability can be calculated, for example, by the following formula: [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 start of measurement] ÷ 2} × 100. The permeability of insulin and glucose in the immunoisolation layer of the present invention is preferably 50% or higher, more preferably 90% or higher, and even more preferably 95% or higher.

[0087] The permeability of the immunoisolating layer of the present invention to humoral factors of the immune system is preferably 30% or less, more preferably 10% or less.

[0088] The substance permeability can be controlled by the pore size of the porous membrane or the gel strength and cross-linking degree of the hydrogel. To achieve immunoisolation function, the pore size of the porous membrane is preferably small enough to block cell permeation, and the hydrogel is preferably able to block the permeation of cells and immune response factors such as antibodies without inhibiting the permeation of physiologically active substances.

[0089] Immune response cells include macrophages, cytotoxic T cells, natural killer cells, dendritic cells, helper T cells, etc., and immune system humoral factors include antibodies, complements, cytokines, etc.

[0090] <Immunoisolation Device> The immunoisolation device of a preferred embodiment of the present invention is in the form of a bag, tube, cylinder, rectangular tube, sphere, cube, rectangular parallelepiped, sheet, or hollow fiber, and encapsulates cell aggregates. In addition to the cell aggregates, physiologically active substances such as enzymes, hormones, cytokines, and drugs may also be encapsulated.

[0091] An immunoisolation device may be fabricated by coating the cell aggregates with an immunoisolation layer, then sealing the periphery of the device by heat sealing and forming it into a pouch. Alternatively, an opening may be formed in a device fabricated with an immunoisolation layer, through which the cell aggregates are inserted, and then the opening may be closed by heat sealing to prevent the entry of immune response cells and humoral factors of the immune system through the opening. Because oxygen and nutrients can permeate through the immunoisolation layer other than the opening, the opening can be closed to prevent the permeation of substances, including nutrients.

[0092] Furthermore, an immunoisolation device may be produced by adhering cell aggregates to the innermost layer of an immunoisolation layer, positioning the immunoisolation layer so that the cell aggregates are inside the device, and sealing the device by heat sealing. Heat sealing can be performed by sandwiching a resin between the immunoisolation layers. By sandwiching a resin, heat sealing can be easily performed even when the innermost layers of the upper and lower immunoisolation layers in the immunoisolation device are both made of hydrogel (when the hydrogels in the upper and lower immunoisolation layers face each other).

[0093] After implanting the immunoisolation device of the present invention in a living body, the impaired functioning transplant is removed and a new functional transplant is introduced, and this procedure can be repeated, and the immunoisolation device can be used repeatedly to introduce transplants, or the immunoisolation device can be removed together with the transplant.

[0094] In a preferred embodiment, the immunoisolation device of the present invention preferably has shape retention to ensure sufficient strength in vivo.

[0095] To maintain the functionality of the device, it is desirable that the fibrous structure, porous membrane, and hydrogel be made of materials that are safe and biocompatible.

[0096] To prevent adhesion with surrounding tissues and fibrosis, it is desirable that the immunoisolation layer be made of a material with excellent biocompatibility. When the immunoisolation layer is made of multiple materials, it is desirable that the material with excellent biocompatibility be the outermost contact surface that comes into contact with the transplant side, i.e., the transplant site of the recipient. An example of a material with excellent biocompatibility is an ethylene-vinyl alcohol copolymer. As the ethylene-vinyl alcohol copolymer, those described in the explanation of the porous membrane can be preferably used.

[0097] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings.

[0098] The schematic diagram of the device shows the immunoisolation layer formed into a bag shape (Fig. 1) or a tube shape (Fig. 2). The bag-shaped device (Fig. 1) is formed by welding (a3) ​​the immunoisolation layers (a1 and a2) consisting of multiple layers shown below, separated by a certain distance (a4) using heat, ultrasound, high frequency, electron beam, etc., to ensure a space for encapsulating the cell aggregates. A spacer may be provided to ensure the certain distance (a4).

[0099] The tubular device (Figure 2) is composed of each immunoisolation layer (b1, b2, b3) formed into a tubular shape, and is formed by enclosing a cell aggregate inside the tubular interior (b4) and welding and sealing both ends of the tubular shape using heat, ultrasound, high frequency, electron beam, etc.

[0100] As shown in FIG. 1 or 2, the conceptual diagram of the device is a bag-like or tubular shape, and the recipient to be transplanted, such as cells or cell masses, is enclosed inside the device as a cell aggregate.

[0101] Figure 3 is a conceptual diagram of a cell sheet, which is composed of cells (11) to be transplanted and an extracellular matrix (10).

[0102] Figure 4 is a conceptual diagram of an immunoisolation device encapsulating a cell sheet. The diagram shows a cell sheet (14) prepared using recipient cells or cell clusters inserted into the device. The immunoisolation device is made in the shape of a pouch using a multi-layer immunoisolation layer, with the outermost layer being a porous membrane (12) and the innermost layer being a hydrogel (13). The porous membrane is preferably made of an ethylene-vinyl alcohol copolymer or the like. Furthermore, the hydrogel in the innermost layer is preferably made of polyvinyl alcohol or the like.

[0103] The immunoisolation layers shown in Figures 5 and 6 are composed of a combination of the following materials. The outermost layer contacts the recipient's tissue at the transplant site, and the innermost layer contacts the cell aggregate. These immunoisolation layers are molded into a bag (Figure 1) or tube (Figure 2), and the cell aggregate containing the immobilized cells and / or cell masses to be transplanted is enclosed inside to form an immunoisolation device.

[0104] Figure 5 shows a multi-layer immunoisolation layer consisting of a porous membrane (15) and a hydrogel (17). The hydrogel (17) is applied or impregnated (16) onto the porous membrane (15), with the outermost surface (18) being made of the porous membrane and the innermost surface (19) being made of the hydrogel.

[0105] Figure 6 shows a multi-layer immunoisolation layer consisting of a fiber structure (20) and a hydrogel (21). The hydrogel (21) is applied to or impregnated into the fiber structure (20), with the outermost surface (22) being made of the fiber structure and the innermost surface (23) being made of the hydrogel.

[0106] The present invention will be described in more detail below with reference to examples.

[0107] <Preparation of Multilayered Immunoisolation Layer> [Preparation Example 1] A porous membrane formed using ethylene-vinyl alcohol copolymer (EVOH) and a hydrogel mainly composed of methacryloyl-modified polyvinyl alcohol (MA-PVA) were multilayered by the following procedure. (1) A porous membrane (average pore size: 1.8 μm, maximum pore size: 3.4 μm, thickness: 100 μm) was prepared by polymer phase separation reaction using EVOH (EVAL® F101A, manufactured by Kuraray Co., Ltd.). (2) A 10% by mass aqueous solution of MA-PVA (average degree of polymerization: 1700, degree of saponification: 98.0-99.0 mol%, methacryloyl-modification rate: 1.2 mol%) was dissolved in phenyl(2,4,6-trimethylbenzoyl)phosphinic acid lithium salt, a water-soluble photoradical polymerization initiator, to a concentration of 0.1% by mass to prepare a sol. (3) This sol was coated onto a PET film using a bar coater to a thickness of 50 μm. (4) A porous membrane was placed on the sol, and the sol and the porous membrane were adhered to each other using a laminator. (5) Then, the sol side was placed facing up, and 365 nm light was irradiated at an intensity of 15 mW / cm. 2 A hydrogel was formed on the porous membrane by irradiating it with light at 4000 kJ / cm for 3 minutes, thereby producing a multi-layered immunoisolation layer. An SEM image of the cross section of the obtained multi-layered immunoisolation layer is shown in Figure 7. It was found that the hydrogel had penetrated into the porous membrane to a depth of approximately 3 to 6 μm.

[0108] [Production Example 2] A hydrogel was formed on a porous membrane using a 4% by mass aqueous solution of MA-PVA (average degree of polymerization: 3500, degree of saponification: 87.0-89.0 mol%, methacryloyl group modification rate: 1.2 mol%) in the same manner as in Production Example 1, to prepare a multilayered immunoisolation layer. <Substance Permeability Test> The permeabilities of glucose, insulin, and IgG were measured for the porous membrane used in Production Example 1 and the multilayered immunoisolation layer obtained in Production Examples 1 and 2, using the following procedure. (1) The porous membrane or multilayered immunoisolation layer was sandwiched between chambers a and b (Figure 8), and 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. Water (65 mL) was placed in chamber b. (2) The solution was stirred at a constant temperature of 37°C with a stirrer, and after 20 hours, a sample was taken from chamber b, and changes in the concentrations of insulin, glucose, and IgG were measured by ELISA. (3) The transmittance of each substance was calculated from the results of (2) (Table 1). [Transmittance of each substance (%)] = [Concentration of each substance in chamber b after 20 hours of measurement] / {[Concentration of each substance in chamber a at the start of measurement] ÷ 2} × 100

[0109]

[0110] Immobilization of a cell adhesive protein on a hydrogel [Production Example 3] Collagen, a cell adhesive protein, was immobilized (covalently bonded) to the surface of a multilayered immunoisolation layer using the following procedure: (1) Twenty multilayered immunoisolation layers (24 mm x 32 mm) were prepared by forming an MA-PVA hydrogel on one side of an EVOH porous membrane using the method described in Production Example 2. (2) 20 mL of MES (2-morpholinoethanesulfonic acid) buffer was placed in a petri dish, and the multilayered immunoisolation layer from (1) was immersed in the buffer and shaken at 100 rpm for 30 minutes. (3) 10 mL of MES buffer was placed in a separate beaker, and 100 mg of N-hydroxysuccinimide (Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as NHS) and 170 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Dojindo Laboratories, Inc., hereinafter referred to as WSC) were dissolved to prepare an NHS / WSC solution. (4) After removing the MES buffer from the dish prepared in (2), 5 mL of NHS / WSC solution was added to the dish and shaken at 100 rpm for 60 minutes at room temperature. (5) The NHS / WSC solution was removed from the dish, and the multilayer immunoisolation layer was immersed in 20 mL of MES buffer and washed by shaking at room temperature. (6) Next, a collagen solution (CellMatrix® Type I-C, Nitta Gelatin Co., Ltd.) was diluted with MES buffer to a concentration of 0.2 mg / mL. The multilayer immunoisolation layer was immersed in 20 mL of this solution and shaken at 100 rpm for 20 hours at room temperature. (7) The multilayer immunoisolation layer was then removed, immersed in 20 mL of distilled water, and washed by shaking at 100 rpm for 5 minutes at room temperature. (8) A release PET film (Cosmo Peel (registered trademark) model E7004, Toyobo Co., Ltd.) was placed on a glass plate, the multilayer immunoisolation layer was placed on top of it, and then a release PET film and a glass plate were placed on top. The multilayer immunoisolation layer was then dried under reduced pressure at room temperature for 20 hours to obtain a multilayer immunoisolation layer in which collagen was immobilized on the surface of the hydrogel.(9) A part of the hydrogel was immersed overnight in excess PBS (PBS tablet (manufactured by Takara Bio Inc.) dissolved in a specified amount of ion-exchanged water), and the amount of immobilized collagen was measured by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (manufactured by Takara Bio Inc.)). The amount of immobilized collagen was found to be 0.5 μg / cm. 2 It was.

[0111] <Preparation of Cell Sheet> [Production Example 4] A co-culture cell sheet (circular, 8 mm diameter, approximately 100 μm thick) of a rat pancreatic β cell line capable of secreting insulin-Gaussia luciferase (iGL cells; Cosmo Bio Co., Ltd.) and human adipose-derived mesenchymal stem cells (hASCs; Lonza Co., Ltd.) was prepared by the following procedure: (1) 45 × 10 iGL cells were placed in a temperature-responsive culture dish (12-well multiwell "Upcell (registered trademark)"; CellSeed Co., Ltd.). 4 40 × 10 hASCs 4 The cells were seeded and cultured for 24 hours in an incubator at 37°C. (2) The temperature-responsive culture dish was then removed from the incubator and allowed to stand at room temperature of about 20°C for 10 minutes, thereby detaching the cells that had adhered and spread to the bottom of the culture dish and obtaining a co-culture cell sheet.

[0112] iGL cells are cells derived from the rat pancreatic β cell line INS-1E, into which a fusion protein of insulin and secretory Gaussia luciferase has been genetically inserted. They react with the luminescent substrate coelenterazine (CTZ) to emit light. The intensity of this luminescence can be used to measure insulin-releasing activity.

[0113] <Preparation of immunoisolation device: Encapsulating cell sheet in immunoisolation layer> [Example 1] The cell sheet prepared in Production Example 4 was placed in the center of a collagen sponge ("Pelnac" (registered trademark), product number PN-S82060, manufactured by Gunze Co., Ltd.) cut into a 1 cm square, and this collagen sponge was covered by sandwiching it with the multi-layered immunoisolation layer prepared in Production Example 2. The periphery of the multi-layered immunoisolation layer was heat-sealed to form a pouch, thereby preparing a rectangular cell sheet encapsulation device measuring 2 cm x 3 cm.

[0114] In the above process, the multilayer immunoisolation layer was sandwiched between the MA-PVA hydrogel side and the EVOH porous membrane side, with the MA-PVA hydrogel side facing the cell sheet (so that the EVOH porous membrane was exposed on the surface of the device). A conceptual diagram of the device is shown in Figure 9. Note that the dashed lines in Figure 9 indicate that part of the device is omitted.

[0115] <Immunoisolation Functionality Test> The cell sheet encapsulation device prepared in Example 1 was implanted into SD rats (wild rats with immune function), and the immunoisolation performance was confirmed by cell engraftment and survival. 1) Immunoisolation Device Implantation The rat was subjected to laparotomy, and the cell sheet encapsulation device was inserted between the liver lobes and fixed using fibrin glue (product name: Bolheal Tissue Adhesion, manufactured by KM Biologics). 2) Functionality Evaluation 2-1) Insulin Release Ability in VITRO The device was removed on the 10th day after implantation, and the cell sheet was then removed. The cell sheet was immersed in a 20 mM glucose solution for 2 hours, and the supernatant was collected. The insulin release ability of iGL cells was compared based on the fluorescence intensity of the cell sheet supernatant.

[0116] As a result, as shown in FIG. 10, it was confirmed that the luminescence intensity of iGL cells in the cell sheet supernatant significantly correlated with insulin release activity.

[0117] 2-2) Histopathological Evaluation Histological sections of the devices were prepared 10 days after implantation and subjected to HE staining and fluorescent immunostaining using an insulin antibody. The results of the histopathological evaluation are shown in Figures 11 and 12.

[0118] In the group implanted with the device produced in Example 1, survival of insulin-producing cells was confirmed, but no inflammatory cell infiltration was observed.

[0119] These results confirmed the immunoisolation effect of the device.

[0120] Example 2 The cell sheet prepared in Production Example 4 was placed on the collagen-immobilized hydrogel surface of the multi-layered immunoisolation layer prepared in Production Example 3, placed in a 60 mm dish, and 5 mL of culture medium (DMEM / F12 GlutaMAX" (registered trademark; Thermo Fisher Scientific, Inc.) was poured into the dish. The 60 mm dish was placed in a 37°C incubator and removed after 24 hours. Visual observation revealed that the cell sheet had adhered to the surface of the multi-layered immunoisolation layer.

[0121] Furthermore, when cells were labeled with a red fluorescent reagent ("Cell Explorer" (registered trademark), Live Cell Tracking Orange Fluorescence, AAT Bioquest) and observed under a stereofluorescence microscope, cells around the cell sheet were observed to adhere to the surface of the multilayered immunoisolation layer and extend. These results demonstrate that by using a collagen-immobilized multilayered immunoisolation layer, it is possible to directly adhere a cell sheet to the multilayered immunoisolation layer without using a collagen sponge, thereby enabling the device to be made thinner.

[0122] The present invention relates to a transplantation device used in cell transplantation therapy and the like, and more particularly to an immunoisolation device for protecting a recipient from immune rejection.

[0123] The immunoisolation device is intended to be used primarily in cell transplantation therapy as a regenerative medicine product, but can also be applied to the transplantation of physiologically active substances other than cells, such as enzymes, hormones, and drugs.

[0124] a1 Immunoisolation layer a2 Immunoisolation layer a3 Welding a4 Fixed distance b1 Immunoisolation layer b2 Immunoisolation layer b3 Immunoisolation layer b4 Tubular interior 10 Extracellular matrix 11 Cell 12 Porous membrane 13 Hydrogel 14 Cell sheet 15 Porous membrane 16 Impregnation and immobilization 17 Hydrogel 18 Outermost layer surface 19 Innermost layer surface 20 Fiber structure 21 Hydrogel 22 Outermost layer surface 23 Innermost layer surface

Claims

1. An immunoisolation device comprising a sheet-shaped cell aggregate containing cells and an extracellular matrix, and an immunoisolation layer covering the cell aggregate.

2. The immunoisolation device of claim 1 , wherein the cell aggregate is a cell sheet composed of cells and extracellular matrix.

3. The immunoisolation device of claim 1 , wherein the cell aggregate has a thickness of 300 μm or less.

4. the immunoisolatory layer comprises a porous membrane or a fibrous structure; The immunoisolation device of claim 1 , wherein the porous membrane or the fibrous structure comprises at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate.

5. The immunoisolation device of claim 4 , wherein the immunoisolation layer is a multi-layer immunoisolation layer comprising the porous membrane or fibrous structure and a hydrogel.

6. The immunoisolation device of claim 5 , wherein the outermost layer of the multi-layered immunoisolation layer is the porous membrane or fibrous structure, and the innermost layer is the hydrogel.

7. The immunoisolation device of claim 5 , wherein the outermost layer of the multi-layered immunoisolation layer is the hydrogel and the innermost layer is the porous membrane or fibrous structure.

8. The immunoisolation device of claim 1, wherein the immunoisolation layer is composed of multiple layers, and the innermost layer of the immunoisolation layer is a hydrogel or porous membrane on which cell adhesion proteins and / or cell adhesion peptides are immobilized.

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