Immunoisolation device

Abstract

The present invention addresses the problem of providing an immunoisolation device: that realizes both a reduction in diffusion distance, which is effective for improving the permeation of physiologically active substance, nutrients, etc., and improved durability that can withstand long-term implantation; and that includes a cell-capturing layer that can prevent the departure of cells or a cluster of cells.　Provided is a immunoisolation device comprising a cell-capturing layer (A) and an immunoisolation layer (B) that covers the cell-capturing layer (A). The cell-capturing layer (A) includes a fibrous structure (a1).

Description

Immunoisolation Device

[0001] 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 in cases of functional decline of transplanted cells, because they allow for specific transplantation sites and device replacement. Required functions of a macroencapsulated immunoisolation device include the ability to disperse and immobilize cells or cell clusters without uniform aggregation, the ability to easily transmit oxygen and nutrients to the transplanted cells, the ability to easily release physiologically active substances (e.g., cytokines, hormones, growth factors) required for therapeutic effects in response to cellular responses, and the ability to be impermeable to immune response cells and immune response factors. Furthermore, it is important that the implanted device is highly biocompatible and unlikely to induce adhesions with surrounding tissues or inflammatory reactions such as granulation tissue.

[0003] Many immunoisolation devices using porous membranes have been investigated to date (Patent Document 1), but one issue has been reduced permeability due to protein adsorption and / or fibrosis of the porous membrane material, as well as 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 maintain the engraftment of embedded cells in the recipient and easily achieve 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.

[0004] Japan Special Table No. 2012-508584

[0005] An object of the present invention is to provide an immunoisolation device that is highly durable for long-term implantation and is suitable for improving the diffusion efficiency of physiologically active substances required for implantation while maintaining the immunoisolation effect.

[0006] Another object of the present invention is to provide an invention that has excellent biocompatibility, maintains immunoisolation, reduces the diffusion distance within the device, and simultaneously achieves improved durability without interfering with the engraftment of transplanted materials such as cells and cell masses.

[0007] A further object of the present invention is to provide an immunoisolation device having a cell-trapping layer that can prevent escape of cells or cell clusters.

[0008] The present invention provides an immunoisolation device comprising:

[0009] [1] An immunoisolation device having a cell-trapping layer (A) and an immunoisolation layer (B) covering the cell-trapping layer (A), wherein the cell-trapping layer (A) comprises a fiber structure (a1). [2] The immunoisolation device according to [1], wherein the fiber structure (a1) has a porosity of 90% or more. [3] The immunoisolation device according to [1] or [2], wherein the fiber structure (a1) has a thickness of 100 to 2000 μm. [4] The immunoisolation device according to any one of [1] to [3], wherein the fiber structure (a1) comprises at least one selected from the group consisting of an ethylene-vinyl alcohol copolymer and cellulose acetate. [5] The immunoisolation device according to any one of [1] to [4], wherein the cell-trapping layer (A) comprises the fiber structure (a1) and a dense fiber structure (a2), wherein the dense fiber structure (a2) is disposed around the fiber structure (a1), and wherein the average pore size of the dense fiber structure (a2) is 35 μm or less. [6] The immunoisolation device according to [5], wherein the thickness of the dense fiber structure (a2) is 1500 μm or less. [7] The immunoisolation device according to [5] or [6], wherein the dense fiber structure (a2) comprises at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate. [8] The immunoisolation device according to [1], wherein the immunoisolation layer (A) is formed by arranging a dense fiber structure (a2) around the fiber structure (a1) and is capable of preventing escape of cells or cell clusters captured by the fiber structure (a1), and the dense fiber structure (a2) has the function of preventing escape of cells or cell clusters. [9] The immunoisolation device according to any of [1] to [8], wherein the immunoisolation layer (B) comprises a porous membrane (b1) or a fiber structure (b2), and the porous membrane (b1) or the fiber structure (b2) comprises at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate.

[10] The immunoisolation device according to [9], wherein the immunoisolation layer (B) is a multilayer immunoisolation layer (B') comprising the porous membrane (b1) or the fibrous structure (b2), and a hydrogel (b3).

[11] The immunoisolation device according to

[10] , wherein the multilayer immunoisolation layer (B') has a thickness of 500 μm or less.

[12] The immunoisolation device according to

[10] or

[11] , wherein the outermost layer of the multilayered immunoisolation layer (B') is the porous membrane (b1) or the fiber structure (b2), and the innermost layer is the hydrogel (b3).

[13] The immunoisolation device according to

[10] or

[11] , wherein the outermost layer of the multilayered immunoisolation layer (B') is the hydrogel (b3), and the innermost layer is the porous membrane (b1) or the fiber structure (b2).

[14] The immunoisolation device according to any of

[10] to

[13] , wherein the hydrogel (b3) contains polyvinyl alcohol, and the average degree of polymerization of the polyvinyl alcohol is 300 to 10,000.

[15] A method for producing an immunoisolation device comprising a cell-trapping layer (A) containing a fiber structure (a1) and a multi-layered immunoisolation layer (B') covering the cell-trapping layer (A), the method comprising the steps of: (1) applying a hydrosol solution to a porous membrane (b1), (2) hydrogelling the hydrosol solution by heat, temperature, light, or chemical action to form the multi-layered immunoisolation layer (B'), and (3) molding the multi-layered immunoisolation layer (B') into a pouch shape by heat welding.

[16] The method for producing an immunoisolation device according to

[15] , wherein the hydrosol solution in step (1) contains polyvinyl alcohol, the degree of polymerization of the polyvinyl alcohol is 300 to 10,000, and the solids concentration of the hydrosol solution is 3 to 15 mass%.

[17] The method for producing an immunoisolation device described in

[15] or

[16] , wherein the heat fusion in step (3) is performed by sandwiching a resin between two multilayered immunoisolation layers (B'), the two multilayered immunoisolation layers (B') face each other with their hydrogel surfaces facing each other, and the resin comprises at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate.

[0010] 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 transplantation, and that has excellent biocompatibility and a cell capture layer that can prevent the escape of cells or cell clusters.

[0011] Perspective view of a bag-shaped immunoisolation device Cross-sectional view of a tubular immunoisolation device Conceptual diagram of an immunoisolation device using a fiber structure (nonwoven fabric) as a cell-trapping layer Conceptual diagram of an immunoisolation device using a fiber structure (nonwoven fabric) as a cell-trapping layer Conceptual diagram of a cell-trapping layer Transmitted light microscopic observation image of a dense fiber structure (a2) Cross-sectional view of an immunoisolation layer with a two-layer structure of a porous membrane and hydrogel Cross-sectional view of an immunoisolation layer with a multi-layer structure of nonwoven fabric and hydrogel SEM image of a cross-section of an immunoisolation layer (B) in Production Example 1 Outline of a substance permeability test

[0012] As used herein, the singular forms (a, an, the, etc.) include both the singular and the plural unless otherwise specified herein or clearly contradictory in context. As used herein, "comprise" is a concept that encompasses "consist essentially of" and "consist of."

[0013] The immunoisolation device of the present invention comprises a cell-trapping layer (A) and an immunoisolation layer (B) capable of trapping recipient cells, and the cell-trapping layer (A) is covered with the immunoisolation layer (B), thereby inhibiting the infiltration of immune cells and cytokines into the cell-trapping layer (A). The cell-trapping layer (A) includes a fiber structure (a1). The immunoisolation layer (B) is preferably composed of a porous membrane (b1) or a fiber structure (b2), and a hydrogel (b3).

[0014] <Cell-trapping layer (A)> (Fiber structure (a1)) The fiber structure (a1) contained in the cell-trapping layer (A) functions as a scaffold for capturing cells or cell clusters as transplant recipients. Examples of the fiber structure (a1) constituting the cell-trapping layer (A) include nonwoven fabrics, woven fabrics, and knitted fabrics, with nonwoven fabrics being preferred.

[0015] The material of the fiber structure (a1) preferably contains at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate, and more preferably contains ethylene-vinyl alcohol copolymer. These may be used alone to form the fiber structure (a1), or two or more may be combined to form the fiber structure (a1). The fiber structure (a1) may also be a cell scaffold material such as collagen fiber.

[0016] Ethylene-vinyl alcohol copolymers can usually be obtained by saponifying an ethylene-vinyl ester copolymer. The production and saponification of the ethylene-vinyl ester copolymer 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.

[0017] 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.

[0018] 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.

[0019] 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-methylene Examples of suitable copolymers include alkenes having an ester group such as propane or saponified products thereof; unsaturated acids such as acrylic acid, methacrylic acid, crotonic acid, and itaconic acid 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 or 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. The ethylene-vinyl alcohol copolymer may also be post-modified by urethanization, acetalization, cyanoethylation, oxyalkylenation, or the like.The ethylene vinyl alcohol copolymer may be used alone or in combination of two or more.

[0020] The fiber structure (a1) may be formed by combining polyester or the like. It may also be formed by a composite fiber having a core-sheath structure in which the fiber core is polyester and the sheath is ethylene-vinyl alcohol copolymer. The cross-sectional structure of the composite fiber may be a parallel type (side-by-side type or multilayer adhesive type) or an eccentric core-sheath type. In this case, it is preferable that the ethylene-vinyl alcohol copolymer covers 50% or more of the fiber surface. Furthermore, the fiber structure (a1) preferably contains 50% or more by mass of the composite fiber, and may be mixed with fibers made of other materials. Examples of other materials include polyester.

[0021] The average fineness of the fibers used in the fiber structure (a1) is preferably 1.0 to 10 dtex. A fineness within the above range is preferred because it is easy to obtain spaces for capturing cells or cell clusters. The average fiber length of the fibers used in the fiber structure (a1) is not particularly limited, and they may be long fibers or short fibers. When producing the fiber structure (a1) by the CAD method, the average fiber length is preferably 32 to 62 mm from the viewpoint of spinnability.

[0022] The fiber structure (a1) may be composed of one type of fiber structure, or may be composed of two or more types of fiber structures laminated together.

[0023] The porosity of the fibrous structure (a1) is preferably 90% or more, more preferably 93% or more, and even more preferably 95% or more. When the voids between the fibers in the fibrous structure (a1) are within the above range, cells or cell clusters are easily captured in the fibrous structure (a1), which is preferable. The upper limit of the porosity of the fibrous structure (a1) is not particularly limited, but examples include preferably 99.9% or less, more preferably 99.5% or less, and even more preferably 99.0% or less. Furthermore, for example, the porosity of the fibrous structure (a1) is preferably 90.0 to 99.9%, more preferably 93.0 to 99.5%, and even more preferably 95.0 to 99.0%. In this specification, the porosity is defined as the basis weight C (g / m ) of the fibrous structure. 2 ), thickness D (cm) and average specific gravity E (g / cm 2 ) can be calculated using the following formula: Porosity (%) = 100 - ((C / D / E) x 10 -4 x100)

[0024] The thickness of the fiber structure (a1) is preferably 100 to 2000 μm, more preferably 300 to 1500 μm, and even more preferably 300 to 1000 μm. A thickness within the above range is preferable because a sufficient number of cells can be retained and the supply of oxygen and the like to the cells is less likely to be inhibited.

[0025] The basis weight of the fiber structure (a1) is 20 to 300 g / m 2 is preferred, and 30 to 200 g / m 2 More preferably, 40 to 100 g / m 2 is more preferable.

[0026] The area of ​​the fiber structure (a1) is not particularly limited, but is preferably 1.5 to 150 cm 2 is preferred.

[0027] The method for producing the fiber structure (a1) is not particularly limited, but it is preferable that bonding points are formed between the fibers by thermal bonding, and the steam jet method is preferably used to provide a space in which cells or cell clusters can be captured and to obtain hardness that can withstand compression.

[0028] (Dense Fiber Structure (a2)) Furthermore, the cell-trapping layer (A) preferably includes, in addition to the fiber structure (a1), a dense fiber structure (a2) having the function of preventing escape of cells or cell clusters captured by the fiber structure (a1). The dense fiber structure (a2) may be laminated on one side of the fiber structure (a1) in the order of dense fiber structure (a2) / fiber structure (a1) or fiber structure (a1) / dense fiber structure (a2), or may be laminated on both sides of the fiber structure (a1) in the order of dense fiber structure (a2) / fiber structure (a1) / dense fiber structure (a2). Furthermore, the dense fiber structure (a2) may be arranged so as to cover part or all of the periphery of the fiber structure (a1). To prevent escape of cells or cell clusters captured by the fiber structure (a1), it is preferable that the layers are stacked in the order of dense fiber structure (a2) / fiber structure (a1) / dense fiber structure (a2), or that the dense fiber structure (a2) is arranged so as to cover the entire periphery of the fiber structure (a1). It is more preferable that the dense fiber structure (a2) is arranged so as to cover the entire periphery of the fiber structure (a1). Furthermore, when the layers are stacked in the order of dense fiber structure (a2) / fiber structure (a1) / dense fiber structure (a2), it is preferable that the layers are stacked in such a way as to cover the entire periphery of the fiber structure (a1). Note that the function of preventing escape of cells or cell clusters means that cells or cell clusters are captured without escaping from the cell-trapping layer. For example, this means that the cell viability evaluated in the Examples described below is 80% or higher.

[0029] Examples of the dense fiber structure (a2) include nonwoven fabrics, woven fabrics, and knitted fabrics, with nonwoven fabrics being preferred because the pore size can be easily controlled under manufacturing conditions. The material of the dense fiber structure (a2) preferably contains at least one selected from the group consisting of ethylene-vinyl alcohol copolymers and cellulose acetate, and more preferably contains an ethylene-vinyl alcohol copolymer. The ethylene-vinyl alcohol copolymers described for the fiber structure (a1) can be preferably used. Alternatively, the fiber may be a composite fiber having a core-sheath structure in which the core of the fiber is polyester and the sheath is an ethylene-vinyl alcohol copolymer.

[0030] The dense fiber structure (a2) preferably has an average pore size of 5 μm or more and 35 μm or less, more preferably 5 μm or more and 25 μm or less. When the dense fiber structure (a2) has an average pore size of 5 μm or more, the supply of oxygen and the like to the cells or cell clusters captured in the fiber structure (a1) is less likely to be hindered. When the dense fiber structure (a2) has an average pore size of 35 μm or less, the cells or cell clusters captured in the fiber structure (a1) are less likely to escape from the fiber structure (a1), which is preferable. In this specification, the average pore size can be measured by the following procedure. Using a stereomicroscope, transmitted light is irradiated and a photograph of the surface of the fiber structure (a2) enlarged 10 times is taken. Using image analysis software, 50 or more bright spots transmitting light are extracted from the micrograph, and the area of ​​each bright spot is measured. The diameter calculated based on the area of ​​a perfect circle is defined as the pore size of the bright spot, and the average value of the pore sizes of the 50 or more bright spots is defined as the average pore size.

[0031] The thickness of the dense fiber structure (a2) is preferably 1500 μm or less, more preferably 1000 μm or less, even more preferably 500 μm or less, and particularly preferably 300 μm or less. The thickness of the dense fiber structure (a2) is preferably 50 μm or more, and preferably 100 μm or more. A thickness of 50 to 1500 μm is preferred because the supply of oxygen and the like to cells is less likely to be inhibited.

[0032] The basis weight of the dense fiber structure (a2) is 3 to 300 g / m 2 It is preferable that the density is 10 to 100 g / m 2 When the basis weight is within the above range, the above-mentioned suitable average pore size and the above-mentioned suitable thickness can be realized, which is preferable.

[0033] The porosity of the dense fiber structure (a2) is preferably less than 90%, more preferably 87% or less, even more preferably 85% or less, and particularly preferably 80% or less. It is also preferably 30% or more, more preferably 50% or more, even more preferably 60% or more, and particularly preferably 70% or more. When the voids between fibers in the dense fiber structure (a2) are within the above range, this is preferred because they tend to have an excellent function of preventing escape of cells or cell aggregates. For example, the porosity of the dense fiber structure (a2) is preferably 30.0 to 89.9%, more preferably 50.0 to 87.0%, even more preferably 60.0 to 85.0%, and particularly preferably 70.0 to 80.0%.

[0034] The area of ​​the dense fiber structure (a2) is not particularly limited, but is preferably 1.0 to 400 cm 2 is preferred, and 1.0 to 200 cm 2 More preferably, 1.5 to 150 cm 2 The area of ​​the dense fiber structure (a2) is preferably larger than that of the fiber structure (a1) to the extent that the dense fiber structure (a2) can cover the fiber structure (a1).

[0035] The method for producing the dense fiber structure (a2) is not particularly limited, but a melt-blowing method is preferably used to achieve a suitable average pore size. The surface of the dense fiber structure (a2) may be subjected to a calendaring process.

[0036] The number of cells retained in the cell-trapping layer (A) is not particularly limited, but is preferably 1×10 ５ ~1 x 10 ７ pieces / cm 3 is preferred, and 1 × 10 6 ~1 x 10 7 pieces / cm 3 is more preferred.

[0037] <Immunoisolation Layer (B)> The immunoisolation device of the present invention includes an immunoisolation layer (B) covering the cell-trapping layer (A). In a typical embodiment, from the viewpoint of obtaining a sufficient immunoisolation effect, it is preferable that the immunoisolation layer (B) covers the entire surface of the cell-trapping layer (A) in the device of the present invention. For the same purpose, it is preferable that the immunoisolation layer (B) does not have any holes such as pinholes penetrating the membrane. It is more preferable that the immunoisolation layer (B) is a multilayer immunoisolation layer (B') including a porous membrane (b1) or a fiber structure (b2), and a hydrogel (b3).

[0038] (Porous membrane (b1)) The porous membrane (b1) constituting the immunoisolation layer (B) is a membrane having a plurality of pores. The fact that it is a porous membrane can be confirmed by a scanning electron microscope (SEM) image or a transmission electron microscope (TEM) image of the membrane cross section.

[0039] The thickness of the porous membrane (b1) 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 (B) while preventing the supply of oxygen and the like to cells from being inhibited.

[0040] The average pore size of the porous membrane (b1) 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 an SEM image or a TEM image. 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 obtain the average pore size.

[0041] The maximum pore size of the porous membrane (b1) 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 and humoral factors of the immune system 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.

[0042] The porous membrane (b1) is required to have the function of suppressing cell infiltration from the recipient and preventing leakage of the transplanted cells, and therefore the average or maximum pore size is preferably 5 μm or less, which is smaller than the cell size.

[0043] The porous membrane (b1) preferably contains a polymer and is substantially composed of a polymer. Examples of the polymer include thermoplastic or thermosetting polymers. Specific examples of the polymer 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), etc. From the viewpoints of solubility, optical properties, electrical properties, strength, elasticity, etc., these may be homopolymers, copolymers, polymer blends, polymer alloys, etc. The polymer constituting the porous membrane may contain a hydrophilic polymer such as polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, polyethylene glycol, etc. Combining hydrophilic and hydrophobic polymers can improve biocompatibility.

[0044] The polymer forming the porous membrane (b1) is preferably a material with excellent biocompatibility that is unlikely to cause adhesion to the recipient's surrounding tissues, inflammation, etc. The porous membrane (b1) preferably contains at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate, more preferably an ethylene-vinyl alcohol copolymer. As the ethylene-vinyl alcohol copolymer, those described in relation to the fiber structure (a1) can be preferably used.

[0045] The porous membrane (b1) 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 (b1) 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. In one preferred embodiment, the porous membrane (b1) is a membrane formed from one composition as a single layer, and in another preferred embodiment, it is not a laminate structure of multiple layers.

[0046] (Fiber structure (b2)) The fiber structure (b2) constituting the immunoisolation layer (B) may be a nonwoven fabric, a woven fabric, or a knitted fabric, with a nonwoven fabric being preferred. The fiber structure is formed by bonding or entangling fibers by thermal, mechanical, or chemical action. The fiber diameter and / or fiber amount can be adjusted to adjust the basis weight (weight per unit area), which in turn makes it possible to control not only strength but also permeability, filtration, etc. The basis weight of the fiber structure (b2) is 10 to 100 g / m 2 The thickness of the fiber structure (b2) is preferably 300 μm or less, and desirably as thin as possible, such as 200 μm or less, in consideration of the diffusion efficiency of the physiologically active substance from the transplant recipient. The lower limit of the thickness of the fiber structure (b2) is not particularly limited, but is preferably 50 μm or more, and more preferably 100 μm or more, for example.

[0047] Examples of fiber materials for the fiber structure (b2) 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, carboxymethylcellulose, and other cellulose acetates, fibroin, and silk. As with the porous membrane (b1), the fiber material for the fiber structure is preferably biocompatible. The fiber structure (b2) preferably contains at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate, and more preferably contains ethylene-vinyl alcohol copolymer. The ethylene-vinyl alcohol copolymer may be the same as that described for the fiber structure (a1). The surface of the fiber structure is preferably smoothed by thermal, mechanical, or chemical treatment.

[0048] (Hydrogel (b3)) Examples of hydrosols for producing the hydrogel (b3) constituting the immunoisolation layer (B) include sols that gel in the presence of metal ions 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 gelling conditions, adjusting the pH to gelling conditions, irradiating them with light that satisfies gelling conditions, or applying a magnetic field that satisfies gelling conditions may be performed depending on the properties of the gel used.

[0049] 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.

[0050] Examples of pH-responsive hydrogels include alginate gel, chitosan gel, carboxymethyl cellulose gel, and acrylic acid-based synthetic gel.

[0051] Examples of photoresponsive hydrogels include synthetic gels combining azobenzene and cyclodextrin in the backbone, gels composed of supramolecules with fumaric acid amide as a spacer, gels crosslinked or bonded via nitrobenzyl groups, and gels composed of modified polyvinyl alcohol. Examples of modified polyvinyl alcohols include (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 polyvinyl alcohol in the presence of a base. Examples of ethylenically unsaturated group-containing compounds include (meth)acrylic acid or derivatives thereof, such as (meth)acrylic acid, (meth)acrylic acid anhydride, (meth)acrylic acid halide, and (meth)acrylic acid ester.

[0052] Preferred examples of the hydrogel (b3) include polyvinyl alcohol, polyethylene glycol, chitosan, alginate, etc. The hydrogel (b3) 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 a vinyl ester monomer and converting the ester groups in the polyvinyl ester to hydroxyl groups.

[0053] 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.

[0054] 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.

[0055] 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 a salt thereof; acrylic acid alkyl ester compounds 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 a salt thereof; methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, and methacrylic acid alkyl ester compounds. methacrylic acid alkyl ester compounds 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 ether compounds 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; nitrile compounds 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.

[0056] 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, even more preferably 1,500 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 material permeability and multilayer handling. 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.

[0057] 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.

[0058] Furthermore, from the viewpoint of suppressing an increase in viscosity of the hydrosol solution and improving the storage stability of the hydrosol solution, the degree of saponification of the polyvinyl alcohol is preferably 99.9 mol% or less, more preferably 99.5 mol% or less, and even more preferably 99.0 mol% or less.

[0059] 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.

[0060] The thickness of the hydrogel (b3) 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.

[0061] The hydrogel (b3) can be crosslinked to adjust the permeability, strength, etc. of nutrients such as glucose, physiologically active substances such as insulin, and humoral factors of the immune system.

[0062] The gel strength of the hydrogel (b3) is preferably 20 to 300 kPa, more preferably 50 to 200 kPa. The gel strength can be measured using a tensile tester by the following procedure. First, the hydrosol solution is poured between glass plates sandwiching a 1 mm thick spacer, and treated under specified gelation conditions to obtain a 1 mm thick gel sheet. Test pieces are cut from this gel sheet using a dumbbell cutter conforming to JIS K-6251-3. The test piece is placed in an Instron tensile tester (Model 5566), and the breaking stress and breaking strain are measured while acquiring image data. The stress at which the test piece breaks is defined as the gel strength.

[0063] (Configuration of immunoisolation layer (B)) The immunoisolation layer (B) is preferably a porous membrane (b1) containing at least one selected from the group consisting of an ethylene-vinyl alcohol copolymer and cellulose acetate, or a fiber structure (b2) containing at least one selected from the group consisting of an ethylene-vinyl alcohol copolymer and cellulose acetate. Furthermore, the immunoisolation layer (B) may have a multilayer structure containing, in addition to the porous membrane (b1) or the fiber structure (b2), a layer other than the porous membrane (b1) and the fiber structure (b2). The layer other than the porous membrane (b1) and the fiber structure (b2) is preferably a hydrogel (b3). In this specification, an immunoisolation layer containing a porous membrane (b1) or the fiber structure (b2) and a hydrogel (b3) may be referred to as a multilayer immunoisolation layer (B').

[0064] The multilayer structure with the hydrogel (b3) enhances the immunoisolation properties and also improves the strength of the immunoisolation layer (B). The compatibility of permeability and immunoisolation properties can be adjusted by the pore size of the porous membrane and the gel strength and / or degree of crosslinking of the hydrogel (b3).

[0065] Although the porous membrane (b1) 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 crosslinking density of the hydrogel (b3) layered on the porous membrane (b1), it becomes possible to suppress the infiltration of humoral immune factors such as IgG antibodies without suppressing the permeability of physiologically active substances.

[0066] Furthermore, with a single layer of fiber structure (b2), 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 (b3) layered on the fiber structure (b2), 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.

[0067] The porous membrane (b1), fiber structure (b2), and hydrogel (b3) 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, the fiber structure (b2) and the hydrogel (b3), may have a clearly separated fiber structure and hydrogel, a layer in which the fiber structure and the hydrogel are mixed between the fiber structure and the hydrogel, or the fiber structure and the hydrogel are completely integrated to form a single layer. An immunoisolation layer containing two types, the fiber structure (b2) and the porous membrane (b1), may have a clearly separated fiber structure and porous membrane, a layer in which the fiber structure and the porous membrane are mixed between the fiber structure and the porous membrane, or the fiber structure and the porous membrane are completely integrated to form a single layer. The immunoisolation layer containing two types of materials, hydrogel (b3) and porous membrane (b1), may have a clearly separated hydrogel and porous membrane, may have a layer of a mixture of hydrogel and porous membrane between the hydrogel and porous membrane, or may have the hydrogel and porous membrane completely integrated to form a single layer.

[0068] A particularly preferred immunoisolation device of the present invention is composed of any of the following three types of multilayer structures (i) to (iii): (i) A porous membrane (b1) is used as a substrate, and a hydrogel (b3) is applied to or impregnated into the porous membrane (b1); (ii) A fibrous structure (b2) is used as a substrate, and a hydrogel (b3) is applied to or impregnated into the fibrous structure (b2); or (iii) A porous membrane (b1) is formed using the fibrous structure (b2) as a substrate, and a hydrogel (b3) is further applied to or impregnated into the porous membrane (b1).

[0069] When the immunoisolation layer (B) 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.

[0070] Specifically, a porous membrane (b1) is used as a substrate, and a hydrosol solution is directly applied to the porous membrane, followed by hydrogelation by heat, temperature, light, or chemical action, thereby forming a multilayer structure. Alternatively, a fiber structure (b2) is used as a substrate, and a hydrosol solution is directly applied to the fiber structure, followed by hydrogelation by heat, temperature, light, or chemical action, thereby forming a multilayer structure. By using a fiber structure (b2) such as a nonwoven fabric coated with a hydrogel (b3) instead of the porous membrane (b1), it is possible to form an immunoisolation layer (B) that is stronger and more permeable to substances than the porous membrane (b1), while ensuring immunoisolation by the hydrogel (b3).

[0071] 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.

[0072] In addition, the porous membrane (b1) can be formed by directly applying a polymer solution, which is the raw material of the porous membrane (b1), onto the substrate using the hydrogel (b3), and solidifying the raw material of the porous membrane (b1) through phase separation, which is a phase transition phenomenon. Depending on the raw material of the porous membrane (b1), a pretreatment may be required in which the hydrogel (b3) is dried in advance to reduce the water content.

[0073] In one preferred embodiment of the present invention, a highly durable fiber structure (b2) is used as a substrate, and a hydrogel (b3) is formed on the fiber structure (b2), or both a porous membrane (b1) and a hydrogel (b3) are formed on the fiber structure (b2). This makes it possible to achieve both a thinner device that improves the diffusion efficiency of physiologically active substances and durability due to increased strength while maintaining the immunoisolation effect.

[0074] The thickness of the immunoisolation layer (B) 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, still more preferably 170 μm or less, and particularly preferably 150 μm or less. Similarly, when the immunoisolation layer (B) is a multi-layered immunoisolation layer (B'), the thickness is preferably within the above range. Taking into consideration the diffusion efficiency of physiologically active substances from the transplant recipient, the thickness of the multi-layered immunoisolation layer (B') is preferably 500 μm or less, more preferably 300 μm or less, and even more preferably 100 μm or less, and is as thin as possible. The area of ​​the immunoisolation layer (B) is not particularly limited, but is preferably 1.0 to 400 cm 2 is preferred, and 1.0 to 200 cm 2 More preferably, 1.5 to 150 cm 2 The area of ​​the immunoisolation layer (B) is preferably equal to or larger than that of the cell-trapping layer (A) so that the immunoisolation layer (B) can cover the cell-trapping layer (A).

[0075] The outermost layer of the immunoisolatory layer (B) is preferably biocompatible to prevent it from being recognized as a foreign body, and is required to have sufficient permeability to allow sufficient oxygen and nutrients to reach the recipient.

[0076] The outermost layer of the immunoisolation layer (B) may be a porous membrane (b1), a fiber structure (b2), or a hydrogel (b3), or a mixture of two or three of these. The innermost layer of the immunoisolation layer (B) may be a porous membrane (b1), a fiber structure (b2), or a hydrogel (b3), or a mixture of two or three of these. In this specification, the outermost layer of the immunoisolation layer (B) refers to the layer of the immunoisolation layer (B) that constitutes the outer portion of the immunoisolation device of the present invention, i.e., the layer that contacts the tissue surrounding the transplantation site (host). The innermost layer of the immunoisolation layer (B) refers to the layer of the immunoisolation layer (B) that constitutes the portion (inner portion) that contacts the cell-trapping layer (A) of the immunoisolation device of the present invention.

[0077] When the outermost layer of the immunoisolation layer (B) is a porous membrane (b1), the porous membrane (b1) is preferably made of a material that is more biocompatible than the hydrogel (b3), and also serves to prevent adhesion of the hydrogel (b3) to the recipient's transplant site tissue, inflammation, etc.

[0078] When the outermost layer of the immunoisolation layer (B) is a fiber structure (b2), the fiber structure (b2) is preferably made of a material that is more biocompatible than the hydrogel (b3), and also plays a role in preventing adhesion of the hydrogel (b3) to recipient tissue, inflammation, etc., and therefore it is desirable to smooth the surface of the fiber structure (b2), which is the outermost layer, by thermal, mechanical, or chemical treatment.

[0079] By suppressing the induction of an inflammatory response by modifying the hydrogel (b3), it is possible to form the outermost layer of the hydrogel (b3) and the innermost layer of the porous membrane (b1). In this case, it is possible to impart functionality such as the induction of angiogenesis by loading a physiologically active substance onto the hydrogel.

[0080] (Substance permeability of immunoisolation layer (B)) The amount of glucose, insulin, immune system humoral factors, etc. permeated through the immunoisolation layer (B) can be measured by sandwiching the immunoisolation layer between the connecting portion 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 water in chamber b. After a certain period of time, the solution is sampled from chamber b under stirring at 37°C, and the amount of insulin or other substances contained in the solution is quantified by ELISA or the like ( Figure 9 ). The liquid volumes in chambers a and b are adjusted to be equal at the time the sample solution is placed in chamber a.

[0081] The permeability of the immunoisolation layer (B) to glucose, insulin, immune system humoral factors, etc. is a percentage value that represents 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

[0082] The permeability of the immunoisolating layer (B) to insulin and glucose is preferably 50% or more, more preferably 90% or more, and even more preferably 95% or more.

[0083] The permeability of the immunoisolating layer (B) to humoral factors of the immune system is preferably 30% or less, more preferably 10% or less.

[0084] The permeability of each substance can be controlled by the pore size of the porous membrane (b1), the fiber basis weight of the fiber structure (b2), or the strength and degree of crosslinking of the hydrogel (b3). To exhibit the immunoisolation function, the pore size of the porous membrane (b1) is preferably equal to or smaller than a size that does not allow cells to pass through, and the hydrogel (b3) is preferably capable of inhibiting the permeation of cells and immune response factors such as antibodies without inhibiting the permeation of physiologically active substances.

[0085] 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.

[0086] <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 a cell-trapping layer (A) inside. A recipient is introduced into the cell-trapping layer (A). The recipient may be a cell, cell cluster, cell sheet, graft, or the like, and physiologically active substances other than cells, such as enzymes, hormones, cytokines, and drugs, can also be used. Preferred cells, cell clusters, or grafts are those that release a physiologically active substance to the outside of the immunoisolation device. That is, in a preferred embodiment, the cells, cell clusters, or grafts include cells that produce a physiologically active substance.

[0087] To introduce the transplant recipient into the cell-trapping layer (A), a suspension of the transplant recipient is uniformly injected and seeded into the fiber structure (a1) using a pipette or the like. After seeding, in order to prevent the transplant recipient from escaping from the fiber structure, the surrounding area can be covered with a dense fiber structure (a2) of different specifications, which has smaller voids. In other words, it is preferable that the porosity of the dense fiber structure (a2) is smaller than the porosity of the fiber structure (a1).

[0088] An immunoisolation device may be produced by covering the periphery of the cell-trapping layer (A) produced by the above method with an immunoisolation layer (B), then sealing the periphery of the device by heat sealing and forming it into a pouch. Alternatively, an opening may be provided in a device previously produced with an immunoisolation layer (B), through which the cell-trapping layer (A) is inserted, and the opening may then 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, nutrients, and the like can permeate the immunoisolation layer (B) from other than the opening, the opening can be closed to prevent the permeation of substances, including nutrients.

[0089] Heat sealing can be performed by sandwiching a resin between the immunoisolation layers (B) (multilayer immunoisolation layers (B')). By sandwiching a resin between the layers, heat sealing can be easily performed even when the innermost layers of the upper and lower immunoisolation layers (B) in the immunoisolation device are both made of hydrogel (when the hydrogels in the upper and lower immunoisolation layers (B) face each other). The resin used for heat sealing is not particularly limited, but is preferably at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate, and more preferably ethylene-vinyl alcohol copolymer. Alternatively, heat sealing can be performed by sandwiching a dense fiber structure (a2) between the immunoisolation layers (B).

[0090] 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.

[0091] The immunoisolation device 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 cell-trapping layer (A), thereby simultaneously inhibiting the infiltration of the recipient inside the cell-trapping layer (A) into the recipient's body. Therefore, it can be used safely even if the recipient is derived from iPS cells, for which there is a concern that the transplant may become cancerous.

[0092] In a preferred embodiment, the immunoisolation device of the present invention preferably has shape retention to ensure sufficient strength in vivo.

[0093] To maintain functionality as an immunoisolation device, the fiber structure (a1) and dense fiber structure (a2) used as the cell-trapping layer (A), and the porous membrane (b1), fiber structure (b2), and hydrogel (b3) used as the immunoisolation layer (B) are preferably made of materials with excellent safety and biocompatibility. To prevent adhesion with surrounding tissues and fibrosis, the immunoisolation layer (B) is preferably made of a material with excellent biocompatibility. When the immunoisolation layer (B) 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 ethylene-vinyl alcohol copolymer.

[0094] The thickness of the immunoisolation device of the present invention varies depending on the recipient tissue, the transplant recipient, etc., and is not particularly limited, but is preferably 400 to 2000 μm. The area of ​​the immunoisolation device of the present invention also varies depending on the recipient tissue, the transplant recipient, etc., and is not particularly limited, but is preferably 1.0 to 200 cm. 2 is preferred.

[0095] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings.

[0096] The device schematic diagram shows the immunoisolation layer (B) molded into a bag shape (FIG. 1) or a tube shape (FIG. 2). The bag-shaped device (FIG. 1) is formed by welding (x3) the immunoisolation layer (B) (x1 and x2) consisting of multiple layers shown below, separated by a certain distance (x4) using heat, ultrasound, high frequency, electron beam, etc., to ensure a space for enclosing the cell-trapping layer (A). A spacer may be provided to ensure the certain distance (x4).

[0097] The tubular device (Figure 2) is composed of each immunoisolation layer (B) (y1, y2, y3) formed into a tubular shape, and is formed by enclosing the cell capture layer (A) inside the tubular interior (y4) and welding and sealing both ends of the tubular shape using heat, ultrasound, high frequency, electron beam, etc.

[0098] 3A and 3B show conceptual diagrams of an immunoisolation device according to one embodiment of the present invention, in which the outermost layer of the immunoisolation layer (B) contacts the implantation site, and the innermost layer contacts the cell-trapping layer (A).

[0099] As shown in Figures 3A and 3B, which are conceptual diagrams of an immunoisolation device, the immunoisolation device has a bag-like or tubular shape and encapsulates a cell-trapping layer containing a dispersed recipient. In this embodiment, the cell-trapping layer (A) is formed by uniformly dispersing and immobilizing recipients, such as cells and cell aggregates, on a fibrous structure (a1) (5) within the cell-trapping layer (A). The fibrous structure (a1) (5) preferably contains at least one material selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate, which have excellent biocompatibility, and may also be a cell scaffold material such as collagen fiber. Both of the fibrous structures (a1) (5) can be sterilized. The fibrous structure (a1) (5) may be present in the cell-trapping layer (A) surrounded by the immunoisolation layer (B) before the introduction of the recipient. Alternatively, the recipient (3) and the fibrous structure (a1) (5) may be subsequently introduced as the cell-trapping layer (A) into an immunoisolation device composed of the immunoisolation layer (B). Furthermore, when dispersing the transplant recipient in the fiber structure (a1) constituting the cell trapping layer (A), polyvinyl alcohol or polyethylene glycol-based hydrogel particles, block-shaped structures, etc. may be suspended with the transplant recipient to prevent aggregation and association of the transplant recipient cells or cell clusters.

[0100] 4 shows a conceptual diagram of the cell-trapping layer (A). The cell-trapping layer (A) preferably comprises two types of fiber structure, a fiber structure (a1) and a dense fiber structure (a2), and has a structure in which the fiber structure (a1) captures the recipient, and the dense fiber structure (a2), which has smaller voids, covers the periphery of the fiber structure (a1) to prevent the captured recipient from escaping from the fiber structure.

[0101] Figure 5 shows a transmitted light microscopic image of the dense fiber structure (a2). This image was acquired, and the area of ​​each bright spot through which light was transmitted was measured using image analysis software. The diameter calculated from this area as the area of ​​a perfect circle was used as the pore size of the bright spot. The average pore size of 50 or more bright spots was used as the average pore size.

[0102] The immunoisolation layer (B) shown in Figures 6 and 7 is 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-trapping layer (A). These immunoisolation layers (B) are formed into a bag (Figure 1) or tube (Figure 2), and the cell-trapping layer (A) with the cells and / or cell clusters to be transplanted fixed therein is enclosed inside to form an immunoisolation device.

[0103] Figure 6 shows the multilayer structure of a porous membrane (b1) (6) and a hydrogel (b3) (8). The hydrogel (b3) (8) is impregnated (7) into the porous membrane (b1) (6), with the outermost surface (9) being made of the porous membrane (b1) and the innermost surface (10) being made of the hydrogel (b3).

[0104] 7 shows a multilayer structure of a fiber structure (b2) (11) and a hydrogel (b3) (12). The hydrogel (b3) (12) is formed by coating or impregnation on the fiber structure (b2) (11), with the outermost surface (13) being made of the fiber structure (b2) and the innermost surface (14) being made of the hydrogel (b3).

[0105] <Production of multi-layered immunoisolation layer (B')> [Production Example 1] A porous membrane made using ethylene-vinyl alcohol copolymer (hereinafter referred to as EVOH) and a hydrogel mainly composed of methacryloyl-modified polyvinyl alcohol (hereinafter referred to as MA-PVA) were multi-layered by the following procedure.

[0106] (1) A porous membrane (average pore size 1.8 μm, maximum pore size 3.4 μm, thickness 100 μm) was prepared using EVOH (EVAL® (registered trademark), F101A, manufactured by Kuraray Co., Ltd.) by polymer phase separation reaction. (2) A 10% by mass aqueous solution of MA-PVA (average degree of polymerization 1700, degree of saponification 98.0-99.0 mol%, methacryloyl group modification rate 1.2 mol%, hereinafter referred to as MA-PVA) was added to a water-soluble photoradical polymerization initiator, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate, to a concentration of 0.1% by mass, and dissolved 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 porous membrane were adhered together using a laminator. (5) Then, the sol surface was turned upside down and 365 nm light was applied at an intensity of 15 mW / cm ２ The porous membrane was irradiated with light at 1000 kJ / cm for 3 minutes to form a hydrogel on the membrane, producing a multi-layered immunoisolation layer (B'). An SEM image of the cross section of the resulting multi-layered immunoisolation layer (B') is shown in Figure 8. It was found that the hydrogel had penetrated into the porous membrane to a depth of approximately 3 to 6 μm.

[0107] [Production Example 2] A hydrogel was formed on a porous membrane using an aqueous solution of MA-PVA (average degree of polymerization 3500, degree of saponification 87.0 to 89.0 mol%, methacryloyl group modification rate 1.2 mol%) at a concentration of 4% by mass in the same manner as in Production Example 1, to prepare a multilayer immunoisolation layer (B').

[0108] <Substance Permeability Test> The permeabilities of glucose, insulin, and IgG for the porous membrane used in Production Example 1 and the multilayered immunoisolation layer (B') obtained in Production Example 1 or 2 were measured using the following procedure. (1) The porous membrane or multilayered immunoisolation layer was sandwiched between chambers a and b (Figure 9), 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 mixture was stirred at a constant temperature of 37°C, 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 permeability of each substance was calculated from the results of (2) (Table 1). [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

[0109]

[0110] <Preparation of Cell-Trapping Layer> [Production Example 3] (Preparation of Fiber Structure (a1)) (1) A core-sheath composite fiber (fineness 3.3 dtex, specific gravity 1.25, product name: "Sophista" S-220, manufactured by Kuraray Co., Ltd., ethylene content of ethylene-vinyl alcohol copolymer 44 mol%, saponification degree 98.4 mol%) was used to prepare a fiber sheet called a web by a carding method. (2) The web was sandwiched between conveyors and molded. (3) Subsequently, the web was passed through a steam jet process, and the fibers were bonded together using heat and a jet of steam to form a web with a basis weight of 50 g / m. 2 A nonwoven fabric sheet with a thickness of 1 mm was obtained. (4) The nonwoven fabric sheet was cut into a circle using a punch with a diameter of 15 mm to prepare a fiber structure (a1) for cell capture (porosity: 96%). The porosity was calculated based on the basis weight C (g / m) of the fiber structure. 2 ), thickness D (cm) and average specific gravity E (g / cm 2 ) was calculated using the following formula: Porosity (%) = 100 - ((C / D / E) x 10 -4 x100)

[0111] [Production Example 4] (Preparation of dense fiber structure (a2)) (1) Using a general meltblown machine, a molten ethylene-vinyl alcohol copolymer (ethylene content 44 mol%, saponification degree 99.3 mol%, "E105" manufactured by Kuraray Co., Ltd.) was extruded from a die to obtain a meltblown nonwoven fabric sheet having a fiber diameter of 8 μm. (2) The meltblown nonwoven fabric sheet was passed through a metal roll and a rubber roll heated to 100° C. at a pressure of 40 kg / cm and a speed of 3 m / min, and subjected to a calendering treatment. The meltblown nonwoven fabric obtained after the treatment had a basis weight of 76.6 g / m. 2 , thickness 0.24 mm, and porosity 73%. The porosity was calculated based on the basis weight C (g / m 2 ), thickness D (cm) and average specific gravity E (g / cm 2 ) was calculated using the following formula: Porosity (%) = 100 - ((C / D / E) x 10 -4 (3) A meltblown nonwoven fabric was cut into 24 mm squares to prepare a dense fiber structure (a2) (average pore diameter 11 μm) for preventing cell deviation. The area of ​​each bright spot transmitting light was measured from a transmitted light microscope image ( FIG. 5 ) of the dense fiber structure (a2) using image analysis software, and the average value of the diameters (50 spots) calculated as the area of ​​a perfect circle was used as the average pore diameter.

[0112] <Cell Preparation> [Production Example 5] MIN6 (a mouse pancreatic islet-like cell line) was used to confirm the cell and cell cluster capturing function of the cell-trapping layer. 2.8 × 10 MIN6 cells were placed in a suspension cell culture flask (manufactured by Sumitomo Bakelite Co., Ltd.) containing DMEM medium. ５ cells / cm ２ The cells were seeded at a density of 5 × 10 to 5 × 10 and cultured at 37°C for 7 days to produce cell clusters with a diameter of approximately 100-200 μm. 1 mL of the cell suspension was collected from the flask and treated with 1 mL of trypsin-EDTA to disperse the cell clusters into single-cell units, and the cell density was measured using a hemocytometer. Based on the measured cell density, when cells were seeded in the following examples, 5 × 10 cells were seeded on the fiber structure. ５ The volume of the liquid was adjusted to give 100 cells, and the cells were used for the test.

[0113] <Preparation and Evaluation of Immunoisolation Device> [Example 1] 1) Seeding of Cells and Encapsulation into Immunoisolation Device (1) The multi-layered immunoisolation layer (B') prepared in Preparation Example 2 was cut into a 24 mm square and placed with the hydrogel side facing up. The dense fiber structure (a2) prepared in Preparation Example 4 was placed on top of it, and the fiber structure (a1) prepared in Preparation Example 3 was placed on top of that in the center. (2) Next, the MIN6 cell mass suspension (5 x 10 cells in 50 μl of DMEM medium) prepared in Preparation Example 5 was added. ５ (including 100 pieces of cell aggregates) were seeded onto the fiber structure (a1) from above using a micropipette and left for 30 seconds to allow the cell aggregate suspension to penetrate the fiber structure (a1). (3) The dense fiber structure (a2) prepared in Production Example 4 was placed on top of the cell-seeded fiber structure (a1), and then the multilayered immunoisolation layer (B') prepared in Production Example 2 was placed on top of it with the hydrogel side facing down. The four sides of the overlapping immunoisolation layer (B') and dense fiber structure (a2) were welded together using a heat sealer (Clip Sealer Z-1, manufactured by Techno Impulse) to produce an immunoisolation device (Figure 3B).

[0114] 2) Evaluation of Cell Viability The immunoisolation device prepared in the previous section was placed in a 6-well plate, 7 mL of DMEM medium was added, and the cells were cultured at 37°C for one week. Half of the medium (3.5 mL) was replaced daily. As a control, 7 mL of DMEM medium was added to an ultra-low attachment surface 6-well plate (Corning Incorporated), and the same number of cell cluster suspensions seeded in the previous section were placed therein. The cells were cultured at 37°C for one week, with half of the medium (3.5 mL) replaced daily. After one week of culture, the entire medium was removed, 1 mL of fresh medium was added, and 1 mL of ATP activity measurement reagent ("CellTiter-Glo" (registered trademark), Promega Corporation) was added. The cells were then shaken on a plate shaker for two minutes. The cells were then left to stand at room temperature for 10 minutes, and 100 μL of the supernatant was transferred to a white 96-well plate. The luminescence intensity was measured using a plate reader (ARVO X2; PerkinElmer). As a result, when the luminescence intensity obtained from the control cell mass was taken as a survival rate of 100%, the luminescence intensity obtained from the device-encapsulated cell mass was a survival rate of 103%.

[0115] [Example 2] The basis weight of the fiber structure (a1) was 100 g / m2 An immunoisolation device was produced in the same manner as in Example 1, except that the porosity was changed to 92%. The cell viability was measured in the same manner as in Example 1 and was found to be 88%.

[0116] [Example 3] The basis weight of the fiber structure (a1) was 25 g / m 2 An immunoisolation device was produced in the same manner as in Example 1, except that the thickness was changed to 0.5 mm (porosity 96%). The cell viability was measured in the same manner as in Example 1 and was found to be 103%.

[0117] [Example 4] Dense fiber structure (a2) with a basis weight of 22.3 g / m 2 An immunoisolation device was fabricated in the same manner as in Example 1, except that the thickness was changed to 0.12 mm (average pore size 23.5 μm, porosity 84%). The cell viability was measured in the same manner as in Example 1 and was found to be 96%.

[0118]

[0119] As is clear from the results in Table 2, in Examples 1 to 4, cell clusters can be captured without escaping from the cell-trapping layer (A). Furthermore, a fiber structure (a1) having an appropriate porosity can capture cell clusters while maintaining a high viability. Furthermore, a dense fiber structure (a2) having an appropriate average pore size can prevent cell clusters from escaping from the fiber structure (a1).

[0120] The present invention relates to a transplantation device used in cell transplantation therapy, etc., and in particular to an immunoisolation device for protecting the recipient from immune rejection. 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.

[0121] x1 Immunoisolation layer (B) x2 Immunoisolation layer (B) x3 Dissolution x4 Certain distance y1 Immunoisolation layer (B) y2 Immunoisolation layer (B) y3 Immunoisolation layer (B) y4 Tubular interior 1 Porous membrane (b1) 2 High density fibrous structure (b3) 3 Implant 4 Dense fibrous structure (a2) 5 fibrous structure (a1) 6 Porous membrane (b1) 7 Impregnation 8 High density fibrous structure (b3) 9 Outermost surface 10 Innermost surface 11 fibrous structure (b2) 12 High density fibrous structure (b3) 13 Outermost surface 14 Innermost surface

Claims

1. An immunoisolation device having a cell-trapping layer (A) and an immunoisolation layer (B) covering the cell-trapping layer (A), An immunoisolation device, wherein the cell-trapping layer (A) comprises a fibrous structure (a1).

2. The immunoisolation device of claim 1 , wherein the fibrous structure (a1) has a porosity of 90% or more.

3. The immunoisolation device of claim 1, wherein the thickness of the fiber structure (a1) is 100 to 2000 μm.

4. The immunoisolation device of claim 1, wherein the fibrous structure (a1) comprises at least one selected from the group consisting of ethylene vinyl alcohol copolymer and cellulose acetate.

5. the cell-trapping layer (A) comprises the fiber structure (a1) and a dense fiber structure (a2), and the dense fiber structure (a2) is disposed around the fiber structure (a1); The immunoisolation device of claim 1, wherein the dense fibrous structure (a2) has an average pore size of 35 μm or less.

6. The immunoisolation device of claim 5, wherein the dense fiber structure (a2) has a thickness of 1500 μm or less.

7. The immunoisolation device of claim 5 or 6, wherein the dense fiber structure (a2) comprises at least one selected from the group consisting of ethylene vinyl alcohol copolymer and cellulose acetate.

8. a cell-trapping layer (A) in which a dense fiber structure (a2) is arranged around the fiber structure (a1) and which can prevent escape of cells or cell aggregates trapped in the fiber structure (a1); The immunoisolation device according to claim 1, wherein the dense fiber structure (a2) has the function of preventing escape of cells or cell clusters.

9. The immunoisolation layer (B) comprises a porous membrane (b1) or a fiber structure (b2), The immunoisolation device of claim 1, wherein the porous membrane (b1) or the fibrous structure (b2) comprises at least one selected from the group consisting of ethylene-vinyl alcohol copolymer and cellulose acetate.

10. The immunoisolation device of claim 9, wherein the immunoisolation layer (B) is a multi-layered immunoisolation layer (B') comprising the porous membrane (b1) or the fibrous structure (b2) and a hydrogel (b3).

11. The immunoisolatory device of claim 10, wherein the multi-layered immunoisolatory layer (B') has a thickness of 500 μm or less.

12. The immunoisolation device described in claim 10 or 11, wherein the outermost layer of the multilayered immunoisolation layer (B') is the porous membrane (b1) or fiber structure (b2), and the innermost layer is the hydrogel (b3).

13. The immunoisolation device described in claim 10 or 11, wherein the outermost layer of the multilayered immunoisolation layer (B') is the hydrogel (b3) and the innermost layer is the porous membrane (b1) or fibrous structure (b2).

14. The immunoisolation device of claim 10 or 11, wherein the hydrogel (b3) comprises polyvinyl alcohol, and the average degree of polymerization of the polyvinyl alcohol is 300 to 10,000.

15. A method for producing an immunoisolation device comprising a cell-trapping layer (A) including a fiber structure (a1) and a multi-layered immunoisolation layer (B') covering the cell-trapping layer (A), comprising: (1) a step of applying a hydrosol solution to a porous membrane (b1); (2) forming a multi-layered immunoisolation layer (B') by hydrogelling the hydrosol solution using heat, temperature, light, or chemical action; and (3) A method for producing an immunoisolatory device, comprising the step of forming the multi-layered immunoisolatory layer (B') into a pouch by heat welding.

16. the hydrosol solution in step (1) contains polyvinyl alcohol; The degree of polymerization of the polyvinyl alcohol is 300 to 10,000, The method for producing an immunoisolation device according to claim 15, wherein the solids concentration of the hydrosol solution is 3 to 15% by mass.

17. The heat fusion in the step (3) is carried out by sandwiching a resin between two multi-layered immunoisolation layers (B'), and the two multi-layered immunoisolation layers (B') face each other with their hydrogel surfaces facing each other. The method for producing an immunoisolation device according to claim 15 or 16, wherein the resin comprises at least one selected from the group consisting of ethylene vinyl alcohol copolymer and cellulose acetate.