Immune isolation device

Abstract

It is an object of the present invention to provide an immune isolation device: which not only achieves a reduction in the diffusion distance to effectively improve the permeability of substances such as physiologically active substances and nutrients, but also achieves an improvement in durability to withstand long-term transplantation; and comprising a cell capture layer capable of preventing cells or cell clusters from escaping. Provided is an immunoisolation device comprising a cell capture layer (A) and an immunoisolation layer (B) covering the cell capture layer (A). The cell capture layer (A) includes a fiber structure (a1).

Description

Technical Field

[0001] The invention relates to an immunoisolation device. Background Art

[0002] Immunoisolation devices have been developed as a means of cell transplantation therapy without the need for the administration of immunosuppressants. In particular, for example, in the transplantation of somatic cells derived from iPS cells where the risk of carcinogenesis is concerned or when the function of transplanted cells is reduced, macroencapsulation immunoisolation devices are considered to be an effective method because the transplantation site can be confirmed and the device can be replaced. The important functions required for macroencapsulation immunoisolation devices are as follows: cells or cell clusters can be uniformly dispersed and fixed without inducing the binding of cells or cell clusters; the device allows oxygen and nutrients to easily penetrate into transplanted cells; the device allows the expected physiologically active substances (cytokines, hormones, growth factors, etc.) required for the therapeutic effect to be easily released by cells according to cell responses; prevent the penetration of immune response cells and immune response factors; the transplant device has excellent biocompatibility, and is less likely to adhere to surrounding tissues, and is less likely to induce inflammatory reactions such as granulation.

[0003] Many immunoisolation devices using porous membranes have been studied (Patent Document (PTL) 1), and have been considered to have a problem of reduced permeability due to protein adsorption to the porous membrane material and / or fibrosis, or reduced permeability due to adhesion to surrounding tissues.

[0004] When the diameter exceeds 500 μm, it is believed that internal necrosis of cell clusters and the like is induced. In order to easily maintain the implantation of embedded cells into the recipient and release physiologically active substances, the total thickness of such an immunoisolation device must be appropriately controlled, and it is desirable that the immunoisolation layer constituting the device is as thin as possible. On the other hand, in order to be transplanted into the body, the strength and durability of the membrane must be improved to prevent the device from breaking or twisting. However, it is not easy to simultaneously achieve a reduction in membrane thickness and an improvement in durability.

[0005] Citation List

[0006] Patent Literature

[0007] PTL 1:JP2012-508584A Summary of the invention

[0008] Technical issues

[0009] An object of the present invention is to provide an immunoisolation device which is highly durable in long-term transplantation and is suitable for improving the diffusion efficiency of physiologically active substances required for transplantation while maintaining the immunoisolation effect.

[0010] Another object of the present invention is to provide an invention that simultaneously achieves a reduction in diffusion distance and an improvement in durability in the device while having excellent biocompatibility and maintaining immunoisolation properties without interfering with the implantation of materials to be transplanted, such as cells and cell clusters.

[0011] Furthermore, another object of the present invention is to provide an immunoisolation device comprising a cell capture layer capable of preventing cells or cell clusters from escaping.

[0012] Solutions to the Problem

[0013] The present invention provides the following immunoisolation device. [1]

[0015] An immunoisolation device comprising

[0016] Cell capture layer (A) and

[0017] an immunoisolation layer (B) covering the cell capture layer (A),

[0018] The cell-trapping layer (A) comprises a fiber structure (a1). [2]

[0020] The immunoisolation device according to [1], wherein the fiber structure (a1) has a porosity of 90% or more. [3]

[0022] The immunoisolation device according to [1] or [2], wherein the fiber structure (a1) has a thickness of 100 to 2000 μm. [4]

[0024] The immunoisolation device according to any one of [1] to [3], wherein the fiber structure (a1) comprises at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate. [5]

[0026] The immunoisolation device according to any one of [1] to [4], wherein

[0027] The cell-trapping layer (A) comprises the fiber structure (a1) and the dense fiber structure (a2), and

[0028] The dense fiber structure (a2) is arranged around the fiber structure (a1) and has an average pore size of 35 μm or less. [6]

[0030] The immunoisolation device according to [5], wherein the dense fiber structure (a2) has a thickness of 1500 μm or less. [7]

[0032] The immunoisolation device according to [5] or [6], wherein the dense fiber structure (a2) comprises at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate. [8]

[0034] An immunoisolation device according to [1], wherein

[0035] The cell-trapping layer (A) comprises a dense fiber structure (a2) arranged around the fiber structure (a1) and capable of preventing cells or cell clusters trapped in the fiber structure (a1) from escaping, and

[0036] The dense fiber structure (a2) has the function of preventing cells or cell clusters from escaping. [9]

[0038] The immunoisolation device according to any one of [1] to [8], wherein

[0039] The immunoisolation layer (B) comprises a porous membrane (b1) or a fiber structure (b2), and

[0040] The porous film (b1) or the fiber structure (b2) includes at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate.

[10]

[0042] The immunoisolation device according to [9], wherein the immunoisolation layer (B) is an immunoisolation multilayer (B') comprising the porous membrane (b1) or the fiber structure (b2) and a hydrogel (b3).

[11]

[0044] The immunoisolation device according to

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

[12]

[0046] The immunoisolation device according to

[10] or

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

[13]

[0048] The immunoisolation device according to

[10] or

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

[14]

[0050] The immunoisolation device according to any one of

[10] to

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

[15]

[0052] A method for producing an immunoisolation device, the immunoisolation device comprising a cell capture layer (A) and an immunoisolation multilayer (B'),

[0053] The cell-capturing layer (A) comprises a fiber structure (a1), and

[0054] The immunoisolation multilayer covers the cell capture layer (A),

[0055] The method comprises the steps of:

[0056] (1) applying a hydrosol solution to a porous membrane (b1);

[0057] (2) converting the hydrosol solution into a hydrogel by applying heat, temperature, light or chemical action to form an immunoisolation multilayer (B'); and

[0058] (3) The immunoisolation multilayer (B') is molded into a bag shape by heat fusion.

[16]

[0060] The method for producing an immunoisolation device according to

[15] , wherein the hydrosol solution in step (1) comprises polyvinyl alcohol,

[0061] The polyvinyl alcohol has a degree of polymerization of 300 to 10,000, and

[0062] The hydrosol solution has a solid concentration of 3 to 15% by mass.

[17]

[0064] The method for producing an immunoisolation device according to

[15] or

[16] , wherein

[0065] The heat fusion in step (3) is performed with the resin interposed between the two sheets of the immuno-isolating multilayer (B'), and the two sheets of the immuno-isolating multilayer (B') are placed with their hydrogel surfaces facing each other, and

[0066] The resin includes at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate.

[0067] Effects of the Invention

[0068] The present invention can provide an immunoisolation device that achieves both a reduction in diffusion distance to effectively improve the permeability of substances such as physiologically active substances and nutrients and an improvement in durability to withstand long-term transplantation, wherein the immunoisolation device has excellent biocompatibility and further comprises a cell capture layer capable of preventing the escape of cells or cell clusters. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 is a perspective view of a bag-shaped immunoisolation device.

[0070] Figure 2 is a cross-sectional view of a tubular immunoisolation device.

[0071] Figure 3A A conceptual diagram of an immunoisolation device using a fiber structure (nonwoven) as a cell capture layer.

[0072] Figure 3B A conceptual diagram of an immunoisolation device using a fiber structure (nonwoven) as a cell capture layer.

[0073] Figure 4 This is a conceptual diagram of the cell capture layer.

[0074] Figure 5 is a transmitted light microscopy image of a dense fiber structure (a2).

[0075] Figure 6 is a cross-sectional view of an immunoisolation layer having a double-layer structure consisting of a porous membrane and a hydrogel.

[0076] Figure 7 is a cross-sectional view of an immunoisolation multilayer formed by nonwoven and hydrogel.

[0077] Figure 8 is a SEM image of a cross section of the immunoisolation layer (B) obtained in Production Example 1.

[0078] Fig. 9 It is an outline of material permeability testing. DETAILED DESCRIPTION

[0079] The use of the singular forms ("a", "an" and "the") in the present specification should be construed to include the singular and the plural, unless the context clearly indicates that only the singular is intended.

[0080] In the present specification, the term "comprising" is a concept covering "consisting essentially of" and "consisting only of."

[0081] The immunoisolation device of the present invention comprises a cell capture layer (A) capable of capturing cells as materials to be transplanted, and an immunoisolation layer (B), wherein the cell capture layer (A) is covered by the immunoisolation layer (B), thereby inhibiting immune cells and cytokines from invading the cell capture layer (A). The cell capture layer (A) comprises 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).

[0082] Cell capture layer (A)

[0083] Fiber structure (a1)

[0084] The fiber structure (a1) contained in the cell-capturing layer (A) serves as a scaffold for capturing cells or cell clusters that are materials to be transplanted. The fiber structure of the immunoisolation layer (a1) of the cell-capturing layer (A) is, for example, a nonwoven, a woven fabric or a knitted fabric, preferably a nonwoven.

[0085] The material of the fiber structure (a1) preferably contains at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate, and more preferably contains ethylene-vinyl alcohol copolymer. Such materials can be used alone to form the fiber structure (a1), or two or more of them can be used in combination to form the fiber structure (a1). The fiber structure (a1) can be a cell scaffold material, such as collagen fiber.

[0086] Ethylene-vinyl alcohol copolymers can generally be obtained by saponifying ethylene-vinyl ester copolymers. The production and saponification of ethylene-vinyl ester copolymers can be carried out by known methods. A representative example of vinyl esters 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 can also be used.

[0087] The ethylene unit content of the ethylene-vinyl alcohol copolymer is preferably 20 mol% or more, more preferably 25 mol% or more. The ethylene unit content of the ethylene-vinyl alcohol copolymer is preferably 60 mol% or less, more preferably 55 mol% or less, and even more preferably 50 mol% or less.

[0088] 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 can be determined by 1 The peak area of ​​hydrogen atoms contained in the vinyl ester structure and the peak area of ​​hydrogen atoms contained in the vinyl alcohol structure were measured and calculated.

[0089] In addition, the ethylene-vinyl alcohol copolymer may contain units derived from other monomers other than ethylene, vinyl esters and saponification products thereof, as long as the purpose of the present invention is not impaired. When the ethylene-vinyl alcohol copolymer contains such other monomer units, the content of such other monomer units is preferably 30 mol% or less, more preferably 20 mol% or less, even more preferably 10 mol% or less, and particularly preferably 5 mol% or less, based on the total monomer units of the ethylene-vinyl alcohol copolymer. When the ethylene-vinyl alcohol copolymer contains units derived from such other monomers, the lower limit of the content may be 0.05 mol% or may be 0.10 mol%. Examples of such other monomers include olefins such as propylene, butene, pentene and hexene; olefins having an ester group such as 3-acyloxy-1-propylene, 3-acyloxy-1-butene, 4-acyloxy-1-butene, 3,4-diacyloxy-1-butene, 3-acyloxy-4-methyl-1-butene, 4-acyloxy-2-methyl-1-butene, 4-acyloxy-3-methyl-1-butene, 3,4-diacyloxy-2-methyl-1-butene, 4-acyloxy-1-pentene, 5-acyloxy-1-pentene, 4,5-diacyloxy-1-pentene, 4-acyloxy-1-hexene, 5-acyloxy-1-hexene, 6-acyloxy-1-hexene, 5,6-diacyloxy-1-hexene and 1,3-diacetoxy-2-methyl-1-butene; -methylene prylene (1,3-diacetoxy-2-methyleneprylene) or its saponification product; unsaturated acids such as acrylic acid, methacrylic acid, crotonic acid and itaconic acid, or their anhydrides, salts or monoalkyl esters 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 methallyl sulfonic acid, or their salts; vinyl silane compounds such as vinyl trimethoxysilane, vinyl triethoxysilane, vinyl tri(β-methoxy-ethoxy) silane and γ-methacryloxypropyl methoxysilane; and alkyl vinyl ethers, vinyl ketones, N-vinyl pyrrolidone, vinyl chloride, vinylidene chloride, etc.

[0090] The ethylene-vinyl alcohol copolymer can be post-modified by amination, acetalization, cyanoethylation, oxyalkyleneization, or the like.

[0091] The ethylene-vinyl alcohol copolymer can be used alone or in combination of two or more.

[0092] The fiber structure (a1) can be formed by combining ethylene-vinyl alcohol copolymer with polyester and the like. The fiber structure (a1) can also be composed of a composite fiber with a core-skin structure, wherein the core of the fiber is polyester and the skin is ethylene-vinyl alcohol copolymer. The cross-sectional structure of the composite fiber can be parallel (parallel or multi-layer bonding) or eccentric core-skin type. In such a case, it is preferred that the ethylene-vinyl alcohol copolymer covers more than 50% of the fiber surface. In addition, the fiber structure (a1) preferably contains more than 50% by mass of the composite fiber, and may further contain fibers made of other materials. Examples of other materials include polyester.

[0093] The fibers used in the fiber structure (a1) preferably have an average fineness of 1.0 to 10 dtex. The fineness within the above range is preferred because spaces for capturing cells or cell clusters are easily obtained.

[0094] There is no particular restriction on the average fiber length of the fibers used in the fiber structure (a1), and the fibers may be long fibers or short fibers. When the fiber structure (a1) is produced by the CAD method, the average fiber length is preferably 32 to 62 mm from the viewpoint of spinnability.

[0095] The fiber structure (a1) may consist of one fiber structure, or a laminate of two or more fiber structures.

[0096] The fiber structure (a1) preferably has a porosity of 90% or more, more preferably 93% or more, and even more preferably 95% or more. The porosity between the fibers in the fiber structure (a1) within the above range is preferred because it makes it easy for cells or cell clusters to be captured in the fiber structure (a1). There is no particular restriction on the upper limit of the porosity of the fiber structure (a1), but it is preferably, for example, 99.9% or less, more preferably 99.5% or less, and even more preferably 99.0% or less. For example, the porosity of the fiber 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%.

[0097] In this specification, the porosity can be expressed by the basis weight C (g / m 2 ), thickness D (cm) and average specific gravity E (g / cm 2 ) is calculated according to the following formula.

[0098] Porosity (%) = 100 - ((C / D / E) x 10 -4 x 100)

[0099] The thickness of the fiber structure (a1) is preferably 100 to 2000 μm, more preferably 300 to 1500 μm, and still more preferably 300 to 1000 μm. The thickness within the above range is preferred because a sufficient number of cells can be retained and oxygen supply to cells is less likely to be hindered.

[0100] The basis weight of the fiber structure (a1) is preferably 20 to 300 g / m 2 , more preferably 30 to 200 g / m 2 , and more preferably 40 to 100 g / m 2 .

[0101] The area of ​​the fiber structure (a1) is not particularly limited, but is preferably 1.5 to 150 cm 2 .

[0102] There is no particular limitation on the method for producing the fiber structure (a1). However, it is preferred to form bonding points between fibers by thermal bonding, and preferably use a steam jet method to provide spaces that can capture cells or cell clusters and obtain hardness against compression.

[0103] Dense fiber structure (a2)

[0104] The cell capture layer (A) preferably includes a dense fiber structure (a2) in addition to the fiber structure (a1), which has the function of preventing the cells or cell clusters captured in the fiber structure (a1) from escaping. The dense fiber structure (a2) can 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 can be laminated on both sides of the fiber structure (a1) to form a multilayer of dense fiber structure (a2) / fiber structure (a1) / dense fiber structure (a2). The dense fiber structure (a2) can be arranged around the fiber structure (a1) so that part or all of the fiber structure (a1) is covered by the dense fiber structure (a2). In order to prevent the cells or cell clusters trapped in the fiber structure (a1) from escaping, it is preferred to form a laminate of dense fiber structure (a2) / fiber structure (a1) / dense fiber structure (a2) or to arrange the dense fiber structure (a2) around the fiber structure (a1) to cover the entire fiber structure (a1). It is more preferred to arrange the dense fiber structure (a2) to cover the entire periphery of the fiber structure (a1). When laminating the dense fiber structure (a2) / fiber structure (a1) / dense fiber structure (a2), it is preferred to laminate the dense fiber structure (a2) on the fiber structure (a1) to cover the entire periphery of the fiber structure (a1).

[0105] The function of preventing cells or cell clusters from escaping means that cells or cell clusters are captured and do not escape from the cell-capturing layer. For example, it means that the cell viability evaluated in the examples below is 80% or more.

[0106] The dense fiber structure (a2) can be a nonwoven, a woven fabric or a knitted fabric, wherein the nonwoven is preferred because the pore size can be easily controlled by production conditions. The material of the dense fiber structure (a2) preferably contains at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate, more preferably contains ethylene-vinyl alcohol copolymer. Examples of ethylene-vinyl alcohol copolymers that can be preferably used are those described in the explanation of the fiber structure (a1). The dense fiber structure (a2) can also be composed of composite fibers with a skin-core structure, wherein the core of the fiber is polyester and the skin is ethylene-vinyl alcohol copolymer.

[0107] 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 average pore size of the dense fiber structure (a2) is 5 μm or more, it is less likely to hinder the supply of oxygen, etc. to the cells or cell clusters captured in the fiber structure (a1). The average pore size of the dense fiber structure (a2) of 35 μm or less is preferred because the cells or cell clusters captured in the fiber structure (a1) are less likely to escape from the fiber structure (a1).

[0108] In this specification, the average pore size can be measured by the following procedure. Use a stereo microscope to take a photo of the surface of the dimensional structure (a2) at a magnification of 10 times under transmitted light. Using image analysis software, 50 or more light-transmitting bright spots are extracted from the microphotographs, and the area of ​​each bright spot is measured. The diameter calculated as the area of ​​a perfect circle is taken as the aperture of the bright spot. The average pore size is determined by calculating the average pore size of 50 or more bright spots.

[0109] The dense fiber structure (a2) preferably has a thickness of 1500 μm or less, more preferably 1000 μm or less, still 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, more preferably 100 μm or more. A thickness of 50 to 1500 μm is preferred because it is less likely to hinder the supply of oxygen to cells, etc.

[0110] The dense fiber structure (a2) preferably has a density of 3 to 300 g / m 2 , more preferably 10 to 100 g / m 2 The basis weight within the above range is preferred because a preferred average pore diameter and a preferred thickness can be achieved.

[0111] The dense fiber structure (a2) preferably has a porosity of less than 90%, more preferably less than 87%, even more preferably less than 85%, and particularly preferably less than 80%. The porosity of the dense fiber structure (a2) is preferably 30% or more, more preferably more than 50%, even more preferably more than 60%, and particularly preferably more than 70%. The porosity between the fibers in the dense fiber structure (a2) within the above range is preferred because it tends to enhance the function of preventing cells or cell clusters from escaping. 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%.

[0112] The area of ​​the dense fiber structure (a2) is not particularly limited, but is preferably 1.0 to 400 cm 2 , more preferably 1.0 to 200 cm 2 , even more preferably 1.5 to 150 cm 2 The area of ​​the dense fiber structure (a2) is preferably larger than the area of ​​the fiber structure (a1) so that the dense fiber structure (a2) can cover the fiber structure (a1).

[0113] The method for producing the dense fiber structure (a2) is not particularly limited, but preferably a melt-blowing method is used to achieve a suitable average pore size. The surface of the dense fiber structure (a2) may be subjected to calendering.

[0114] The number of cells retained in the cell-trapping layer (A) is not particularly limited, but is preferably 1×10 5 Up to 1×10 7 cells / cm 3 , more preferably 1×10 6 Up to 1×10 7 cells / cm 3 .

[0115] Immune isolation layer (B)

[0116] The immunoisolation device of the present invention comprises an immunoisolation layer (B) covering a cell-capturing layer (A). In a typical embodiment, from the perspective of obtaining a sufficient immunoisolation effect, it is preferred that the immunoisolation layer (B) in the device of the present invention covers the entire surface of the cell-capturing layer (A). For the same reason, the immunoisolation layer preferably does not have any pores, such as pinholes, that penetrate the membrane.

[0117] The immunoisolation layer (B) is preferably an immunoisolation multilayer (B') comprising a porous membrane (b1) or a fiber structure (b2) and a hydrogel (b3).

[0118] Porous membrane (b1)

[0119] The porous membrane (b1) of the immunoisolation layer (B) is a membrane having a plurality of pores. It can be confirmed that the membrane is a porous membrane by a scanning electron microscope (SEM) or transmission electron microscope (TEM) image of a membrane cross section. The porous membrane is preferably a semipermeable membrane.

[0120] 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 preferred because it is less likely to hinder the supply of oxygen and other substances to cells while maintaining the strength of the immunoisolation layer (B).

[0121] The average pore size of the porous film (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 a SEM image or a TEM image. For example, the surface of the porous film is observed using SEM, and 50 pores are randomly selected from the pores formed on the surface. The major diameter of each pore is measured, and the average value of the major diameters of the 50 pores is calculated to obtain the average pore size.

[0122] The maximum pore size of porous membrane (b1) is not particularly limited, but preferably 0.01 μm to 10 μm, more preferably 0.01 μm to 5 μm, and more preferably 0.01 μm to 4 μm. When the maximum pore size is within the above range, immune response cells and immune system humoral factors can be inhibited from invading the device, while allowing nutrients such as carbon sources such as amino acids, vitamins, inorganic salts, glucose and physiologically active substances such as oxygen, carbon dioxide, cytokines, hormones and insulin to fully penetrate. The maximum pore size can be determined by SEM images or TEM images. For example, the surface of the porous membrane is observed using SEM, and 50 pores are randomly selected from the pores formed on the surface. The major diameter of each pore is measured, and the maximum value in 50 major diameters is defined as the maximum pore size.

[0123] Since it is necessary to have the function of inhibiting cell infiltration from the recipient and preventing leakage of cells as materials to be transplanted, the average pore size or maximum pore size of the porous membrane (b1) is desirably smaller than the cell size and is 5 μm or less.

[0124] The porous membrane (b1) comprises a polymer. Preferably, the porous membrane (b1) consists essentially of a polymer.

[0125] The example of this polymer includes thermoplastic resin or thermosetting resin.The specific example of this polymer includes ethylene-vinyl alcohol copolymer, polysulfone, cellulose acetate class such as cellulose acetate, nitrocellulose, sulfonated polysulfone, polyether sulfone, polyacrylonitrile, styrene-acrylonitrile copolymer, styrene-butadiene copolymer, polyvinyl alcohol, polycarbonate, organosiloxane-polycarbonate copolymer, polyester carbonate, organopolysiloxane, polyphenylene oxide, polyamide, polyimide, polyamide-imide, polybenzimidazole and polytetrafluoroethylene (PTFE).Considering solubility, optical property, electrical property, intensity, elasticity etc., this polymer can be homopolymer or can be copolymer, polymer blend, polymer alloy etc.The polymer constituting porous membrane can contain hydrophilic polymer, such as polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose or polyethylene glycol.Combination hydrophilic polymer and hydrophobic polymer can improve biocompatibility.

[0126] The polymer used to form the porous membrane (b1) is preferably a highly biocompatible material that is unlikely to cause adhesion to the recipient tissue around the transplant site or to cause inflammation, etc. The porous membrane (b1) preferably contains at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate, and more preferably contains ethylene-vinyl alcohol copolymer. Examples of ethylene-vinyl alcohol copolymers that can be preferably used are those described in the explanation of the fiber structure (a1).

[0127] The porous membrane (b1) may be composed of one type of porous membrane, or may be formed by a laminate of two or more types of porous membranes. When the porous membrane (b1) comprises two or more porous membranes, these porous membranes may be directly laminated, or a hydrogel or fiber structure may be sandwiched between the two porous membranes.

[0128] In a preferred embodiment, the porous membrane is a single layer formed from one composition. In a preferred embodiment, the porous membrane does not have a laminated structure comprising a plurality of layers.

[0129] Fiber structure (b2)

[0130] The fibrous structure (b2) of the immunoisolation layer (B) is, for example, a nonwoven, a woven fabric or a knitted fabric. The fibrous structure (b2) is preferably a nonwoven. The fibers are bonded or entangled together to form the fibrous structure by heat, mechanical or chemical action. The basis weight (weight per unit area) can be adjusted by adjusting the fiber diameter and / or the fiber amount, so that in addition to strength, permeability, filtration, etc. can also be controlled. The fibrous structure (b2) preferably has a molecular weight of 10 to 100 g / m 2The thickness of the fiber structure (b2) is preferably 300 μm or less, more preferably 200 μm or less and as thin as possible, taking into account the diffusion efficiency of the physiologically active substance from the material to be transplanted. There is no particular restriction on the lower limit of the thickness of the fiber structure (b2), but for example, it is preferably 50 μm or more, more preferably 100 μm or more.

[0131] The example of the fiber material of fiber structure (b2) includes gelatin, collagen, chitin, chitosan, fibronectin, dextran, cellulose, polyethylene (PE), polypropylene (PP), polyurethane, polyamide, polyester, polyvinyl alcohol (PVA), ethylene-vinyl alcohol copolymer, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, PVA (such as methacrylic acid modified PVA and acrylic acid modified PVA) modified with monomer, polycaprolactone, polyglycerol sebacate, polyhydroxyalkanoic acid, polybutylene succinate, polymethylene carbonate, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, cellulose acetate such as carboxymethylcellulose, silk core protein and silk. As with porous membrane (b1), the fiber material of this fiber structure is preferably biocompatible. Fiber structure (b2) preferably contains at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate, more preferably contains ethylene-vinyl alcohol copolymer. The example of ethylene-vinyl alcohol copolymer that can be preferably used includes those described in the explanation of fiber structure (a1). The surface of the fibrous structure is preferably smoothed by thermal, mechanical or chemical treatment.

[0132] Hydrogel (b3)

[0133] Examples of hydrosols for producing the hydrogel (b3) of the immunoisolation layer (B) include sols that gel in the presence of metal ions to form hydrogels; sols that gel in response to pH to form hydrogels; and sols that gel in response to light to form hydrogels. Metal ions and pH are examples of chemical actions. In order to gel these hydrosols, operations can be performed according to the characteristics of the gel used, such as contacting metal ions with the hydrosol, adjusting the temperature to the gelling condition, adjusting the pH to the gelling condition, applying light under the gelling condition, or providing a magnetic field under the gelling condition.

[0134] Examples of hydrogels obtainable by gelation in the presence of metal ions include alginate gels obtainable by gelation in the presence of divalent or trivalent metal ions, preferably alkaline earth metal ions, such as calcium ions or magnesium ions; carrageenan gels obtainable by gelation in the presence of calcium ions and / or potassium ions; and acrylic acid-based synthetic gels obtainable by gelation in the presence of sodium ions.

[0135] Examples of pH-responsive hydrogels include alginate gel, chitosan gel, carboxymethyl cellulose gel, and acrylic acid-based synthetic gel.

[0136] Examples of photoresponsive hydrogels include synthetic gels in which azobenzene and cyclodextrin are incorporated in the backbone, gels composed of supramolecules having fumaric acid amide as spacers, gels obtainable by crosslinking or bonding through nitrobenzyl groups, and gels composed of modified polyvinyl alcohol.

[0137] The modified polyvinyl alcohol may be, for example, (meth)acryloyl-modified polyvinyl alcohol. The (meth)acryloyl group may be introduced by subjecting the hydroxyl group on the side chain of the polyvinyl alcohol to an esterification reaction or an ester exchange reaction with a compound containing an ethylenically unsaturated group in the presence of a base. Examples of compounds containing an ethylenically unsaturated group include (meth)acrylic acid or derivatives thereof, such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acryloyl halide, and (meth)acrylate.

[0138] Preferred examples of hydrogel (b3) include polyvinyl alcohol, polyethylene glycol, chitosan, alginate, etc. Hydrogel (b3) preferably contains at least one member selected from polyvinyl alcohol and polyethylene glycol, more preferably contains polyvinyl alcohol. Polyvinyl alcohol-based polymers can be produced, for example, by saponification of polyvinyl ester and conversion of ester groups in polyvinyl ester into hydroxyl groups, and the polyvinyl ester can be obtained by polymerization of vinyl ester monomers.

[0139] Examples of vinyl ester monomers include aliphatic vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate. These vinyl ester monomers can be used alone or in combination of two or more.

[0140] Among the vinyl ester monomers, aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of production cost. In other words, the polyvinyl ester is preferably polyvinyl acetate obtainable by polymerization of vinyl acetate.

[0141] Polyvinyl ester may optionally contain structural units derived from monomers other than vinyl ester monomers as long as the effects of the present invention are not impaired. Examples of such other monomers include α-olefins such as ethylene, propylene, n-butene and isobutylene; acrylic acid or its salts; acrylic acid alkyl ester compounds such as methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate and octadecyl acrylate; methacrylic acid or its salts; methacrylic acid alkyl ester compounds such as methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate and octadecyl methacrylate; acrylamide derivatives such as acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetoneacrylamide, acrylamidopropanesulfonic acid or its salts, acrylamidopropyldimethylamine or its salts or quaternary salts, and N-hydroxymethyl acrylamide or its derivatives; methacrylamide derivatives such as methacrylamide, N-methyl methacrylamide, N-ethyl methacrylamide, methacrylamidopropanesulfonic acid or its salts, methacrylamidopropyl dimethylamine or its salts or quaternary salts, N-hydroxymethyl methacrylamide or its derivatives; N-vinylamide derivatives such as N-vinyl formamide and N-vinylacetamide; vinyl ether compounds such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, isopropyl vinyl ether, n-butyl vinyl ether, isobutyl vinyl ether, tert-butyl vinyl ether, dodecyl vinyl ether and stearyl vinyl ether; 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 its salts, esters or anhydrides; vinyl silyl compounds such as vinyl trimethoxysilane; and isopropenyl acetate. These may be used alone or in combination of two or more.

[0142] The average polymerization degree of polyvinyl alcohol is preferably 300 to 10000, more preferably 500 to 5000, still more preferably 1000 to 5000, still more preferably 1500 to 5000, particularly preferably 2000 to 5000. The thickness within the above range is preferred in view of the permeability of substances and ease of handling in multilayer formation.

[0143] The average degree of polymerization of polyvinyl alcohol in this specification refers to the average degree of polymerization measured in accordance with JIS K 6726: 1994. Specifically, the average degree of polymerization can be determined from the intrinsic viscosity measured in 30° C. water after the raw material PVA has been saponified and purified.

[0144] From the viewpoint of improving the water solubility of polyvinyl alcohol, the saponification degree of polyvinyl alcohol is preferably 50 mol % or more, more preferably 60 mol % or more, and still more preferably 65 mol % or more.

[0145] From the viewpoint of suppressing the hydrosol solution from having high viscosity and improving the storage stability of the hydrosol solution, the saponification degree of the polyvinyl alcohol is preferably 99.9 mol % or less, more preferably 99.5 mol % or less, and still more preferably 99.0 mol % or less.

[0146] In this specification, the saponification degree of polyvinyl alcohol refers to the ratio (mol %) of the number of moles of vinyl alcohol units relative to the total number of moles of structural units (e.g., vinyl acetate units) and vinyl alcohol units in the raw material PVA that can be converted into vinyl alcohol units by saponification. The saponification degree can be measured according to JIS K 6726:1994.

[0147] The thickness of the hydrogel (b3) is not particularly limited, but is preferably 1 to 300 μm, more preferably 5 to 200 μm, and still more preferably 10 to 100 μm.

[0148] Cross-linking of the hydrogel (b3) enables regulation of permeability, strength, etc. of nutrients such as glucose, physiologically active substances such as insulin, and humoral factors of the immune system.

[0149] 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 by the following procedure using a tensile tester.

[0150] First, the hydrosol solution was poured between two glass plates separated from each other by inserting a 1 mm thick spacer between them, and processed under predetermined gelling conditions to obtain a gel sheet with a thickness of 1 mm. A test piece was cut from the gel sheet using a dumbbell cutter specified in JIS K 6251-3. The test piece was set on a tensile tester (model 5566) produced by Instron Co., Ltd., and the fracture stress and fracture strain were measured while collecting image data. The stress when the test piece fractured was defined as the gel strength.

[0151] Structure of immune isolation layer (B) etc.

[0152] The immunoisolation layer (B) is preferably a porous membrane (b1) comprising at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate, or a fiber structure (b2) comprising at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate. In addition, the immunoisolation layer (B) may have a multilayer structure, which contains one or more layers different from the porous membrane (b1) and the fiber structure (b2) in addition to the porous membrane (b1) or the fiber structure (b2). A preferred example of a layer different from the porous membrane (b1) and the fiber structure (b2) is a hydrogel (b3). In the present specification, the immunoisolation layer comprising a porous membrane (b1) or a fiber structure (b2) and a hydrogel (b3) may be referred to as an immunoisolation multilayer (B').

[0153] Forming a multilayer with hydrogel (b3) not only improves immunoisolation, but also increases the strength of the immunoisolation layer (B). The permeability and immunoisolation can be adjusted by adjusting the pore size of the porous membrane and the gel strength and / or crosslinking degree of the hydrogel (b3).

[0154] Cell infiltration and cell leakage can be suppressed by the porous membrane (b1). However, it is not easy to suppress the infiltration of immune system humoral factors such as IgG antibodies without reducing the permeability of essential physiologically active substances. Therefore, by adjusting the gel strength or cross-linking density of the hydrogel (b3) formed with the porous membrane (b1) to form a multilayer, the infiltration of immune system humoral factors such as IgG antibodies can be suppressed without reducing the permeability of physiologically active substances.

[0155] When the fiber structure layer (b2) used is a single layer, it is not easy to suppress the infiltration of immune system humoral factors such as IgG antibodies and cell infiltration and cell leakage without reducing the permeability of necessary physiologically active substances. Therefore, by adjusting the gel strength or cross-linking density of the hydrogel (b3) formed with the porous membrane (b2) to form a multilayer, it is possible to suppress the infiltration of immune system humoral factors such as IgG antibodies and cell infiltration and cell leakage without reducing the permeability of physiologically active substances.

[0156] The porous membrane (b1), the fiber structure (b2) and the hydrogel (b3) each form a layer, and the layers may be clearly separated at their boundaries, or the boundary between two adjacent layers may not be clearly distinct, or two or three different types of layers may be integrated to form one layer.

[0157] For example, when the immunoisolation layer comprises two types of layers, namely, a fibrous structure (b2) and a hydrogel (b3), the immunoisolation layer may have a structure in which the fibrous structure (b2) and the hydrogel (b3) are clearly separated, or there may be a mixed layer of the fibrous structure and the hydrogel between the fibrous structure and the hydrogel, or the fibrous structure and the hydrogel may be completely integrated into one layer.

[0158] When the immunoisolation layer comprises two types of layers, namely, a fibrous structure (b2) and a porous membrane (b1), the immunoisolation layer may have a structure in which the fibrous structure and the porous membrane are clearly separated, or there may be a mixed layer of the fibrous structure and the porous membrane between the fibrous structure and the hydrogel, or the fibrous structure and the porous membrane may be completely integrated into one layer.

[0159] When the immunoisolation layer comprises two types of layers, namely, a hydrogel (b3) and a porous membrane (b1), the immunoisolation layer may have a structure in which the hydrogel and the porous membrane are clearly separated, or a mixed layer of the hydrogel and the porous membrane may exist between the hydrogel and the porous membrane, or the hydrogel and the porous membrane may be completely integrated into one layer.

[0160] The immunoisolation device according to a particularly preferred embodiment of the present invention is composed of any one of the following three multilayer structures (i) to (iii):

[0161] (i) A porous film (b1) is used as a substrate, and the hydrogel (b3) is applied to the porous film (b1) or the porous film (b1) is impregnated with the hydrogel (b3) to form a multilayer structure.

[0162] (ii) The fiber structure (b2) is used as a substrate, and the hydrogel (b3) is applied to the fiber structure (b2) or the fiber structure (b2) is impregnated with the hydrogel (b3) to form a multilayer structure.

[0163] (iii) Using the structural fiber (b2) as a substrate, and forming a porous film (b1) on the structural fiber (b2). In addition, applying the hydrogel (b3) to the porous film (b1) or impregnating the porous film (b1) with the hydrogel (b3) to form a multilayer structure.

[0164] When the immunoisolation layer (B) comprises a plurality of layers, the layers may be bonded by adhesives, heat, pressure, etc. When adjacent layers are formed of highly compatible materials, the layers may be bonded by sequentially forming the layers.

[0165] Specifically, porous membrane (b1) is used as substrate, and hydrosol solution is directly applied to the porous membrane and formed into hydrogel by heat, temperature, light or chemical action, to form a multilayer structure. Alternatively, fiber structure (b2) is used as substrate, and hydrosol solution is directly applied to the fiber structure and formed into hydrogel by heat, temperature, light or chemical action, to form a multilayer structure. When using fiber structure (b2) coated with hydrogel (b3) such as nonwoven fabric to replace porous membrane (b1), it is possible to form an immunoisolation layer (B) with a material permeability higher than porous membrane (b1) and higher strength, while hydrogel (b3) ensures immunoisolation.

[0166] The hydrosol solution preferably has a solid concentration of 3 to 15% by mass, more preferably 3 to 10% by mass, still more preferably 3 to 8% by mass, and particularly preferably 3 to 5% by mass. A solid concentration within the above range is preferred because the permeability of immune system fluid factors such as IgG can be suppressed while maintaining the permeability of substances such as glucose and insulin.

[0167] Alternatively, a hydrogel (b1) is used as a substrate, and a polymer solution as a raw material of the porous membrane (b1) is directly applied to the substrate. The raw material of the porous membrane (b1) solidifies by phase separation (which is a phase change phenomenon), thereby forming the porous membrane (b1). Some of the raw materials of the porous membrane (b1) may require pretreatment to reduce the water content by pre-drying the hydrogel (b3).

[0168] In a preferred embodiment of the present invention, a fiber structure (b2) with excellent durability is used as a substrate; and a separate hydrogel (b3) or a porous membrane (b1) and a hydrogel (b3) are formed on the fiber structure, thereby achieving a reduction in the thickness of the device to improve the diffusion efficiency of physiologically active substances, and achieving durability due to increased strength while maintaining an immunoisolation effect.

[0169] There is no particular limitation on the thickness of the immunoisolation layer (B), but it is preferably 10 μm or more and 500 μm or less, more preferably 300 μm or less, even more preferably 200 μm or less, even more preferably 170 μm or less, and particularly preferably 150 μm or less. Even when the immunoisolation layer (B) is an immunoisolation multilayer (B'), the preferred thickness of the immunoisolation layer is within the above range. Considering the mass diffusion efficiency of the physiologically active substance from the material to be transplanted, the thickness of the immunoisolation multilayer (B) is preferably 500 μm or less, more preferably 300 μm or less, even more preferably 100 μm or less, and as thin as possible.

[0170] The area of ​​the immunoisolation layer (B) is not particularly limited, but is preferably 1.0 to 400 cm 2 , more preferably 1.0 to 200 cm 2 , even more preferably 1.5 to 150 cm 2 The area of ​​the immunoisolation layer (B) is preferably equal to or larger than the area of ​​the cell-trapping layer (A) so as to cover the cell-trapping layer (A).

[0171] The outermost layer of the immunoisolation layer (B) is preferably biocompatible to prevent the immunoisolation layer from being recognized as a foreign body. The immunoisolation layer needs to have the necessary permeability to allow sufficient penetration of oxygen and nutrients into the material to be implanted present inside the device.

[0172] The outermost layer of the immunoisolation layer (B) can be any one of a porous membrane (b1), a fibrous structure (b2) or a hydrogel (b3), or a mixture of two or three thereof. The innermost layer of the immunoisolation membrane (B) can be any one of a porous membrane (b1), a fibrous structure (b2) or a hydrogel (b3), or a mixture of two or three thereof. In the present specification, the outermost layer of the immunoisolation layer (B) refers to the layer constituting the outside of the immunoisolation device of the present invention in the immunoisolation layer (B), i.e., the layer contacting the tissue around the transplantation site (host), and the innermost layer of the immunoisolation layer (B) refers to the layer constituting the part (inner part) of the immunoisolation layer (B) contacting the cell capture layer (A) in the immunoisolation device of the present invention.

[0173] When the outermost layer of the immunoisolation layer (B) is the porous membrane (b1), the porous membrane (b1) is ideally made of a material that is more biocompatible than the hydrogel (b3) and is also used to prevent the hydrogel (b3) from adhering to the recipient's tissue or causing inflammation, etc.

[0174] When the outermost layer of the immunoisolation layer (B) is a fiber structure (b2), the fiber structure (b2) is preferably a material having higher biocompatibility than the hydrogel (b3). Since the outermost layer is also used to prevent the hydrogel from adhering to the tissue of the recipient at the transplantation site and causing inflammation, etc., it is ideal to smooth the surface of the fiber structure (b2) as the outermost layer by heat, mechanical or chemical treatment.

[0175] By modifying the hydrogel (b3) to suppress the induction of inflammatory response, it is also possible to use the hydrogel (b3) as the outermost layer and the porous membrane (b1) as the innermost layer. In this case, it is possible to load physiologically active substances on the hydrogel and exert functions such as inducing angiogenesis.

[0176] Material permeability of the immunoisolation layer (B)

[0177] The amount of glucose, insulin, and humoral factors of the immune system that permeate through the immune isolation layer (B) can be measured by inserting the immune isolation layer (B) into the connection point between two glass chambers having the same volume, pouring a sample solution of insulin, etc. of a known concentration into chamber a, pouring water into chamber b, and quantifying the amount of insulin, etc. contained in the liquid sampled from chamber b by ELISA, etc. at 37° C. under stirring after a period of time ( Fig. 9 ). It is important to adjust the liquid amounts in chambers a and b to be equal when pouring the sample solution into chamber a.

[0178] The permeability of glucose, insulin, immune system humoral factors, etc. through the immune isolation layer is expressed as a percentage, which represents the amount of each substance that has penetrated into chamber b as measured by the above method after 20 hours relative to the concentration at equilibrium, that is, half of the concentration of the substance poured into chamber a. More specifically, for example, the permeability can be obtained according to the following formula:

[0179] Permeability of each substance (%) = concentration of each substance in chamber b after 20 hours of measurement / (concentration of each substance in chamber a at the beginning of measurement / 2) × 100

[0180] The insulin and glucose permeability of the immunoisolation layer (B) of the present invention is preferably 50% or more, more preferably 90% or more, and even more preferably 95% or more.

[0181] The immune system humoral factor permeability of the immunoisolation layer (B) of the present invention is preferably 30% or less, more preferably 10% or less.

[0182] The permeability of each material can be controlled by the pore size of the porous membrane (b1), the fiber basis weight of the fiber structure (b2), or the gel strength and crosslinking degree of the hydrogel (b3). In order to exhibit the immune isolation function, it is desired that the pore size of the porous membrane (b1) is equal to or less than a size that does not allow cells to penetrate. It is desired that the hydrogel (b3) does not inhibit the penetration of physiologically active substances, but can inhibit the penetration of immune response factors such as cells and antibodies.

[0183] Examples of immune response cells include macrophages, cytotoxic T cells, natural killer cells, dendritic cells, and helper T cells.Examples of humoral factors of the immune system include antibodies, complement, and cytokines.

[0184] Immunoisolation device

[0185] The immunoisolation device according to a preferred embodiment of the present invention is in the form of a bag, a tube, a cylinder, a rectangular tube, a sphere, a cube, a cuboid, a sheet or a hollow fiber, and the cell capture layer (A) is encapsulated in the device. The material to be transplanted is introduced into the cell capture layer (A). Examples of materials to be transplanted include cells, cell clusters, cell sheets, transplants, etc. Other examples of materials to be transplanted include non-cellular physiologically active substances, such as enzymes, hormones, cytokines and drugs. Preferred cells, cell clusters and implants are those that release physiologically active substances from the immunoisolation device. That is, in a preferred embodiment, cells, cell clusters and transplants include cells that produce physiologically active substances.

[0186] In order to introduce the material to be transplanted into the cell capture layer (A), a suspension of the material to be transplanted is uniformly injected and inoculated into the fiber structure (a1) using a pipette or the like. After inoculation, a dense fiber structure (a2) of different specifications with smaller pores is arranged around the fiber structure (a1) so that the fiber structure (a1) is covered by the dense fiber structure (a2), thereby preventing the material to be transplanted from escaping from the cell capture layer. That is, the porosity of the dense fiber structure (a2) is preferably smaller than the porosity of the fiber structure (a1).

[0187] The immunoisolation device can be produced, for example, in the following manner. After the immunoisolation layer (B) is arranged around the cell capture layer (A) prepared by the above method so that the cell capture layer (A) is covered by the immunoisolation layer (B), the peripheral portion is sealed by heat fusion to mold the device into a bag shape. Alternatively, after an opening is provided in a device prepared in advance using the immunoisolation layer (B) and the cell capture layer (A) is inserted from the opening, the opening is closed to thereby block the invasion of immune response cells and immune system humoral factors from the opening. Since oxygen, nutrients, etc. can penetrate the immunoisolation layer in the portion outside the opening, the opening can be closed to inhibit the penetration of substances including nutrients.

[0188] In heat fusion, the resin can be sandwiched between the immunoisolation layers (B) (immunoisolation multiple layers (B')), and then heat fusion can be performed. By performing heat fusion in a state where the resin is sandwiched, heat fusion can be easily performed even when the innermost layers of the upper and lower immunoisolation layers (B) in the immunoisolation device are both hydrogels (when the hydrogels of the upper and lower immunoisolation layers (B) face each other). There is no particular limitation on the resin used in heat fusion, but it is preferably at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate, and more preferably ethylene-vinyl alcohol copolymer. Alternatively, heat fusion can be performed with the dense fiber structure (a2) sandwiched between the immunoisolation layers (B).

[0189] After the immunoisolation device of the present invention is implanted in a living body, the transplant material whose function has been reduced can be taken out, and new functional material to be transplanted can be introduced. This operation can be repeated. The immunoisolation device can be repeatedly used to introduce material to be transplanted, or the immunoisolation device can be taken out together with the transplant material.

[0190] Since the immunoisolation device of the present invention has sufficient strength, it can stably exist in the recipient's body, and can inhibit the invasion of immune-competent cells into the cell capture layer (A), and can also simultaneously inhibit the invasion of the material to be transplanted present in the cell capture layer (A) into the recipient's body. Therefore, even iPS-derived cells (for which there are concerns about the risk of canceration of the material to be transplanted) can be used safely.

[0191] In a preferred embodiment, the immunoisolation device of the present invention preferably has shape retention to ensure sufficient strength in vivo.

[0192] In order to maintain functionality as an immunoisolation device, it is desired that the fiber structure (a1) and dense fiber structure (a2) used as the cell capture layer (A) and the porous membrane (b1), fiber structure (b2) and hydrogel (b3) used as the immunoisolation layer (B) are all made of safe and highly biocompatible materials.

[0193] In order to prevent adhesion to surrounding tissues and prevent fibrosis, the immunoisolation layer (B) is ideally composed of a material with excellent biocompatibility. When the immunoisolation layer (B) is composed of a variety of materials, it is desirable to arrange the material with excellent biocompatibility on the transplant side, that is, on the contact surface of the outermost layer in contact with the transplant site in the recipient. An example of a material with excellent biocompatibility is ethylene-vinyl alcohol copolymer.

[0194] The thickness of the immunoisolation device of the present invention varies with the tissue of the recipient, the material to be transplanted, 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 with the tissue of the recipient, the material to be transplanted, etc., and is not particularly limited, but is preferably 1.0 to 200 cm 2 .

[0195] Preferred embodiments of the present invention will be described below with reference to the accompanying drawings.

[0196] Figure 1 is a schematic diagram of a device made by molding an immunoisolation layer (B) into a bag shape. Figure 2 Schematic diagram of a device made by molding an immunoisolation layer into a tubular shape. A bag-shaped device ( x3 ) is molded by applying heat, ultrasound, high frequency or electron beam to fuse two sheets ( x1 and x2 ) of an immunoisolation multilayer (B) shown below ( x3 ). Figure 1 ), there is a certain distance (x4) between the two sheets to ensure a space for encapsulating the cell-capturing layer (A) therein. A spacer may be provided to ensure the certain distance (x4).

[0197] Tubular device ( Figure 2 ) is composed of immunoisolation layers (y1, y2 and y3) each molded into a tubular shape. A cell-capturing layer (A) is encapsulated inside a tubular portion (y4) and heat, ultrasound, high frequency, electron beam, etc. are applied to seal both ends of the tube by fusion, thereby molding a tubular device.

[0198] Figure 3A and 3B is a conceptual diagram of an immunoisolation device according to one embodiment of the present invention. Figure 3A and 3BIn the embodiment, the outermost layer of the immunoisolation layer (B) contacts the transplantation site, while the innermost layer contacts the cell capture layer (A).

[0199] like Figure 3A and 3B As shown in Fig. 1 (which are conceptual diagrams of immunoisolation devices), each immunoisolation device has a bag shape or a tubular shape, and a cell capture layer in which a material to be transplanted is dispersed is enclosed in the device. In this embodiment, it is shown that the cell capture layer (A) contains a material to be transplanted, such as cells or cell clusters, so that the material to be transplanted is uniformly dispersed and fixed to the fiber structure (a1) (5) present in the cell capture layer (A). The fiber structure (a1) (5) preferably contains at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate, which has excellent biocompatibility, or may be a cell scaffold material, such as collagen fiber.

[0200] As the fiber structure (a1)(5), any sterilized fiber structure can be used. The fiber structure (a1)(5) can be present in the cell capture layer (A) surrounded by the immunoisolation layer (B) before the material to be transplanted is introduced into the cell capture layer (A), or the material to be transplanted and the fiber structure (a1)(5) can be introduced later as the cell capture layer (A) into the immunoisolation device composed of the immunoisolation layer (B).

[0201] Furthermore, when the material to be transplanted is dispersed in the fiber structure (a1) constituting the cell capturing layer (A), hydrogel particles based on polyvinyl alcohol or polyethylene glycol, blocked structures, etc. may be suspended together with the material to be transplanted to prevent aggregation or binding between cells or cell clusters as the material to be transplanted.

[0202] Figure 4 : is a conceptual diagram of a cell capture layer (A). The cell capture layer (A) preferably comprises two types of components, namely a fiber structure (a1) and a dense fiber structure (a2), and has a structure in which a dense fiber structure (a2) having relatively small pores is arranged around a fiber structure (a1) for capturing a material to be transplanted so that the fiber structure (a1) is covered by the dense fiber structure (a2) to prevent the captured material to be transplanted from escaping from the fiber structure.

[0203] Figure 5 is a transmitted light microscopy image of a dense fiber structure (a2). Figure 5 The image shown in is taken and the area of ​​each bright spot through which light is transmitted is measured using image analysis software. The diameter calculated under the assumption that the obtained area is the area of ​​a perfect circle is taken as the aperture of the bright spot. The average value of the apertures of 50 or more bright spots is defined as the average aperture.

[0204] Figure 6 and7 The immunoisolation layer (B) shown in FIG. 1 is composed of a combination of multiple materials. The outermost layer is to be in contact with the recipient's tissue at the transplantation site, and the innermost layer is to be in contact with the cell capture layer (A). Such an immunoisolation layer is formed into a bag shape ( Figure 1 ) or tubular ( Figure 2 ), and a cell capture layer (A) on which cells or cell clusters as materials to be transplanted are fixed is encapsulated therein, thereby making an immunoisolation device.

[0205] Figure 6 The formation of a multilayer of a porous membrane (b1) (6) and a hydrogel (b3) (8) is shown. The porous membrane (b1) (6) is impregnated with a hydrogel (b3) to form a multilayer (7) wherein the outermost surface (9) consists of the porous membrane (b1) and the innermost surface (10) consists of the hydrogel (b3).

[0206] Figure 7 The formation of multiple layers of fibrous structure (b2)(11) and hydrogel (b3)(12) is shown. The hydrogel (b3)(12) is applied to the fibrous structure (b2)(11), or the fibrous structure (b2)(11) is impregnated with the hydrogel (b3)(12) to form multiple layers (7), wherein the outermost surface (13) is composed of the fibrous structure (b2) and the innermost surface (14) is composed of the hydrogel (b3).

[0207] Example

[0208] Production of immunoisolation multilayer (B')

[0209] Production Example 1

[0210] A porous film formed using an ethylene-vinyl alcohol copolymer (hereinafter referred to as EVOH) and a hydrogel containing a methacryloyl-modified polyvinyl alcohol (hereinafter referred to as MA-PVA) as a main component were laminated to form a multilayer by the following procedure.

[0211] (1) A porous film (average pore diameter: 1.8 μm, maximum pore diameter: 3.4 μm, thickness: 100 μm) was produced by subjecting EVOH (EVAL (registered trademark), F101A, produced by Kuraray Co., Ltd.) to a polymer phase separation reaction.

[0212] (2) Phenyl (2,4,6-trimethylbenzoyl) lithium phosphinate was added as a water-soluble photoradical polymerization initiator to a 10 mass % aqueous solution of MA-PVA (average degree of polymerization: 1700, degree of saponification: 98.0 to 99.0 mol %, methacryloyl modification rate: 1.2 mol %, hereinafter referred to as MA-PVA) to achieve a concentration of 0.1 mass %, thereby preparing a sol.

[0213] (3) This sol was applied to a PET film to a thickness of 50 μm using a bar coater.

[0214] (4) A porous film is placed on the sol, and the sol and the porous film are closely adhered to each other using a laminator.

[0215] (5) Place the sol surface on top and use 365nm light at 15mW / cm 2 The film was irradiated at an intensity of 1000 nm for 3 minutes to form a hydrogel on the porous membrane, thereby obtaining an immunoisolation multilayer (B'). Figure 8 A SEM image of a cross section of the resulting immunoisolation multilayer (B') is shown. The hydrogel was found to have penetrated the porous membrane to a depth of approximately 3 to 6 μm.

[0216] Production Example 2

[0217] Using 4 mass % of MA-PVA (average degree of polymerization: 3500, saponification degree: 87.0 to 89.0 mol %, methacryloyl modification rate: 1.2 mol %) aqueous solution, a hydrogel was formed on a porous membrane in the same manner as in Production Example 1 to prepare an immunoisolation multilayer (B').

[0218] Material permeability test

[0219] The permeabilities of glucose, insulin and IgG of the porous membrane prepared in Production Example 1 and the immunoisolation multilayer (B') obtained in Production Example 1 or 2 were measured in the following manner.

[0220] (1) Place a porous membrane or an immunoisolation multilayer between chambers a and b respectively ( Fig. 9 ). An aqueous solution (65 mL) containing insulin (30 U / L), glucose (5 mg / mL) and IgG (0.5 μg / mL) was prepared in chamber a, while water (65 mL) was placed in chamber b.

[0221] (2) Stirring was performed with a stirrer at a constant temperature of 37° C. After 20 hours, the liquid was sampled from chamber b, and changes in the concentrations of insulin, glucose, and IgG were measured by ELISA.

[0222] (3) The permeability of each substance was calculated from the results of (2) (Table 1).

[0223] Permeability of each substance (%) = concentration of each substance in chamber b after 20 hours of measurement / (concentration of each substance in chamber a at the beginning of measurement / 2) × 100

[0224] Table 1

[0225]

[0226] Preparation of cell capture layer

[0227] Production Example 3

[0228] Preparation of fiber structure (a1)

[0229] (1) A fiber sheet called a web was produced by a card method using a sheath-core type composite fiber comprising a polyester as a core material and an ethylene-vinyl alcohol copolymer as a sheath material (fineness: 3.3 dtex, specific gravity: 1.25, trade name: "Sophista" S-220, produced by Kuraray Co., Ltd., ethylene content of the ethylene-vinyl alcohol copolymer: 44 mol%, saponification degree: 98.4 mol%).

[0230] (2) Molding the web while the web is sandwiched between a pair of conveyor rolls.

[0231] (3) Subsequently, the molded web was subjected to steam jetting to bond the fibers together by heat and steam jets, thereby obtaining a web having a thickness of 50 g / m 2 A non-woven fabric sheet with a basis weight of 1 mm and a thickness of 1 mm.

[0232] (4) The nonwoven fabric sheet was cut into a circular shape using a puncher with a diameter of 15 mm to prepare a fiber structure (a1) for capturing cells (porosity: 96%). The basis weight C (g / m 2 ), thickness D (cm) and average specific gravity E (g / cm 2 )The porosity is calculated according to the following formula:

[0233] Porosity (%) = 100 - ((C / D / E) x 10 -4 x 100)

[0234] Production Example 4

[0235] Preparation of dense fiber structure (a2)

[0236] (1) Using a general melt-blowing facility, a molten ethylene-vinyl alcohol copolymer (ethylene content: 44 mol%, saponification degree: 99.3 mol%, "E105" produced by Kuraray Co., Ltd.) was extruded from a die to obtain a melt-blown nonwoven fabric sheet having a fiber diameter of 8 μm.

[0237] (2) The melt-blown nonwoven fabric sheet was calendered by passing the sheet through a metal roll and a rubber roll heated at 100° C. at a pressure of 40 kg / cm and a speed of 3 m / min. The melt-blown nonwoven fabric obtained after the calendering process had a viscosity of 76.6 g / m 2 The basis weight of the dense fiber structure is C (g / m2 ), thickness D (cm) and average specific gravity E (g / cm 2 )The porosity is calculated according to the following formula:

[0238] Porosity (%) = 100 - ((C / D / E) x 10 -4 x 100)

[0239] (3) The meltblown nonwoven fabric was cut into 24 mm squares to prepare a dense fiber structure (a2) (average pore size: 11 μm) for preventing cell escape. The image analysis software was used to analyze the transmission light microscopy image ( Figure 5 ) The area of ​​each bright spot through which light is transmitted is measured. The average diameter (of 50 points) calculated under the assumption that the measured area is the area of ​​a perfect circle is defined as the average pore size.

[0240] Cell preparation

[0241] Production Example 5

[0242] MIN6 (mouse pancreatic islet-like cell line) was used to verify the cell and cell cluster capture function of the cell capture layer. MIN6 was cultured at 2.8 x 10 5 cells / cm 2 The cells were inoculated at a density of 500 μm in a suspension cell culture flask containing DMEM medium (produced by Sumitomo Bakelite Co., Ltd.) and cultured at 37°C for 7 days to produce cell clusters with a diameter of about 100 to 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 cells, and the cell density was measured using a hemocytometer. Based on the measured cell density, when inoculating cells in the following examples, the amount of liquid was adjusted to 5 x 10 5 cells were seeded in the fiber constructs and tested.

[0243] Production and evaluation of immunoisolation devices

[0244] Example 1

[0245] 1) Cell seeding and cell encapsulation into immunoisolation devices

[0246] (1) The immunoisolation multilayer (B') prepared in Preparation Example 2 was cut into 24 mm squares and placed with the hydrogel surface side facing upward. The dense fiber structure (a2) prepared in Production Example 4 was placed thereon, and the fiber structure (a1) prepared in Example 3 was further placed thereon at the center.

[0247] (2) Then, the MIN6 cell cluster suspension prepared in Production Example 5 (containing 5 x 10 5 Cells) were inoculated onto the fiber structure (a1) from the top and allowed to stand for 30 seconds to allow the cell cluster suspension to penetrate the fiber structure (a1).

[0248] (3) The dense fiber structure (a2) prepared in Production Example 4 was placed on top of the fiber structure (a1) in which the cells had been seeded, and then the immunoisolation multilayer (B') prepared in Production Example 2 was placed on top with the hydrogel surface side facing downward. The four edges of the superimposed immunoisolation layer (B') and the dense fiber structure (a2) were fused together using a heat sealer (Clip Sealer Z-1, manufactured by Technoimpulse Co., Ltd.) to produce an immunoisolation device ( Figure 3B ).

[0249] 2) Assessment of cell viability

[0250] The immunoisolation device made in the previous part is placed in a 6-well plate, 7 mL of DMEM culture medium is added, and the device is cultivated for 1 week at 37 ° C. Half of the culture medium (3.5 mL) is replaced every day. As a comparative control, 7 mL of DMEM culture medium is added to a 6-well plate (produced by Corning) with an ultra-low adhesion surface, and the cell cluster suspension prepared in the previous part is added to the 6-well plate in an amount equal to the amount in the test device by cell number, and cultivated for 1 week at 37 ° C, while replacing half of the culture medium (3.5 mL) every day.

[0251] After 1 week of cultivation, the culture medium was completely removed. After adding 1 mL of fresh culture medium, 1 mL of ATP activity assay reagent (CellTiter-Glo (registered trademark) is added, produced by Promega KK). The plate was vibrated for 2 minutes using a flatbed oscillator. The mixture was then allowed to stand at room temperature for 10 minutes, 100 μL of supernatant was sampled and transferred to a white 96-well plate. Plate reader (ARVO X2; PerkinElmer production) was used to measure the luminous intensity. As a result, when the luminous intensity obtained from the control cell cluster was defined as 100% vigor, the luminous intensity obtained from the cell cluster encapsulated in the device showed 103% vigor.

[0252] Example 2

[0253] An immunoisolation device was produced in the same manner as in Example 1, except that the basis weight of the fiber structure (a1) was changed to 100 g / m 2 (Porosity: 92%). Cell viability was measured in the same manner as in Example 1 and found to be 88%.

[0254] Example 3

[0255] An immunoisolation device was produced in the same manner as in Example 1, except that the basis weight of the fiber structure (a1) was changed to 25 g / m 2 And the thickness was changed to 0.5 mm (porosity: 96%). The cell viability was measured in the same manner as in Example 1 and found to be 103%.

[0256] Example 4

[0257] An immunoisolation device was produced in the same manner as in Example 1, except that the basis weight of the dense fiber structure (a2) was changed to 22.3 g / m 2 And the thickness was changed to 0.12 mm (average pore diameter 23.5 μm, porosity 84%). Cell viability was measured in the same manner as in Example 1 and found to be 96%.

[0258] Table 2

[0259]

[0260] As is clear from the results in Table 2, in Examples 1 to 4, cell clusters could be captured without escaping from the cell-capturing layer (A).

[0261] In addition, the fiber structure (a1) with appropriate porosity can capture cell clusters while maintaining high viability. In addition, the dense fiber structure (a2) with appropriate average pore size can prevent cell clusters from escaping from the fiber structure (a1).

[0262] Industrial Applicability

[0263] The present invention relates to a transplantation device for cell transplantation therapy, and more particularly to an immunoisolation device for protecting materials to be transplanted from immune rejection.

[0264] The immunoisolation device is believed to be mainly used as a regenerative medicine product for cell transplantation therapy. However, the immunoisolation device can also be used for the transplantation of non-cellular physiologically active substances such as enzymes, hormones and drugs.

[0265] Explanation of Reference Symbols

[0266] x1 Immune Isolation Layer (B)

[0267] x2 Immune Isolation Layer (B)

[0268] x3 Fusion

[0269] x4 A certain distance

[0270] y1 Immune isolation layer (B)

[0271] y2 Immune isolation layer (B)

[0272] y3 Immune isolation layer (B)

[0273] y4 tubular interior

[0274] 1 Porous membrane (b1)

[0275] 2 Hydrogel (b3)

[0276] 3 Materials to be transplanted

[0277] 4 Dense fiber structure (a2)

[0278] 5 Fiber structure (a1)

[0279] 6 Porous membrane (b1)

[0280] 7 Impregnation and molding

[0281] 8 Hydrogel (b3)

[0282] 9 Outermost surface

[0283] 10 Innermost surface

[0284] 11 Fiber structure (b2)

[0285] 12 Hydrogel (b3)

[0286] 13 Outermost surface

[0287] 14 Innermost surface

Claims

1. An immunoisolation device comprising Cell capture layer (A) and an immunoisolation layer (B) covering the cell capture layer (A), The cell-trapping layer (A) comprises a fiber structure (a1).

2. The immunoisolation device according to claim 1, wherein the fiber structure (a1) has a porosity of more than 90%.

3. The immunoisolation device according to claim 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 claims 1 to 3, wherein the fiber structure (a1) comprises at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate.

5. The immunoisolation device according to any one of claims 1 to 4, wherein The cell-trapping layer (A) comprises the fiber structure (a1) and the dense fiber structure (a2), and The dense fiber structure (a2) is arranged around the fiber structure (a1) and has an average pore size of 35 μm or less.

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

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

8. The immunoisolation device according to claim 1, wherein The cell-trapping layer (A) comprises a dense fiber structure (a2) arranged around the fiber structure (a1) and capable of preventing cells or cell clusters trapped in the fiber structure (a1) from escaping, and The dense fiber structure (a2) has the function of preventing cells or cell clusters from escaping.

9. The immunoisolation device according to any one of claims 1 to 8, wherein The immunoisolation layer (B) comprises a porous membrane (b1) or a fiber structure (b2), and The porous film (b1) or the fiber structure (b2) includes at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate.

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

11. The immuno-isolating device according to claim 10, wherein the immuno-isolating multilayer (B') has a thickness of 500 μm or less.

12. The immunoisolation device according to claim 10 or 11, wherein The outermost layer of the immunoisolation multilayer (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 claim 10 or 11, wherein The outermost layer of the immunoisolation multilayer (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 one of claims 10 to 13, wherein The hydrogel (b3) comprises polyvinyl alcohol, and The average polymerization degree of the polyvinyl alcohol is 300 to 10,000.

15. A method for producing an immunoisolation device, the immunoisolation device comprising a cell-capturing layer (A) comprising a fiber structure (a1) and an immunoisolation multilayer (B') covering the cell-capturing layer (A), The method comprises the steps of: (1) applying a hydrosol solution to a porous membrane (b1); (2) converting the hydrosol solution into a hydrogel by heat, temperature, light or chemical action to form an immunoisolation multilayer (B'); and (3) The immunoisolation multilayer (B') is molded into a bag shape by heat fusion.

16. The method for producing an immunoisolation device according to claim 15, wherein The hydrosol solution in step (1) comprises polyvinyl alcohol, The polyvinyl alcohol has a degree of polymerization of 300 to 10,000, and The hydrosol solution has a solid concentration of 3 to 15% by mass.

17. The method for producing an immunoisolation device according to claim 15 or 16, wherein The heat fusion in step (3) is performed with the resin interposed between the two sheets of the immunoisolation multilayer (B'), placing two sheets of said immunoisolation multilayer (B') with the hydrogel surfaces facing each other, and The resin includes at least one member selected from ethylene-vinyl alcohol copolymer and cellulose acetate.

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