Hydrogels for cell embedding, immunoisolation devices, and implantation materials

Abstract

The present invention addresses the problem, in long-term transplantation of a macroencapsulated immunoisolation device in which a porous membrane, etc., is used, of simultaneously improving the efficiency of diffusion of a physiologically active substance released from a transplanted object, improving the efficiency of diffusion of nutrients to cells, etc., that are the transplanted object, and efficiently fixing the transplanted object in a dispersed manner so that, inter alia, the cells included in the macroencapsulated immunoisolation device are uniform, while maintaining an immunoisolation effect. The present invention provides a hydrogel for embedding a transplanted object, the hydrogel containing water and a polymer crosslinked by visible light.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims priority based on Japanese Patent Application No. 2020 - 180920 filed on October 28, 2020, the entire disclosure of which is incorporated herein by reference. The present invention relates to a hydrogel for cell encapsulation, an immunoisolation device, and a transplantation material.

Background Art

[0002] As a means for performing cell transplantation therapy without the need for administration of immunosuppressive agents, immunoisolation devices have been developed. In particular, in the case of somatic cell transplantation derived from iPS cells where there are concerns such as the risk of canceration, and in the case of functional decline of transplanted cells, etc., the macroencapsulation immunoisolation device is regarded as an effective method in terms of being able to identify the transplantation site and replace the device.

[0003] Functions required for a macroencapsulation immunoisolation device include the ability to disperse and fix cells or cell aggregates without uniformly aggregating them, the ability to easily permeate oxygen and / or nutrient components to transplanted cells, the ability to easily release physiologically active substances (such as cytokines, hormones, growth factors, etc.) released by the cells required for the therapeutic effect in response to cell responses, and the ability to prevent the permeation of immune response cells and immune response factors, and it is important that the transplanted device has excellent biocompatibility and is unlikely to cause inflammatory reactions such as adhesion to surrounding tissues and granulation.

[0004] Many immunoisolation devices using porous membranes and the like have been studied so far, but problems such as protein adsorption to the porous membrane material, reduction in permeability due to fibrosis, and reduction in permeability due to adhesion to surrounding tissues have been an issue. The inventor has achieved reduction of fibrosis by using a porous membrane made of an ethylene - vinyl alcohol copolymer (EVOH) with excellent biocompatibility.

[0005] On the other hand, for long-term engraftment of grafts such as cells, it is important to suppress contact between immune response cells and immune response factors such as IgG and the graft, prevent aggregation (cell clumping) of graft cells within the device, and shorten the diffusion distance from the recipient's bodily fluids and blood, which are sources of oxygen and nutrients for the cells, to the graft.

[0006] Internal necrosis of cell aggregates is known to occur when the diameter exceeds 500 μm. Therefore, to easily maintain the engraftment of embedded cells in the graft and release bioactive substances, it is necessary to appropriately control the overall thickness of the device. For this reason, the selection of substrates for dispersing and immobilizing cells, and the cell immobilization techniques are extremely important.

[0007] Methods for dispersing and fixing cells and other tissues include using fibrous materials such as nonwoven fabrics, woven fabrics, and meshes; hydrogels such as alginate and gelatin; or biomaterials such as collagen. However, when using fibrous materials, it is not easy to uniformly disperse cells and other tissues, and the hydrogels and biomaterials mentioned above are gradually biodegraded in the body over long periods, making them unsuitable as materials for long-term stable retention of cells and other tissues.

[0008] Furthermore, to maintain the functionality of cells and other tissues, it is desirable to appropriately control the thickness of the fixation support layer and minimize the diffusion distance of the material as much as possible, in addition to uniformly dispersing and fixing the cells and other tissues. Therefore, an immunoisolation membrane is an important component of an immunoisolation device that is made of a material that does not adversely affect the cells and other tissues that are grafts, is safe, allows for easy and uniform dispersion and fixation of cells, forms an irreversibly stable hydrogel, has excellent long-term physical and chemical stability, and simultaneously maintains the functionality of cells and other tissues by controlling permeability and diffusion distance. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2007-314736 [Patent Document 2] Polymer Papers, vol69, No.10, pp.555-566(Oct,2012) [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to simultaneously achieve, in the long-term transplantation of macroencapsulated immune isolation devices using porous membranes, etc., improved diffusion efficiency of physiologically active substances released from the transplanted tissue, improved diffusion efficiency of nutrients to the transplanted tissue (cells, etc.), and uniform and efficient dispersion and immobilization of the transplanted tissue (cells, etc.) contained within the macroencapsulated immune isolation device, while maintaining the immune isolation effect. [Means for solving the problem]

[0011] As a solution, a modified polyvinyl alcohol hydrogel (hereinafter referred to as MPVAG), which has good biocompatibility and can be easily hydrogelled in the visible light region without adversely affecting cells, was used as a cell immobilization material (Patent Documents 1 and 2).

[0012] By adjusting the degree of crosslinking of the photocrosslinking agent in MPVAG and the concentration of MPVAG, it is possible to control substance permeability, thereby suppressing the permeability of immune response factors such as immune response cells and antibodies without suppressing the permeability of target physiologically active substances.

[0013] Furthermore, as a method for uniformly dispersing and fixing cells, a cell dispersion obtained by suspending and dispersing cells in a sol solution of MPVAG is seeded onto, for example, a microdimple sheet having depressions with a diameter of 50-500 μm or a microdevice having slits with a width of 50-500 μm. By then photocrosslinking with visible light of 360 nm or higher, it is possible to produce a hydrogel sheet without cells associating, maintaining a certain distance, and without causing internal necrosis of the cell aggregate. Such microdimple sheets and hydrogel sheets can be used as transplantation materials as they are, but they can also be used as transplantation materials by introducing them into a macroencapsulated immunoisolation device covered with an immunoisolation membrane.

[0014] The present invention provides the following hydrogels and implantation materials for embedding grafts. [1] A hydrogel for implantation containing a polymer crosslinked with visible light and water. [2] The hydrogel for embedding a graft according to [1], wherein the polymer is a polyvinyl alcohol-based polymer. [3] An immunoisolation device comprising the hydrogel for implantation of a graft as described in [1] or [2]. [4] A graft material comprising embedding the graft in the hydrogel described in [1] or [2]. [5] A transplant material comprising a transplant embedded in a hydrogel containing a visible light-crosslinked polymer and water, within an embedding chamber covered by an immunoisolation membrane of a macroencapsulated immunoisolation device. [6] The implantation material according to [5], wherein the immunoisolation membrane comprises a hydrogel. [7] The implantation material according to [5] or [6], wherein the wavelength of the visible light is 360 nm or more. [8] The transplant material according to any one of items [5] to [7], wherein the immune isolation membrane is capable of removing 50% or more of immune response cells and immune system humoral factors. [9] The transplantation material according to any one of [5] to [8], wherein the immune isolation membrane allows insulin and glucose to permeate at a permeability rate of 95% or more without allowing immune response cells to permeate, and allows immune system humoral factors to permeate at a permeability rate of 10% or less. 〔10〕 The transplantation material according to any one of [5] to [9], wherein the thickness of the immune isolation membrane is from 20 μm to 500 μm, and the graft is a plurality of cells or cell aggregates, and the plurality of cells or cell aggregates embedded in the hydrogel are arranged at intervals of 10 μm or more and 500 μm or less without contacting each other.

Advantages of the Invention

[0015] According to the present invention, there is provided a hydrogel for embedding a graft, such as cells and cell aggregates, which can wrap the graft and restrict its movement and spacing. This hydrogel not only protects adjacent cells and / or cell aggregates by keeping them at a distance, but also prevents contact between immune response cells and cells or cell aggregates, and can prevent attack on the transplanted cells or cell aggregates by immune response cells. Furthermore, by introducing the hydrogel embedding of the graft into an embedding chamber surrounded by an immune isolation membrane, the influence of immune response cells and immune system humoral factors can be excluded.

[0016] The hydrogel for embedding a graft of the present invention may be used alone as an immune isolation device, or may be arranged in an embedding chamber surrounded by an immune isolation membrane to be used as an immune isolation device.

Brief Description of the Drawings

[0017] [Figure 1] Perspective view of a bag-shaped immune isolation device [Figure 2] Cross-sectional view of a tubular immune isolation device [Figure 3] Cross-sectional view of a three-layer immune isolation membrane having a joint between a porous membrane and a hydrogel [Figure 4] ]>An example of a mold for producing a hydrogel for an immune isolation membrane by a microfabrication technique for uniformly dispersing and fixing cells and the like is shown. [Figure 5] A conceptual diagram of a hydrogel sheet containing cells, etc., prepared using the said mold. [Figure 6] This shows the overall structure of the mold. [Figure 7] Microscopic images of the hydrogel sheet taken in Example 2 are shown. [Modes for carrying out the invention]

[0018] The hydrogel for implantation of the present invention contains a polymer crosslinked with visible light and water. The visible light can be, for example, light with wavelengths of 360 nm to 830 nm or 360 to 800 nm, and also includes light with wavelengths of 500 to 600 nm. The hydrogel for implantation can be prepared by irradiating a composition (e.g., a suspension) containing a hydrosol and the implant with visible light. The temperature of the hydrosol during visible light irradiation is not limited, but is, for example, about 4 to 40°C. The irradiation time is also not limited, but is, for example, about 10 seconds to 15 minutes.

[0019] The thickness of the graft material, in which the graft subject is embedded in hydrogel, is not particularly limited, but is preferably 100 μm to 500 μm, and more preferably 150 μm to 300 μm.

[0020] The tensile breaking strength of the embedding hydrogel is preferably 0.01 to 10 MPa, more preferably 0.03 to 1 MPa.

[0021] Polymers that can be crosslinked with visible light can be obtained by crosslinking polymers that can be crosslinked with visible light. Polymers that can be crosslinked with visible light have a hydrophilic backbone capable of forming a hydrogel in the presence of water and polymerizable groups that can be crosslinked with visible light. Polyvinyl alcohol-based polymers are preferred as polymers that can be crosslinked with visible light. Polyvinyl alcohol-based polymers have ethylenically unsaturated groups.

[0022] The polyvinyl alcohol-based polymer used in the present invention is not particularly limited as long as it has ethylenically unsaturated groups and contains more than 50 mol% of vinyl alcohol-derived structural units in the polymer, and may also contain vinyl ester-derived structural units. The total amount of vinyl alcohol-derived structural units and vinyl ester-derived structural units relative to the total structural units constituting the polyvinyl alcohol-based polymer is preferably 80 mol% or more, more preferably 90 mol% or more, and even more preferably 95 mol% or more.

[0023] There are no particular restrictions on the ethylenically unsaturated group, and it can be freely selected, but a group that can form crosslinks between polyvinyl alcohol polymer chains in visible light is preferred. It is more preferable to use a radical polymerizable group as the ethylenically unsaturated group, and examples include cyclic unsaturated hydrocarbon groups such as vinyl group, (meth)acryloyloxy group, (meth)acryloylamino group, vinylphenyl group, cyclohexenyl group, cyclopentenyl group, norbornel group, and derivatives thereof. These ethylenically unsaturated groups may be present in either the side chains or terminals of the vinyl alcohol polymer chain.

[0024] In this specification, the term "vinyl group" includes not only the ethenyl group, but also chain-like unsaturated hydrocarbon groups such as allyl groups and alkenyl groups, as well as vinyloxycarbonyl groups.

[0025] Among the radical polymerizable groups, at least one selected from the group consisting of vinyl groups, (meth)acryloyloxy groups, (meth)acryloylamino groups, vinylphenyl groups, norborneyl groups, and derivatives thereof is preferred from the viewpoint of improving the mechanical strength of hydrogel particles. Furthermore, from the viewpoint of reactivity, functional groups having terminal unsaturated carbon bonds are preferred, and (meth)acryloyloxy groups are more preferred.

[0026] The average degree of polymerization of the polyvinyl alcohol polymer is not limited to a specific range, but is, for example, 300 to 10,000, more preferably 450 to 5,000, even more preferably 500 to 3,000, and most preferably 500 to 2,500. Note that two or more vinyl alcohol polymers with different average degrees of polymerization may be mixed and used. From the viewpoint of suppressing embrittlement of the hydrogel of the present invention, an average degree of polymerization of 300 or higher is preferred. Furthermore, from the viewpoint of keeping the viscosity of the aqueous solution of the vinyl alcohol polymer within a range that is easy to handle, an average degree of polymerization of 10,000 or less is preferred, more preferably 5,000 or less, and even more preferably 3,000 or less.

[0027] In this specification, the average degree of polymerization of vinyl alcohol-based polymers refers to the average degree of polymerization measured in accordance with JIS K 6726:1994. Specifically, since the degree of polymerization of vinyl alcohol-based polymers and the PVA used as a raw material (described later) can be considered the same, it can be determined from the intrinsic viscosity measured in water at 30°C after purifying the PVA used as a raw material.

[0028] <Method for producing vinyl alcohol-based polymers> The vinyl alcohol polymer used in the present invention, that is, the polyvinyl alcohol polymer having an ethylenically unsaturated group, can be produced by introducing an ethylenically unsaturated group via the side chains, terminal functional groups, etc., of the raw material polyvinyl alcohol (hereinafter sometimes abbreviated as "raw material PVA"); or by copolymerizing a vinyl ester monomer with another polymerizable monomer other than a vinyl ester monomer that has reactive substituents other than hydroxyl groups during the production process of the raw material PVA, and then introducing an ethylenically unsaturated group by reacting the reactive substituent in the copolymer with a compound having an ethylenically unsaturated group.

[0029] The aforementioned PVA raw material can be produced by saponifying a polyvinyl ester obtained by polymerizing vinyl ester monomers, and converting the ester groups in the polyvinyl ester into hydroxyl groups.

[0030] Examples of the 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 may be used individually or in combination of two or more.

[0031] Among the vinyl ester monomers, aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of manufacturing cost. That is, the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.

[0032] Furthermore, the polyvinyl ester may optionally contain structural units derived from monomers other than vinyl ester monomers, as long as it does not impair the effects of the present invention.Other monomers include, for example, α-olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or its salts; alkyl acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, i-propyl acrylate, n-butyl acrylate, i-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate, and octadecyl acrylate; methacrylic acid or its salts; methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, methacrylate Alkyl methacrylate esters such as i-propyl methacrylate, n-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, octadecyl methacrylate; acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetone acrylamide, acrylamidepropanesulfonic acid or its salts, acrylamidopropyldimethylamine or its salts or quaternary salts, N-methylolacrylamide or its Examples of acrylamide derivatives include: methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, methacrylamidepropanesulfonic acid or its salts, methacrylamidepropyldimethylamine or its salts or quaternary salts, N-methylolmethacrylamide or its derivatives; N-vinylamide derivatives such as N-vinylformamide and N-vinylacetamide; vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, i-propyl vinyl ether, n-butyl vinyl ether, i-butyl vinyl ether, t-butyl vinyl ether, dodecyl vinyl ether, and stearyl vinyl ether; nitriles such as acrylonitrile and methacrylonitrile; vinyl halides such as vinyl chloride and vinyl fluoride; vinylides such as vinylidene chloride and vinylidene fluoride; allyl compounds such as allyl acetate and allyl chloride; maleic acid or its salts, esters, or acid anhydrides; vinylsilyl compounds such as vinyltrimethoxysilane; and isopropenyl acetate. These may be used individually or in combination of two or more types.

[0033] If the polyvinyl ester contains structural units derived from other monomers, the content of structural units derived from other monomers is preferably 20 mol% or less, more preferably 10 mol% or less, and even more preferably 5 mol% or less, relative to the total structural units constituting the polyvinyl ester.

[0034] There are no particular restrictions on the method for saponifying the polyvinyl ester, but it can be carried out in the same way as conventional methods. For example, alcohol decomposition using an alkaline or acid catalyst, hydrolysis, etc., can be applied. Among these, a saponification reaction using methanol as a solvent and caustic soda (NaOH) as a catalyst is simple and preferred.

[0035] In this specification, the average degree of polymerization of the raw material PVA refers to the average degree of polymerization measured in accordance with JIS K 6726:1994, as mentioned above. Specifically, it can be determined from the intrinsic viscosity measured in water at 30°C after saponification and purification of the raw material PVA.

[0036] From the viewpoint of improving the water solubility of the raw PVA, the degree of saponification of the raw PVA is preferably 50 mol% or more, more preferably 60 mol% or more, and even more preferably 65 mol% or more.

[0037] Furthermore, from the viewpoint of suppressing the increase in viscosity of the uncured gel solution, which will be described later, and improving the storage stability of the uncured gel solution, the degree of saponification of the raw material PVA is preferably 99 mol% or less.

[0038] In this specification, the degree of saponification of the raw material PVA 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) and vinyl alcohol units that can be converted to vinyl alcohol units by saponification in the raw material PVA, and can be measured in accordance with JIS K 6726:1994.

[0039] The 4% by mass viscosity of the raw PVA at 20°C is preferably 0.5 to 100 mPa·s, more preferably 1 to 80 mPa·s, and even more preferably 2 to 60 mPa·s. When the viscosity is within the above range, the ease of hydrogel production is improved, and the strength of the hydrogel can be increased.

[0040] In this specification, viscosity refers to the viscosity of an aqueous solution containing 4% by mass of raw material PVA, measured at a temperature of 20°C using a Type B viscometer (rotation speed 12 rpm) in accordance with the rotational viscometer method of JIS K 6726:1994.

[0041] The introduction of the ethylenically unsaturated group into the raw material PVA is preferably carried out via the side chains, terminal functional groups, etc., of the raw material PVA, and it is more preferable to react a compound containing an ethylenically unsaturated group (hereinafter sometimes abbreviated as "ethylenically unsaturated group-containing compound") with the hydroxyl group of the side chain of the raw material PVA.

[0042] Examples of ethylenically unsaturated group-containing compounds that react with the hydroxyl group in the side chain of the raw material PVA include (meth)acrylic acid or its derivatives such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acrylic acid halide, and (meth)acrylic acid ester. A (meth)acryloyl group can be introduced by esterification or transesterification of these compounds in the presence of a base.

[0043] Furthermore, examples of ethylenically unsaturated group-containing compounds that react with the hydroxyl group in the side chain of the raw material PVA include compounds containing both an ethylenically unsaturated group and a glycidyl group in their molecule, such as glycidyl (meth)acrylate and allyl glycidyl ether. By etherifying these compounds in the presence of a base, a (meth)acryloyl group and / or an allyl group can be introduced to the raw material PVA.

[0044] Furthermore, examples of ethylenically unsaturated group-containing compounds to react with the 1,3-diol group of the raw material PVA include compounds containing both an ethylenically unsaturated group and an aldehyde group in their molecules, such as acrylaldehyde (acrolein), methacrylaldehyde (methacrolein), 5-norbornene-2-carboxyaldehyde, 7-octenal, 3-vinylbenzaldehyde, and 4-vinylbenzaldehyde. By carrying out an acetalization reaction of these compounds in the presence of an acid catalyst, an ethylenically unsaturated group can be introduced to the raw material PVA. More specifically, for example, by carrying out an acetalization reaction of 5-norbornene-2-carboxyaldehyde, 3-vinylbenzaldehyde, 4-vinylbenzaldehyde, etc., norborneyl groups and / or vinylphenyl groups can be introduced to the raw material PVA. Furthermore, it is possible to introduce (meth)acryloylamino groups into the raw material PVA by reacting it with N-(2,2-dimethoxyethyl)(meth)acrylamide or the like.

[0045] Methods for introducing ethylenically unsaturated groups into the PVA raw material can be other than the exemplified reaction, and two or more reactions may be used in combination.

[0046] Another method for introducing the ethylenically unsaturated group is to copolymerize a vinyl ester monomer with another polymerizable monomer other than a vinyl ester monomer that has reactive substituents other than hydroxyl groups during the manufacturing process of the raw material PVA, and then saponify the copolymer to obtain copolymer-modified polyvinyl alcohol (hereinafter sometimes abbreviated as "copolymer-modified PVA"), and then react the reactive substituents such as carboxyl groups and amino groups present in the copolymer-modified PVA with an ethylenically unsaturated group-containing compound. Note that copolymer-modified PVA having a carboxyl group is sometimes called "carboxylic acid-modified PVA," and copolymer having an amino group is sometimes called "amino-modified PVA."

[0047] Monomers that constitute carboxylic acid-modified PVA include α,β-unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, and itaconic acid; alkyl (meth)acrylates such as methyl (meth)acrylate and ethyl (meth)acrylate; α,β-unsaturated carboxylic acid anhydrides such as maleic anhydride and itaconic anhydride, and their derivatives. Carboxylic acid-modified PVA can be produced, for example, by copolymerizing a vinyl ester monomer with an α,β-unsaturated carboxylic acid anhydride or its derivative, then saponifying the mixture, and then reacting the introduced carboxyl group with, for example, glycidyl methacrylate under acidic conditions to generate an ester bond and introduce a methacryloyl group.

[0048] Furthermore, amino-modified PVA can be prepared by copolymerizing a vinyl ester monomer with N-vinylformamide, then saponifying the mixture, and introducing an acryloylamino group to the introduced amino group by amidation of, for example, acrylic anhydride in the presence of a base. Alternatively, a vinyloxycarbonyl group can be introduced to the amino group of the amino-modified PVA by amidation of, for example, divinyl adipate. Methods for introducing ethylenically unsaturated groups via copolymerized PVA can be carried out using reactions other than those exemplified above, and two or more reactions may be used in combination.

[0049] As polyvinyl alcohol polymers having ethylenically unsaturated groups, from the viewpoint of ease of manufacture, polyvinyl alcohol polymers in which ethylenically unsaturated groups are introduced via hydroxyl groups in the side chains of raw material PVA, such as 1,3-diol groups, are preferred. More preferably, these are vinyl alcohol polymers obtained by esterifying or transesterifying (meth)acrylic acid or its derivatives to the hydroxyl groups in the side chains of raw material PVA, or polyvinyl alcohol polymers obtained by acetalizing the 1,3-diol groups of raw material PVA with a compound containing both ethylenically unsaturated groups and aldehyde groups in the molecule.

[0050] [Introducing rate of ethylenically unsaturated groups] From the viewpoint of suppressing embrittlement of hydrogel particles, the introduction rate of ethylenically unsaturated groups is preferably 10 mol% or less, more preferably 5 mol% or less, and even more preferably 3 mol% or less, of the total structural units constituting the vinyl alcohol-based polymer. Furthermore, from the viewpoint of promoting the crosslinking reaction, rapidly forming hydrogel particles, and improving the elastic modulus of the resulting hydrogel particles, it is preferably 0.01 mol% or more, more preferably 0.1 mol% or more, and even more preferably 0.5 mol% or more. The preferred range is 0.01 to 10 mol%, more preferably 0.1 to 5 mol%, and even more preferably 0.5 to 3 mol%.

[0051] Other polymers that can be used in combination with polyvinyl alcohol-based polymers include polyvinyl alcohol, collagen, chitosan, carboxymethylcellulose, pullulan, poly(2-hydroxyethyl acrylate), poly(2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), polyacrylic acid, polymethacrylic acid, cellulose derivatives, polyvinylpyrrolidone, or copolymers using at least one monomer that constitutes these polymers.

[0052] Examples of "graft material" include cells, cell aggregates, and grafts, with multiple cells or cell aggregates being preferred. Graft material such as cell aggregates is preferably 500 μm or smaller in size to ensure sufficient oxygen and / or nutrients are supplied.

[0053] When a graft embedded in an embedding hydrogel is to be transplanted into the body alone, the embedding hydrosol containing the graft, such as cells, is placed in a mold, and visible light is irradiated to create a hydrogel embedding material with the required intensity and permeability, which can then be transplanted into the body. Alternatively, when using a macroencapsulation immunoisolation device instead of a mold, the embedding hydrosol containing the graft is placed into an embedding chamber surrounded by an immunoisolation membrane using a syringe or similar device, and visible light is irradiated from outside the immunoisolation membrane into the chamber to convert the hydrosol into a hydrogel, which is then transplanted into the body.

[0054] A preferred immunoisolation membrane comprises at least one selected from the group consisting of fibrous structures, porous membranes, and hydrogels. The thickness of the immunoisolation membrane is not particularly limited, but is preferably between 20 μm and 500 μm.

[0055] Examples of "fiber structures" include nonwoven fabrics, woven fabrics, and knitted fabrics, with nonwoven fabrics being preferred. Examples of materials for the fiber structure include gelatin, collagen, chitin, chitosan, fibronectin, dextran, cellulose, polyethylene (PE), polypropylene (PP), polyurethane, polyamide, polyester, polyvinyl alcohol (PVA), polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polyvinyl alcohol, polycaprolactone, polyglycerol sebaciate, polyhydroxyalkanoic acid, polybutylene succinate, polymerylene carbonate, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, carboxymethylcellulose, fibroin, and silk. The fiber structure layer may consist of one type of fiber structure layer, or two or more types of fiber structure layers may be laminated to form one fiber structure layer. Furthermore, if the immunoisolation device of the present invention includes two or more fibrous structural layers, these fibrous structural layers may be directly laminated, or a porous membrane layer or hydrogel layer may be interposed between the two fibrous structural layers.

[0056] The thickness of the fibrous structure layer is not particularly limited, but is preferably 200 μm or less, and more preferably 10 μm to 100 μm.

[0057] Examples of hydrosols used to produce "hydrogels" include sols that gel in the presence of metal ions to form hydrogels, sols that gel in response to temperature to form hydrogels, sols that gel in response to pH to form hydrogels, and sols that gel in response to light to form hydrogels. Metal ions and pH are examples of chemical 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 meets gelling conditions, or applying a magnetic field that meets gelling conditions can be performed depending on the properties of the gel used.

[0058] Examples of hydrogels that gel in the presence of metal ions include alginate gel that gels in the presence of divalent or trivalent metal ions, preferably alkaline earth metal ions such as calcium ions and magnesium ions; carrageenan gel that gels in the presence of calcium ions and / or potassium ions; and acrylic acid-based synthetic gel that gels in the presence of sodium ions.

[0059] Examples of temperature-responsive hydrogels include a temperature-responsive hydrogel made of poly(N-isopropylacrylamide) crosslinked with polyethylene glycol (commercial name: Mebiol Gel), methylcellulose, hydroxypropylcellulose, copolymers of lactic acid and ethylene glycol, triblock copolymers of polyethylene glycol and polypropylene oxide (commercial names: Pluronic®, Poloxamer), agarose, and polyvinyl alcohol.

[0060] Examples of pH-responsive hydrogels include alginate gel, chitosan gel, carboxymethylcellulose gel, and acrylic acid-based synthetic gel.

[0061] Examples of photoresponsive hydrogels include synthetic gels with azobenzene and cyclodextrin in their backbone, gels made of supramolecules with fumarate amide as a spacer, and gels that are crosslinked or bonded via nitrobenzyl groups.

[0062] The thickness of the hydrogel layer is not particularly limited, but is preferably 10 μm to 300 μm, more preferably 20 μm to 200 μm, and even more preferably 30 to 150 μm.

[0063] The hydrogel layer may consist of one type of hydrogel layer, or two or more types of hydrogel layers may be laminated together to form a single hydrogel layer. Furthermore, if the immunoisolation device of the present invention includes two or more hydrogel layers, these hydrogel layers may be directly laminated together, or a porous membrane layer or a fibrous structure layer may be interposed between the two hydrogel layers.

[0064] Hydrogels can have their permeability and strength adjusted by crosslinking to control the permeability and strength of nutrients such as glucose, physiologically active substances such as insulin, and immune system humoral factors. The gel concentration of the hydrogel is not limited, but from the viewpoint of gel strength, it is preferably 2% by mass or more, and more preferably 4% by mass or more. The upper limit of the gel concentration of the hydrogel is also not limited, but from the viewpoint of viscosity that is easy to handle, it is preferably 12% by mass or less, and more preferably 8% by mass or less.

[0065] A "porous membrane" is a membrane that has multiple pores, and its porous nature can be confirmed by scanning electron microscope (SEM) or transmission electron microscope (TEM) images of the membrane cross-section, or by the removal rate of model particles (such as latex).

[0066] The thickness of the porous membrane is not particularly limited, but is preferably 500 μm or less, more preferably 50 μm or less, and even more preferably 20 μm or less.

[0067] The average pore size of the porous membrane is not particularly limited, but is preferably 0.001 μm to 10 μm, and more preferably 0.01 μm to 3 μm.

[0068] The maximum pore size of the porous membrane is not particularly limited, but is preferably 0.01 μm to 10 μm, more preferably 0.01 μm to 3 μm. If the maximum pore size is within the above range, it is possible to suppress the entry of immune response cells into the embedding chamber and to allow sufficient permeability 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 average pore size or maximum pore size can be determined from SEM or TEM images.

[0069] The porous membrane preferably contains a polymer and is substantially composed of a polymer. The polymer forming the porous membrane is preferably biocompatible.

[0070] Examples of polymers include thermoplastic or thermosetting polymers. The polymers may also be biocompatible. Specific examples of polymers include ethylene-vinyl alcohol copolymers, polysulfones, cellulose acylates such as cellulose acetate, nitrocellulose, sulfonated polysulfones, polyethersulfones, polyacrylonitrile, styrene-acrylonitrile copolymers, styrene-butadiene copolymers, saponified ethylene-vinyl acetate copolymers, polyvinyl alcohol, polycarbonates, organosiloxane-polycarbonate copolymers, polyester carbonates, organopolysiloxanes, polyphenylene oxide, polyamides, polyimides, polyamideimides, polybenzimidazoles, and polytetrafluoroethylene (PTFE). These may be homopolymers, copolymers, polymer blends, polymer alloys, etc., from the viewpoint of solubility, optical properties, electrical properties, strength, elasticity, etc. The polymers constituting the porous membrane may include hydrophilic polymers such as polyvinylpyrrolidone, hydroxypropylcellulose, and hydroxyethylcellulose. By combining hydrophilic and hydrophobic polymers, biocompatibility can be improved.

[0071] The porous membrane is preferably a membrane formed from a single composition as a single layer, and is preferably not a laminated structure of multiple layers. Specifically, there is a method of forming a film using the phase separation reaction of polymers. For example, after applying a solution of porous film constituent polymers to a substrate such as glass, the polymers are precipitated and solidified by exposing the substrate to a poor solvent in which they are insoluble or to a low-temperature environment, thereby forming a porous film on the substrate.

[0072] The porous membrane layer may consist of one type of porous membrane layer, or two or more types of porous membrane layers may be laminated to form a single porous membrane layer. Furthermore, if the immunoisolation device of the present invention includes two or more porous membrane layers, these porous membrane layers may be directly laminated, or a hydrogel layer or a fibrous structure layer may be interposed between the two porous membrane layers.

[0073] The amount of glucose, insulin, and immune system humoral factors that permeate through an immunoisolation membrane can be measured by enclosing a sample solution containing insulin of known concentration dissolved in PBS in an embedding chamber covered with an immunoisolation membrane, immersing it in a certain amount of PBS, and then quantifying the amount of each substance eluted outside the immunoisolation membrane by ELISA after a certain period of time at 37°C.

[0074] In this specification, the permeability of the immunoisolation membrane for glucose, insulin, immune system humoral factors, etc., is expressed as a percentage of the amount of each substance that permeated after 24 hours as measured by the method described above, representing the proportion of the amount initially enclosed in the embedding chamber covered with the immunoisolation membrane.

[0075] The 24-hour permeability of the immunoisolation membrane of the present invention to insulin and glucose is preferably 90% or more, more preferably 95% or more.

[0076] The permeability of the immunoisolation membrane of the present invention to immune system humoral factors after 24 hours is preferably 30% or less, more preferably 10% or less. In the present invention, the immunoisolation membrane is preferably capable of removing 50% or more of immune response cells and immune system humoral factors, and more preferably capable of removing 90% or more. The percentage of immune response cells removed can be measured by a cell infiltration test using the Boyden chamber method, in which the immunoisolation membrane is placed in an insert well and cell migration is measured. The percentage of immune system humoral factors removed can be measured by a permeability test in which a solution containing immune system humoral factors is filtered or diffused through the immunoisolation membrane.

[0077] The hydrogel for embedding according to the present invention can prevent immune response cells from attacking the graft even if they pass through the immune isolation membrane and enter the embedding chamber. Hereinafter, one embodiment of the present invention will be described with reference to the drawings.

[0078] A schematic diagram of a macroencapsulated immunoisolation device, in which an embedding hydrogel is enclosed in an embedding chamber surrounded by an immunoisolation membrane, shows the immunoisolation membrane molded into a bag shape (Figure 1) or a tubular shape (Figure 2). The bag-shaped device (Figure 1) is formed by welding (a3) ​​the multilayered immunoisolation layers (a1 and a2) shown below, separated by a certain distance (a4), using heat, ultrasound, high frequency, electron beam, etc., to secure a space (embedding chamber) for embedding the cell fixation layer. A spacer may be provided to ensure a certain distance (a4).

[0079] The tubular device (Figure 2) is composed of tubularly formed immunoisolation layers (b1, b2, b3), with an immunoisolation membrane embedded inside the tubular structure (b4). The ends of the tubular structure are then sealed by welding using heat, ultrasound, high frequency, electron beam, etc.

[0080] Figure 3 shows an example of a conceptual diagram of the isolation layer of the above device. A porous membrane (1) is used as the outer layer, and a hydrogel layer (5) is multilayered as the inner layer via a junction (6). The outermost layer (7) is in contact with the host's implantation site, and the innermost layer (8) is in contact with the immunoisolation membrane.

[0081] Figure 4 shows an example of a mold for producing a hydrogel for immunoisolation membranes using microfabrication technology, for dispersing and fixing cells and other organisms in a nearly uniform manner. It has a certain height (2) and width (3) and has microdimples separated by walls (4). Figure 6 shows the overall view of the mold. Figure 5 is a conceptual diagram of a hydrogel sheet containing cells and other organisms, produced using the mold. In Figure 5, 9 is the overall height of the device, 10 is the height of the cell embedding section, 11 is the width of the cell embedding section, 12 is the distance between cell embedding sections, and 13 indicates the embedded cells.

[0082] This invention relates to transplantation devices and transplantation materials used in cell transplantation therapy and the like, and more particularly to immune isolation devices and transplantation materials for protecting the transplanted tissue from immune rejection.

[0083] As shown in Figure 1 or Figure 2, the device concept diagram consists of a bag-like or tubular shape, and the graft material to be transplanted, such as cells or cell aggregates, is embedded inside the device in a space surrounded by an immunoisolation membrane. The immunoisolation membrane represents the graft material, such as cells or cell aggregates, which is dispersed and fixed almost uniformly in the hydrogel inside the embedding chamber.

[0084] In addition to uniformly fixing cells, the immunoisolation membrane can also be given the function of preventing the entry of immune system humoral factors such as antibodies into the graft by adjusting the crosslinking strength and / or gel strength of the fixation material such as hydrogel. Furthermore, the hydrogel for embedding grafts as a cell fixation material can suppress the permeation of immune system humoral factors without suppressing the release of physiologically active substances from cells, etc., by adjusting the degree of gel crosslinking and / or gel strength. Therefore, the hydrogel for embedding grafts of the present invention may be used as a graft material by embedding the graft without using an immunoisolation membrane, or the graft may be embedded in the hydrogel and then covered with an immunoisolation membrane to be used as a graft material.

[0085] To maintain the engraftment and functionality of transplanted tissues such as cells after transplantation, it is important to adjust the gel strength and other factors, uniformly disperse and fix the transplanted tissues such as cells embedded in the gel without aggregation or localization, maintain a constant spacing between cells, and maintain a constant thickness of the gel layer, as these are crucial elements for supplying oxygen and / or nutrients to the cells.

[0086] While it is possible to disperse and fix cells by suspending and dispersing them in a hydrosol solution and, for example, gelling them into a sheet, it is not easy to suppress cell aggregation and maintain a certain distance between cells. Therefore, by fabricating a plastic sheet with numerous microdimples on the bottom surface, for example, with a diameter of 50-500 μm and a depth of 100-500 μm, and with the spacing between individual microdimples being 50 μm or less, using microfabrication technology, and then using this as a template to fabricate a hydrogel sheet, it becomes possible to efficiently and uniformly disperse and fix cells while maintaining a certain distance between them, without causing them to aggregate or localize.

[0087] From the perspective of ensuring sufficient diffusion of oxygen and / or nutrients to the inner cells by maintaining a certain distance between cells and suppressing cell aggregation, it is desirable to maintain a distance of 10 μm or more between cells. Furthermore, from the perspective of reducing the volume of the embedding chamber while encapsulating the number of cells necessary to obtain a therapeutic effect, and thereby reducing the size of the transplant material, it is desirable to maintain a distance of 500 μm or less between cells and other tissues.

[0088] This gelation process can be easily achieved by photocrosslinking through irradiation with visible light in the visible light region of 360 nm or higher (e.g., 365 nm) for one minute or more, which does not adversely affect cells, etc.

[0089] Alternatively, instead of hydrogel sheets, microcapsule beads with a diameter of 500 μm or less may be used, which are obtained by microencapsulating cells or other materials suspended and dispersed in the same hydrogel solution using a microreactor method, and then gelling them by photocrosslinking. [Examples]

[0090] The present invention will be described in more detail below using examples, but it goes without saying that the present invention is not limited to these examples. Example 1 (Gel preparation and functional control by gel concentration) 1) Synthesis of photocurable vinyl alcohol polymers 40 g of PVA117 (polyvinyl alcohol (product name "PVA117", saponified polyvinyl acetate, average degree of polymerization 1700, degree of saponification approximately 98.0-99.0 mol%, viscosity (4%, 20℃) 25.0-31.0 mPa·s, manufactured by Kuraray Co., Ltd.)) was used and dissolved in 350 mL of dimethyl sulfoxide (DMSO) at 80°C for 4 hours. Then, 2.1 g (18.7 mmol) of vinyl methacrylate was added and the mixture was stirred at 80°C for another 3 hours. After cooling, the reaction solution was poured into 2 L of methanol while stirring. The stirring was stopped and the mixture was left to stand for 1 hour. After collecting the obtained solid, it was washed by immersing it in 1 L of methanol for another 1 hour. This washing process was repeated a total of three times. The collected solid was vacuum-dried overnight at room temperature to obtain methacryloylated PVA117. The introduction rate of ethylenically unsaturated groups (methacryloyloxy groups) in the methacryloylated PVA117 was 2.0 mol% relative to the repeating units of the raw material PVA (hereinafter abbreviated as "MA-PVA117(2.0)").

[0091] 12 g of MA-PVA117(2.0) was dissolved in 88 mL of deionized water at 80°C for 4 hours while stirring. After cooling to room temperature, L0290 (lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (photoradical polymerization initiator, trade name "L0290", manufactured by Tokyo Chemical Industry Co., Ltd.)), a water-soluble photoradical polymerization initiator, was added to this MA-PVA117(2.0) aqueous solution to a concentration of 0.1% by mass and dissolved to prepare an uncured gel solution (hereinafter abbreviated as "photocured PVA117").

[0092] Using the same procedure, uncured gel solutions with different degrees of polymerization (hereinafter abbreviated as "photocurable PVA124" and "photocurable PVA235") were prepared using PVA124 (polyvinyl alcohol (product name "PVA124", saponified polyvinyl acetate, average degree of polymerization 2400, degree of saponification approximately 98.0-99.0 mol%, viscosity (4%, 20℃) 40.0-48.0 mPa·s, manufactured by Kuraray Co., Ltd.)) and PVA235 (polyvinyl alcohol (product name "PVA235", average degree of polymerization 3500, degree of saponification approximately 87.0-89.0 mol%, viscosity (4%, 20℃) 45.0-52.0 mPa·s, manufactured by Kuraray Co., Ltd.)).

[0093] 2) Preparation of PVA hydrogels and evaluation of material permeability The photocurable PVA obtained by the above synthesis procedure was adjusted to concentrations of 8% by mass and 4% by mass in PBS. 0.1 ml of each hydrosol was poured into a mold container measuring W16 mm × D12.5 mm × H0.5 mm, and irradiated with 365 nm visible light for 3 minutes to produce hydrogels of Sample A (photocurable PVA117, concentration 8% by mass), Sample B (photocurable PVA117, concentration 4% by mass), Sample C (photocurable PVA124, concentration 4% by mass), and Sample D (photocurable PVA235, concentration 4% by mass).

[0094] Next, protein permeability tests were performed on samples A, B, C, and D. PVA hydrosol solutions containing insulin (30 U / L), glucose (5 mg / mL), and IgG (0.5 μg / mL) were photocured by photocrosslinking. These samples were then immersed in 20 mL of PBS solution, and 0.5 mL of the PBS solution was collected at various points in time. The amount of each substance in the PBS solution was measured by ELISA. The results are shown in Table 1. Samples A, B, C, and D all show a glucose transmittance of 100% after 24 hours. Samples A, B, C, and D all show an insulin transfer rate of 95% or higher after 24 hours. Samples A, B, C, and D all show an IgG transmittance of 10% or less after 24 hours. Samples B, C, and D, with a gel concentration of 4% by mass, exhibit higher permeability than sample A, with a gel concentration of 8% by mass. I confirmed that.

[0095] [Table 1]

[0096] 3) Functionality evaluation (cell embedding and fixation) Sample A solution, containing mouse fibroblasts NIH / 3T3, was placed in a 24-well multi-well plate and hydrogelized by irradiation with 365 nm visible light for 3 minutes. The cells were then cultured at 37°C and 5% CO2 using Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 15% fetal bovine serum, 100 U / ml penicillin, and 100 μl / ml streptomycin. After 24 hours, the cells were removed and observed under a phase-contrast microscope. The cells maintained their spherical shape, and embedding and immobilization of NIH / 3T3 cells into PVA hydrogel was possible.

[0097] Furthermore, mouse fibroblasts NIH / 3T3 (2*10) were added to the hydrogel. 6 A device containing (1) cells was prepared and cultured in the wells of a 6-well plate. On day 7, the culture surface was observed, and the cells adhering to the wells and the culture supernatant were collected and the cell nuclei were counted. No cell invasion was observed in samples A, B, and C.

[0098] Following the sample preparation conditions in 2), photocurable PVA117 was used in a sterile environment. Rat islet cells (1,000 islets) were suspended in each hydrosol solution, which was adjusted to a concentration of 8% by mass using Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 15% fetal bovine serum, 100 U / ml penicillin, and 100 μl / ml streptomycin. These solutions were then injected into a W16mm × D12.5mm × H0.5mm (0.1ml) mold container and irradiated with 365nm visible light for 3 minutes to produce hydrogels embedded with islet cells. Next, a blood glucose reduction study was conducted by intraperitoneal transplantation (n=4) of this hydrogel into STZ diabetic model mice. As a result, the hydrogel transplantation group maintained normal blood glucose levels from 440-520 mg / dl to 180-240 mg / dl for one week starting the day after transplantation.

[0099] Example 2 (Micro-dimple sheet) Mouse pancreatic β-cell tumorigenic cell line MIN6 was seeded in a suspension cell culture flask (manufactured by Sumitomo Bakelite) and cultured for one week to obtain more than 2000 cell aggregates with a diameter of approximately 150 μm. Approximately 2300 of the MIN6 cell aggregates obtained above were suspended in Sample C (4% MA-PVA124PVA solution, 1 mL) similar to that in Example 1. 250 μL of the PVA solution containing the cell aggregates was injected into a 24-well SBS standard size container (W128 × D85 × H20 mm) with a micro-dimple sheet having a diameter of 500 μm, a height of 400 μm, and a pore spacing of 30 μm. A hydrogel sheet with a thickness of 500 μm was prepared by irradiating with 365 nm visible light for 3 minutes.

[0100] When the obtained hydrogel sheets were immersed in PBS and observed under an inverted microscope, it was confirmed that cell aggregates were embedded in the gel within microdimples, thus enabling the creation of hydrogel sheets in which cells are arranged at appropriate intervals. (Figure 7) [Explanation of Symbols]

[0101] a1 immunoisolation layer a2 immunoisolation layer a3 welding a4 fixed distance b1 immunoisolation layer b2 immunoisolation layer b3 immunoisolation layer b4 tubular interior 1 Porous membrane 2 Height 3 width 4. Thickness 5 Hydrogel layer 6 Joint 7 Outermost layer 8. Innermost layer 9. Overall height of the device 10. Height of the cell embedding area 11 Width of cell embedding area 12 Distance between cell embedding areas 13. Embedded cells

Claims

1. A graft material comprising embedding a graft in a hydrogel for graft embedding containing a cross-linked polymer and water, The polymer is a polyvinyl alcohol-based polymer, and is a polymer obtained by crosslinking a polymer having a hydrophilic backbone capable of forming a hydrogel in the presence of water and polymerizable groups that can be crosslinked with visible light, and the graft is a plurality of cells or cell aggregates. A transplant material in which multiple cells or cell clusters embedded in a hydrogel are arranged at intervals of 10 μm to 500 μm without touching each other.

2. A transplant material comprising a transplant subject enclosed by an immunoisolation device, The aforementioned immunoisolation device comprises a hydrogel for implantation containing a crosslinked polymer and water, The polymer is a polyvinyl alcohol-based polymer, and is a polymer obtained by crosslinking a polymer having a hydrophilic backbone capable of forming a hydrogel in the presence of water and polymerizable groups that can be crosslinked with visible light. The aforementioned transplant is a plurality of cells or a cell mass, A transplant material in which multiple cells or cell clusters contained within an immune isolation device are arranged at intervals of 10 μm to 500 μm without touching each other.

3. A transplant material comprising a transplant embedded in a hydrogel containing a cross-linked polymer and water, in an embedding chamber covered with an immunoisolation membrane of a macroencapsulated immunoisolation device, The implant material is a polyvinyl alcohol-based polymer, and is a polymer obtained by crosslinking a polymer having a hydrophilic backbone capable of forming a hydrogel in the presence of water and polymerizable groups that can be crosslinked with visible light.

4. The implantation material according to claim 3, wherein the immunoisolation membrane comprises a hydrogel.

5. The implantation material according to claim 3 or 4, wherein the wavelength of the visible light is 360 nm or more.

6. The transplant material according to any one of claims 3 to 5, wherein the immune isolation membrane is capable of removing 50% or more of immune response cells and immune system humoral factors.

7. The transplant material according to any one of claims 3 to 6, wherein the immunoisolation membrane prevents the permeation of immune response cells, the permeability of insulin and glucose is 95% or more, and the permeability of immune system humoral factors is 10% or less.

8. The implantation material according to any one of claims 3 to 7, wherein the thickness of the immunoisolation membrane is 20 μm or more and 500 μm or less, and the graft is a plurality of cells or cell aggregates, and the plurality of cells or cell aggregates embedded in the hydrogel are arranged at intervals of 10 μm or more and 500 μm or less without contact with each other.

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