JP2022071773A - Immune isolation device capable of inducing angiogenesis - Google Patents

Abstract

To provide an immune isolation device capable of inducing angiogenesis that promotes angiogenesis around the device outermost layer without the use of a special growth factor or a drug while maintaining the immune isolation property using a constituent material having excellent biocompatibility without interfering with the engraftment of transplanted cells.SOLUTION: Provided is an immune isolation device that includes an embedding room for an object to be transplanted, and in which the embedding room is structured by covering with a multilayer immune isolation membrane, and the outermost layer of the multilayer immune isolation membrane is a non-woven fabric layer and the average fuzz length on the outermost layer surface is 1 to 1000 μm.SELECTED DRAWING: Figure 1

Description

The present invention relates to an immunological isolation device capable of inducing angiogenesis.

Immunoisolation devices have been developed as a means of performing cell transplantation therapy without the need for administration of immunosuppressive agents. Macro-encapsulated immuno-isolation device in that the transplantation site can be identified and the device can be exchanged especially in the case of iPS cell-derived somatic cell transplantation or functional deterioration of transplanted cells, which are concerned about the risk of canceration. Is considered to be an effective method.

The functions required for a macroencapsulated immunoseparation device are that cells or cell clusters can be dispersed and fixed without being uniformly associated, that oxygen and nutrients can be easily permeated into transplanted cells, and that cells required for therapeutic effects. It is possible to easily release the physiologically active substances (cytokines, hormones, growth factors, etc.) intended to be released by the cell in response to the cellular response, and the immune response cells and the immune response factors are not allowed to permeate, and the transplanted device is used. It is important that it has excellent biocompatibility and is less likely to cause inflammatory reactions such as adhesion with surrounding tissues and granulation.

On the other hand, subcutaneous transplantation is the method of transplantation, assuming that the invasion of the recipient due to transplantation surgery is reduced, and if canceration or abnormality occurs, or if the transplanted cells become necrotic and require retransplantation. It can be said that it is appropriate. However, the subcutaneous tissue is poor in blood vessels, and it is not always appropriate considering the supply of oxygen and nutrients to the transplanted cells and the absorption of the physiologically active substance from the transplanted cells into the body.

From the above, it is very important to promote angiogenesis around the transplant site and secure the blood flow required for the transplanted cells in the vicinity. So far, methods for promoting angiogenesis in the subcutaneous tissue in advance by impregnating and releasing angiogenic factors such as bFGF in hydrogels and the like have been studied.

In the present invention, in cell transplantation using a macroencapsulated immunoseparation device, subcutaneous is desirable as the transplantation site in consideration of invasion by transplantation surgery to the recipient, replacement of the transplantation device, and the like. However, the subcutaneous tissue has poor blood flow, and is not suitable as a transplant site in view of the supply of oxygen and nutrients to the transplanted cells and the release and diffusion of physiologically active substances from the transplanted cells. As an angiogenesis technique in the subcutaneous tissue, many studies have been studied to promote engraftment of transplanted cells by ensuring blood flow by pretreatment by releasing growth factors such as bFGF. Considering sustainability, there is a big problem in terms of cost.

The present invention uses a constituent material with excellent biocompatibility without interfering with the engraftment of transplanted cells, maintains immunoisolation, and does not use special growth factors or drugs, and is the outermost layer of the device. The purpose is to promote angiogenesis around the body.

The present invention provides the following immunological isolation devices.
[1] An embedding chamber of an object to be transplanted is provided, the embedding chamber is covered with a multi-layer immunoisolation membrane, and the outermost layer of the multi-layer immunoisolation membrane is a non-woven layer, and the average of the outermost layer surface. An immunological isolation device having a fluff length of 1 to 1000 μm.
[2] The immunoisolation device according to [1], wherein the multi-layer immunoisolation membrane includes at least one layer selected from the group consisting of a porous membrane layer and a hydrogel layer together with a non-woven fabric layer as an outermost layer.
[3] The immunological isolation device according to [1] or [2], wherein the fluff on the outermost layer surface can promote angiogenesis around the immune isolation device.

Even in transplantation sites where blood vessels are scarce, such as subcutaneous transplantation, it is possible to promote angiogenesis without using special growth factors or drugs, and in addition, it becomes difficult to remove the device due to adhesions, etc. It can solve the problem that the device is damaged and it is difficult to control the thickness of the device suitable for improving the diffusion efficiency of the physiologically active substance required for transplantation while maintaining the immunoisolation effect.

By using a non-woven fabric as the outermost layer of the device and providing appropriate fluff on the surface of the non-woven fabric, it is possible to promote angiogenesis. The angiogenesis promoting effect and the adhesion prevention of the device can be adjusted to an appropriate state by the structure of the non-woven fabric surface and the non-woven fabric material. For example, by using ethylene vinyl alcohol copolymer, which is a material with excellent biocompatibility, and adjusting the fluffing condition of the non-woven fabric surface by melt blow conditions and post-processing, it promotes angiogenesis and is fibrous. It is possible to prevent device adhesion due to tissues and the like.

Alternatively, as a pre-transplant treatment, a device having a non-woven fabric having a large amount of fluff and a high angiogenic effect as the outermost layer was transplanted, and after promoting angiogenesis, surface-treated cells having no fluff were embedded. Long-term engraftment can be achieved by replacing the non-woven fabric with a device having the outermost layer.

Perspective view of a bag-shaped immunological isolation device Cross section of tubular immunoisolation device Cross-sectional view of an immunoisolation membrane with a two-layer structure consisting of a non-woven fabric layer (outer layer) and a porous membrane (inner layer) Cross-sectional view of an immunoseparator membrane in which a non-woven fabric layer (outer layer) and a hydrogel layer (inner layer) are multi-layered.

The present invention relates to a transplant device used for cell transplantation therapy and the like, and more particularly to an immunoisolation device for protecting against immune rejection of a transplant piece due to transplantation. Since the device of the present invention can induce angiogenesis in the surroundings, oxygen and nutrient components can be supplied from blood vessels to the implant inside the device, and physiological activities such as insulin, growth factors, and cytokines released from the implant can be supplied. The substance can be supplied systemically through the blood vessels around the device.

Nonwoven fabric is made by adhering or entwining fibers by thermal, mechanical or chemical action, and by adjusting the texture (weight per unit area) according to the fiber diameter and amount, it is permeable in addition to strength. It is possible to control the filterability. FIG. 3 shows a conceptual diagram in which a non-woven fabric, which is an outer layer, a porous film, and a non-woven fabric are bonded and fixed by thermal, mechanical, or chemical action to form a multi-layered structure. FIG. 4 shows a conceptual diagram in which a non-woven fabric, which is an outer layer, a porous film, and a non-woven fabric are bonded and fixed by thermal, mechanical, or chemical action to form a multi-layered structure.

The thickness of the nonwoven fabric is preferably as thin as possible of 100 μm or less in consideration of the material diffusion efficiency of the physiologically active substance from the graft, but the main purpose of the multilayering of the nonwoven fabric is the durability of the isolation layer including the porous membrane. It is necessary to fully consider that it is an improvement.

Further, as the fiber material of the non-woven fabric, a material having excellent biocompatibility is desirable as in the case of the porous film, but since the outermost layer is a non-woven fabric, the fiber material of the non-woven fabric is also preferably a material having excellent biocompatibility. ..

When subcutaneous transplantation, which is less surgically invasive, is selected as the transplantation site, it is extremely necessary to secure blood flow near the device in supplying oxygen and nutrients to the transplanted cells and releasing and diffusing physiologically active substances from the cells. It is an important factor. However, the subcutaneous tissue is poor in blood vessels and is not suitable as a transplant site from the viewpoint of ensuring blood flow. Therefore, it is necessary to induce angiogenesis around the transplant device in the subcutaneous tissue. As a method for inducing angiogenesis, a method in which a growth factor such as bFGF or HGF is administered in advance or impregnated into a device and released is reported.

The present invention promotes the migration of inflammatory cytokines by physically stimulating the host-side transplantation site by providing a non-woven fabric on the outermost layer of the device and controlling the average fluff length on the surface of the non-woven fabric from 1 μm to 1,000 μm. It is an invention that can promote angiogenesis. The average fluff length is preferably 10 to 100 μm as an effect of promoting angiogenesis. If the fluff length is too long, an inflammatory reaction is strongly evoked, granulation tissue is formed around it, and if the fluff length is too short, angiogenesis is not sufficiently evoked. The surface of the device may be fluffy overall, but some fluffy portions of the surface may induce angiogenesis and others may suppress fluffing.

As a method of controlling the fluffing condition of the outermost layer of the non-woven fabric, in addition to adjusting the melt blow conditions (temperature, pressure, etc.), embossing and calendar processing, which are post-processing, or adjusting the conditions of spunlace (water flow entanglement), and physical It can be controlled by surface friction treatment.

Materials for the non-woven fabric include gelatin, collagen, chitin, chitosan, fibronectin, dextran, cellulose, polyethylene (PE), polypropylene (PP), polyurethane, polyamide, polyester, polyvinyl alcohol (PVA), polylactic acid, polyglycolic acid, and poly. Lactic acid-polyglycolic acid copolymer, polyvinyl alcohol, polycaprolactone, polyglycerol sebacic acid, polyhydroxyalkanoic acid, polybutylene succinate, polymerylene carbonate, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, Examples thereof include carboxymethyl cellulose, fibroin, silk and the like, and polyvinyl alcohol is preferable. The non-woven fabric layer may be composed of one kind of non-woven fabric layer, or two or more kinds of non-woven fabrics may be laminated to form one non-woven fabric layer. Further, one nonwoven fabric layer may be formed from two or more kinds of materials.

The thickness of the nonwoven fabric layer is not particularly limited, but is preferably 500 μm or less, more preferably 10 μm to 100 μm.

The immunoseparator membrane of the present invention may be a single-layer immunoseparator membrane having a non-woven fabric layer as an outer surface and is composed of a non-woven fabric layer, and is at least one selected from the group consisting of a porous membrane layer and a hydrogel layer. It may be a layer in which the porous membrane and the hydrogel are integrated, including the layer of. Preferred hydrogels can be produced by gelling the hydrosol. In addition to the immune isolation membrane, hydrogel fills the embedding chamber with the implant to physically protect the implant and protect the implant from immune-responsive cells that have invaded through the immunoisolation membrane. You may.

Examples of the hydrosol for producing a "hydrogel" include a sol that gels in the presence of metal ions to form a hydrogel, a sol that gels in response to temperature to form a hydrogel, and a sol that responds to pH. Examples thereof include a sol that gels to form a hydrogel, a sol that forms a hydrogel in response to light, and an MPC (2-methacryloyloxyethyl phosphorylcholine) polymer that forms a sol with sorbitol. Metal ions and pH are examples of chemical action. In order to gel these hydrosols, depending on the characteristics of the gel used, contact with metal ions, adjust the temperature to gelling conditions, adjust the pH to gelling conditions, and use light under gelation conditions. Operations such as irradiating and applying a magnetic field under gelation conditions may be performed.

Hydrogels that gel in the presence of metal ions include alginate gels and calcium ions that gel in the presence of divalent or trivalent metal ions, preferably alkaline earth metal ions such as calcium ions and magnesium ions. Examples thereof include carrageenan gel that gels in the presence of potassium ion, acrylic acid-based synthetic gel that gels in the presence of sodium ion, and the like.

As the temperature-responsive hydrogel, a temperature-responsive hydrogel (trade name: Mobiol gel) obtained by cross-linking poly (N-isopropylacrylamide) with polyethylene glycol, methyl cellulose, hydroxypropyl cellulose, a copolymer of lactic acid and ethylene glycol, and polyethylene glycol. And a triblock copolymer of polypropylene oxide (commercially available names: pluronic, poloxamer), agarose, polyvinyl alcohol and the like can be mentioned.

Examples of the pH-responsive hydrogel include alginic acid gel, chitosan gel, carboxymethyl cellulose gel, acrylic acid-based synthetic gel and the like.

Examples of the photoresponsive hydrogel include a synthetic gel in which azobenzene and cyclodextrin are combined in a skeleton, a gel composed of supramolecules using fumaric acid amide as a spacer, and a gel cross-linked or bonded via a nitrobenzyl group. ..

The thickness of the hydrogel layer is not particularly limited, but is preferably 5 μm to 200 μm, and more preferably 5 μm to 50 μm.

The hydrogel layer may be composed of one kind of hydrogel layer, or two or more kinds of hydrogel layers may be laminated to form one hydrogel layer.

Hydrogel can regulate the transmittance, intensity, etc. of nutritional substances such as glucose, physiologically active substances such as insulin, and humoral factors of the immune system by cross-linking.

The "porous film" is a film having a plurality of pores, and the porous film can be confirmed by a scanning electron microscope (SEM) image or a transmission electron microscope (TEM) image of the cross section of the film. ..

The thickness of the porous membrane is not particularly limited, but is preferably 200 μm or less, more preferably 50 μm or less.

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.

The maximum pore size of the porous membrane is not particularly limited, but is preferably 0.01 μm to 10 μm, and more preferably 0.01 μm to 3 μm. When the maximum pore size is within the above range, the invasion of immune response cells into the embedding chamber is suppressed, and nutritional substances such as carbon sources such as amino acids, vitamins, inorganic salts and glucose, oxygen, carbon dioxide and cytokines. , Hormones, insulin and other physiologically active substances can be sufficiently permeated.

It is preferable that the porous membrane contains a polymer and is substantially composed of the polymer. The polymer forming the porous membrane is preferably biocompatible.

Examples of polymers include thermoplastic or thermosetting polymers. The polymer may be biocompatible. Specific examples of the polymer include ethylene-vinyl alcohol copolymer, polysulfone, cellulose acylate such as cellulose acetate, nitrocellulose, sulfonated polysulfone, polyethersulfone, polyacrylonitrile, styrene-acrylonitrile copolymer, and styrene-butadiene copolymer. , Ethylene-vinyl acetate copolymer kenide, polyvinyl alcohol, polycarbonate, organosiloxane-polycarbonate copolymer, polyester carbonate, organopolysiloxane, polyphenylene oxide, polyamide, polyimide, polyamideimide, polybenzimidazole, polytetrafluoroethylene (PTFE), etc. Can be mentioned. These may be homopolymers, copolymers, polymer blends, polymer alloys, etc. from the viewpoints of solubility, optical properties, electrical properties, strength, elasticity and the like. The polymer constituting the porous membrane may contain a hydrophilic polymer such as polyvinylpyrrolidone, hydroxypropyl cellulose, and hydroxyethyl cellulose. Biocompatibility can be improved by combining a hydrophilic polymer and a hydrophobic polymer.

The immunoisolation device is expected to be used mainly for cell transplantation therapy as a product for regenerative medicine.

As shown in FIG. 1 or 2, the device conceptual diagram has a bag-like or tubular shape, and a graft to be transplanted such as a cell or a cell mass is embedded in the device as a cell fixation layer. The cell fixation layer indicates a cell, a cell mass, or the like in which a graft is uniformly dispersed and fixed to a fixing material inside the fixation layer. Possible fixing materials include hydrogels such as alginic acid, chitosan, and polyvinyl alcohol, and three-dimensional structures such as extracellular matrix composed of collagen, gelatin, proteoglycan, and the like, as well as non-woven fabrics, textiles, and meshes.

The cell fixation layer can impart a role of preventing the invasion of immune system humoral factors such as antibodies into the graft, in addition to uniformly fixing the cells by adjusting a fixing material such as hydrogel.

Preferred embodiments of the present invention will be described with reference to the drawings.

As a schematic diagram of the device, a bag-shaped (FIG. 1) or tubular (FIG. 2) molded device is shown. The sac-like device (Fig. 1) is a multi-layer immunoisolation membrane (a1 and a2) shown below, with spacers and heat, separated by a certain distance (a4) to secure a space for embedding the implant. It is molded by welding (a3) with ultrasonic waves, high frequencies, electron beams, etc.

The tubular device (Fig. 2) is composed of each isolation layer (b1, b2, b3) formed into a tubular shape, and the cell fixation layer is embedded in the tubular interior (b4), and both ends of the tubular tube are heated by heating. It is molded by welding and sealing with ultrasonic waves, high frequencies, electron beams, and the like.

In all figures, the outermost layer is in contact with the site of transplantation and the innermost layer is in contact with the embedding chamber or implant.

FIG. 3 has a two-layer structure in which the nonwoven fabric (2) is the outer layer and the porous membrane (1) is the inner layer, the outermost layer (3) is in contact with the transplanted site, and the innermost layer (4) is the transplanted object. In contact with.

FIG. 4 shows the multi-layering of the nonwoven fabric (5) and the hydrogel (6). The hydrogel (6) is impregnated and fixed to the nonwoven fabric (5), the outermost layer surface (7) is composed of the nonwoven fabric (5), and the innermost layer surface (8) is composed of the hydrogel (6). ..

The average pore diameter is preferably 5 μm or less, which is smaller than the cell diameter, because the porous membrane needs to have a function of suppressing cell infiltration from the recipient and preventing leakage of the cells as a graft. Further, as the membrane material, a material having excellent biocompatibility that does not easily cause adhesion or inflammation with the tissue surrounding the transplantation of the recipient is desirable, and for example, ethylene vinyl alcohol copolymer or cellulose is desirable.

The film thickness is preferably as thin as possible, which is 100 μm or less, considering the material diffusion efficiency of the physiologically active substance from the implant.

With a single layer of non-woven fabric, it is not easy to suppress not only cell infiltration and cell leakage but also the permeability of necessary bioactive substances and the infiltration of immune system humoral factors such as IgG antibody. Therefore, by adjusting the gel strength or crosslink density of the hydrogel impregnated and fixed in the non-woven fabric, in addition to cell infiltration and cell leakage, an immune system such as an IgG antibody is used without suppressing the permeability of physiologically active substances. It is possible to suppress infiltration of humoral factors. Examples of hydrogels include polyvinyl alcohol-based polymers, chitosan, alginate and the like.

A non-woven fabric is used as a base material, and a hydrosol solution is directly applied to a porous membrane and hydrogelized by heat, temperature, light or chemical action to form a multi-layer.

Hereinafter, the present invention will be described in more detail based on examples.
Example 1
1) Preparation of multi-layered device A porous film formed by a polymer phase separation reaction using an ethylene vinyl alcohol copolymer and a non-woven fabric prepared by a melt blow method using an ethylene vinyl alcohol copolymer. Was multi-layered.

For multi-layering, the bonding site with the porous membrane of the non-woven fabric was treated with 10 ppm of dimethyl sulfoxide solution at 40 ° C for 30 seconds under pressure, and the porous membrane was overlaid, and the pressure of 100 kPa from the non-woven fabric side was 15 It was pressed for a second and crimped.

By setting the temperature of the thermocompression bonding (embossing) from 30 ° C to 60 ° C and the pressure from 50 to 300 kPa, a multi-layer film having a thickness of 50 to 100 μm was produced.
Further, on the surface of the non-woven fabric of the outermost layer, a fiber fluff length of 10 to 500 μm (average fluff length of 150 μm) could be confirmed.

For the trial production, a 100 μm thick porous membrane and a 200 μm thick non-woven fabric were used. The tensile strength of each was 0.1 MPa for the porous membrane and 10 MPa for the non-woven fabric.

By the multi-layering, the tensile strength was improved from 0.1MPa of the porous membrane single layer to 10MPa.

2) Functionality test 2-1) VITRO Vascular Endothelial Growth Factor (VEGF) induction Gamma-ray sterilized multi-layered device with both non-woven outermost layer a and porous membrane outermost layer b as scaffolding material at the bottom of a 35 mm petri dish installed. Using Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 15% fetal bovine serum, 100 U / ml penicillin, 100 μl / ml streptomycin under the conditions of 37 ° C, 5% CO ₂ with human fibroblasts NHDF. , HUVEC vascular endothelial cells were inoculated into the petri dish at 104 cells / ml, ^respectively , and the amount of vascular endothelial cell growth factor (VEGF) released when contact-cultured by co-culture was measured by the ELISA method.

As a result, it was confirmed that the amount of VEGF released on Day 5 was significantly higher at a: 0.68 pg / μg protein and a than at b: 0.25 pg / μg protein.

2-2) Both the outermost non-woven fabric layer a and the outermost porous membrane layer b of the gamma-ray sterilized multi-layer device were installed at the bottom of a 35 mm petri dish as scaffolding materials. Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 15% fetal bovine serum, 100 U / ml penicillin, 100 μl / ml streptomycin was used and adjusted to DMEM medium under the conditions of 37 ° C and 5% CO ₂ . ⁴ cells / ml human vascular endothelial cells HUVEC were seeded, contact-cultured with a multi-layered device, and the amount of vascular endothelial cell growth factor (VEGF) released in the culture medium was measured by the ELISA method over time. .. As a result, it was confirmed that the amount of VEGF released on Day 5 was significantly higher at a: 0.68 pg / μg protein and a than at b: 0.25 pg / μg protein.

2-3) VIVO transplantation test
Under the skin of Balb / c mice, the non-woven fabric outermost layer a (fluff length of about 30-80 μm) and the porous membrane outermost layer b adjusted to a size of 1 * 2 cm were transplanted, and one or two weeks after the transplantation. Later, a subcutaneous incision was made and gross findings around the device were observed (n = 3). We also performed immunohistochemical staining with the von Willebrand factor (vWF) to evaluate new blood vessels. As a result, significant angiogenesis was confirmed in the non-woven fabric outermost layer device transplantation group as compared with the untreated group and the porous membrane outermost layer device transplantation group.

a1 Immune isolation membrane a2 Immune isolation membrane a3 Welding a4 Fixed distance b1 Isolation layer b2 Isolation layer b3 Isolation layer b4 Tubular inside 1 Porous membrane 2 Non-woven fabric 3 Outermost layer 4 Outermost layer 5 Non-woven fabric 6 Hydrogel 7 Outermost layer surface 8 Outermost layer surface

Claims (3)

It is provided with an embedding chamber of the implant, the embedding chamber is covered with a multi-layer immunoisolation membrane, the outermost layer of the multi-layer immunoisolation membrane is a non-woven layer, and the average fluff length on the outermost layer surface is. Immune isolation device, 1-1000 μm. The immunological isolation device according to claim 1, wherein the multi-layer immunoisolation membrane includes at least one layer selected from the group consisting of a porous membrane layer and a hydrogel layer together with a non-woven fabric layer as an outermost layer. The immunological isolation device according to claim 1 or 2, wherein the fluff on the outermost layer surface can promote angiogenesis around the immune isolation device.

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