JP2017196150A - Transplant device and artificial bio-organ - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology for an artificial bio-organ, in which cells in the artificial bio-organ is separated from immune system and time dependent reduction in substance permeability between inside and outside of the artificial bio-organ arranged in a living body is suppressed.SOLUTION: An embodiment of the present invention is a transplant device and an artificial bio-organ comprising cells. The transplant device comprises a semipermeable membrane for separating the cells from outer environment and gel for separating the cells from liquid immunity, which may maintain the cells in the gel in a distributed state. The transplant device is consisting of a semipermeable membrane containing resin and a cell fixing substrate enclosed in the semipermeable membrane. Albumin adhesion volume is 10 μG/CMor less under a condition where semipermeable membrane of 1 square centimeter is immersed in 0.1% albumin solution for 90 minutes. The cell fixing substrate is hydrogel.SELECTED DRAWING: Figure 2A

Description

  The present invention relates to a transplant device and a bioartificial organ.

  In recent years, bioartificial organs made by combining artificial materials and cells have been studied. A bioartificial organ is a device intended to prevent or treat a patient's disease. A bioartificial organ contains cells and biological tissues within itself, thereby providing part or all of the functions of a patient's organ or tissue temporarily or semi-permanently. Recent research on bioartificial organs aims to take advantage of each other's strengths of artificial materials and cells.

  In the bioartificial organ, the types of cells constituting the bioartificial organ can be selected according to the type of disease to be treated. An example of a bioartificial organ is a bioartificial islet. Bioartificial islets are composed of insulin-secreting cells, such as cells within the islets. In bioartificial islets, insulin hormone secreted from cells in the islets is supplied to patients with abnormal blood glucose levels. For this reason, correction of a patient's blood glucose level is achieved.

  Another bioartificial organ produces blood clotting factors. Such a bioartificial organ has hepatocytes that produce blood coagulation factors. As blood coagulation factors, there are factor VIII and factor IX. Such bioartificial organs are used for the treatment of hemophilia patients who exhibit blood coagulation disorders due to the lack of these blood coagulation factors.

  Another bioartificial organ produces growth hormone. Such bioartificial organs have human growth hormone (hGH) secreting cells. Such bioartificial organs are used for the treatment of pituitary dwarfism caused by, for example, insufficient secretion of growth hormone.

  Another bioartificial organ contains cells that secrete parathyroid hormone. Such bioartificial organs can be used for the treatment of hypoparathyroidism. Another bioartificial organ contains cells that secrete erythropoietin. Such bioartificial organs can be used to treat anemia.

  Another bioartificial organ contains hepatocytes. Such bioartificial organs can be used for the treatment of congenital enzyme deficiency, inborn metabolic diseases, and the like. A hepatic cell has a urea cycle, which converts toxic ammonia produced in the intestinal tract into non-toxic urea. In the case of ornithine transcarbamylase deficiency, which is one of the enzyme deficiencies in the urea cycle, the urea cycle works in such transplanted bioartificial organs and synthesizes non-toxic urea to treat hyperammonemia. There is expected.

  A bioartificial organ is a device that is implanted in the body of a patient. The transplantation of a bioartificial organ has an advantage over the living organ transplantation. Living organ transplantation may not be suitable for long-term efficacy maintenance. This is because, in living organ transplantation, the transplanted cells are rejected from the patient's body by the defense mechanism of the living body. As a defense mechanism of a living body, acute rejection that occurs mainly by the latter half of the transplantation and chronic rejection that progresses in the following years. Such problems also occur in allogeneic transplants.

  In living organ transplantation, it is difficult to isolate the transplanted cells from the defense mechanism of the living body. The defense mechanism of a living body functions by cellular immunity such as antibody proteins and complements involved in humoral immunity, or phagocytes such as macrophages and helper T cells. Acute rejection in allogeneic transplantation is largely due to cellular immunity, and it is considered important to isolate transplanted cells from cellular immunity. In contrast, bioartificial organs have an immune isolation membrane as an artificial material. Immunoisolation membranes can protect the cells that make up bioartificial organs from these immunities.

  In the research of bioartificial organs, an immune isolation membrane having permselectivity has been searched for macromolecules involved in humoral immunity. The immunoisolation membrane can sequester cells constituting the bioartificial organ from humoral immunity due to permselectivity.

  Even in a bioartificial organ, there is a problem that it is difficult to maintain long-term efficacy. This is due to the clogging of permeation sites in the immune isolation membrane or the membrane surface covering with a fibrous coating. Clogging of the permeation site is due to physical clogging of the protein into the permeation site. Alternatively, because the biocompatibility of the material is not good, the protein is adsorbed on the surface of the material, and the cells adhere to the surface so that the material is covered with a fibrous coating. When the above phenomenon occurs, the movement of the permeating substance inside and outside the membrane is suppressed, and the efficacy cannot be maintained.

  As a method of isolating cells in a bioartificial organ, (1) a method of encapsulating cells in a polymer gel (sometimes referred to as entrapping immobilization method or encapsulation) has been attempted. Also, (2) a method of isolating cells with a polymer semipermeable membrane has been attempted. Further, (3) a method in which cells are sealed in a polymer gel and the polymer gel is isolated with a polymer semipermeable membrane has been attempted. Method (3) can be said to be a method in which (1) and (2) are combined. Thus, the cells in the bioartificial organ are isolated from the body fluid. For this reason, the blood in the body and the cells in the bioartificial organ are separated from each other through a polymer semipermeable membrane or the like. However, among body fluids containing blood, components that permeate a polymer gel or semipermeable membrane may come into contact with cells in a bioartificial organ. Hereinafter, such technical features are simply referred to as isolation or separation.

  The following is an example of a bioartificial pancreas as an example of a bioartificial organ. In the bioartificial pancreas, insulin-secreting cells are isolated from the body fluid by containment or encapsulation. (1) Patent Document 1 describes a method of encapsulating insulin-secreting cells in a polymer gel. In such a method, an alkali metal salt of alginic acid or gelatin is used as the polymer gel. Patent Document 2 and Non-Patent Document 1 propose a method of encapsulating insulin-secreting cells in a capsule obtained from an agarose degradation product. Patent Document 3 proposes a method for encapsulating insulin-secreting cells in a gel of polyvinyl alcohol.

  Patent Document 4 and Non-Patent Documents 2 and 3 describe (2) a method of isolating insulin-secreting cells with a polymer semipermeable membrane. In such a method, a module for bioartificial pancreas that is externally mounted or transplanted into the body is used. Such a module includes a polymer semipermeable membrane made of an ethylene-vinyl alcohol copolymer. Patent Document 5 describes a module including a polymer semipermeable membrane made of polyglycolic acid (PGA) and polycaprolactone (PCL).

  Patent Document 6 describes (3) a method of encapsulating insulin-secreting cells contained in pancreatic islets in a polymer gel and further isolating the polymer gel with a polymer semipermeable membrane. In such a method, the islets of living islets are encapsulated with agar gel. In addition, such encapsulated cells are included in a semipermeable membrane. Patent Document 7 shows an example in which encapsulation is not performed. In Patent Document 7, instead of encapsulation, collagen sponge and gelatin microspheres are enclosed in a PET mesh bag, and then islets are transplanted into the bag.

JP-A-55-157502 JP-A-62-215530 JP 2004-331643 A JP 2004-275718 A JP 2004-307505 A Japanese National Patent Publication No. 11-508477 JP 2001-299908 A

Hiroo Iwata, et al., Artificial organs, 16, 1263-1266, 1987 Naoya Kobayashi, 1 other person, “Built-in biomedical pancreas”, [online], December 19, 2011, Japan Science and Technology Agency, [searched January 15, 2016], Internet < URL: http://www.jst.go.jp/chiiki/ikusei/seika/h22/h22_hiroshima01.pdf> Shuhei Nakaji, 8 others, “Development of novel diabetes treatment using implantable bioartificial pancreas”, [online], Okayama University of Science External Collaboration Promotion Office, [searched on January 15, 2016], Internet <URL: http: //www.ous.ac.jp/renkei/wp-content/uploads/2012/01/C-5.pdf>

  The bioartificial pancreas can provide a certain metabolic function. However, in the bioartificial pancreas, it is difficult to stably supply insulin according to changes in blood glucose level. Similar problems are seen in organs other than artificial pancreas. That is, with existing bioartificial organs, it is difficult to provide metabolic functions according to changes in the living body.

  The inventors of the present invention have found that the above problem is caused by the immune system of a living body into which a bioartificial organ is transplanted and the substance permeability of the bioartificial organ. An object of the present invention is to isolate cells in a bioartificial organ from the immune system in bioartificial organ technology. Another object of the present invention is to suppress a decrease in material permeability over time between the inside and outside of a bioartificial organ placed in a living body.

[1] A semipermeable membrane containing a resin and a cell-fixing base material wrapped in the semipermeable membrane,
A device for transplantation in which the amount of adsorbed albumin is 10 μg / cm ² or less when the 1-cm square semipermeable membrane is immersed in a 0.1% albumin solution for 90 minutes.
[2] The device for transplantation according to [1], wherein the cell fixing substrate is a hydrogel.
[3] The transplantation device according to [1] or [2], wherein in the semipermeable membrane, the albumin permeability in 30 minutes is 30% or more.
[4] The transplant device according to any one of [1] to [3], wherein in the transplant device, the albumin permeability in 30 minutes is 30% or more.
[5] When the semipermeable membrane sucks water through the semipermeable membrane at a negative pressure of about 3 ± 0.2 kPa, the amount of water permeation is represented by 1,000 L / (m ² · hr) or more. The device for transplantation in any one of 1]-[4].
[6] Any of [1] to [5], wherein when the semipermeable membrane is brought into contact with serum, a reduction rate of complement value (CH50) in the serum by the semipermeable membrane is 25% or less. A transplant device.
[7] When the semipermeable membrane is contacted with serum, the increase rate of complement protein activity in the serum by the semipermeable membrane is 30% or less,
The increase in activity is due to the amount of one or more complements of C1, C2, C3, C4, C5, C6, C7, C8 and C9, the amount of protein produced by degradation of the complement, and the complement. The implantable device according to any one of [1] to [6], which is calculated based on an amount of measurement of one or more of the body complexes.
[8] The transplant device according to any one of [1] to [7], wherein the thickness of the semipermeable membrane is 50 μm or more and 200 μm or less.
[9] The transplant device according to any one of [1] to [8], wherein an average surface pore diameter is 0.1 μm or more and 3 μm or less when the semipermeable membrane on the surface of the transplant device is measured.
[10] The implantable device according to any one of [1] to [9], wherein the semipermeable membrane contains 50% by mass or more of an ethylene-vinyl alcohol copolymer.
[11] For isolating the cells from the living environment outside the transplant device by fixing the cells with a cell fixing substrate and transplanting the cells together with the transplant device into a living body. [10] The transplant device according to any one of [10].
[12] Cell-fixing substrate made of at least one polymer selected from the group consisting of collagen, heparin, chitin, chitosan, alginate, agarose, agar, thrombin, vinyl alcohol polymer, gellan gum, and resin A method of manufacturing a transplant device by wrapping in a bag made of a semipermeable membrane,
When the 1-cm square semipermeable membrane is immersed in a 0.1% albumin solution for 90 minutes, the amount of albumin adsorbed is 10 μg / cm ² or less, and the permeability of albumin in 30 minutes in the semipermeable membrane is 30% or more. A method for manufacturing an implantable device.
[13] A dispersion in which at least one polymer and cells are dispersed, selected from the group consisting of collagen, heparin, chitin, chitosan, alginate, agarose, agar, thrombin, vinyl alcohol polymer, gellan gum, A bioartificial organ is produced, which is wrapped in a bag made of a semipermeable membrane containing a resin and gels the polymer in a bag made of a semipermeable membrane containing a resin, thereby fixing the cells in the gel. A method,
When the 1-cm square semipermeable membrane is immersed in a 0.1% albumin solution for 90 minutes, the amount of albumin adsorbed is 10 μg / cm ² or less, and the permeability of albumin in 30 minutes in the semipermeable membrane is 30% or more. A method for producing a bioartificial organ.
[14] The method for producing a bioartificial organ according to [13], wherein the cell is an islet.

  The present invention can isolate cells in a bioartificial organ from the immune system in bioartificial organ technology. The present invention can also suppress a temporal decrease in substance permeability between inside and outside of a bioartificial organ placed in a living body.

It is a schematic diagram of the bioartificial organ which consists of a semipermeable membrane. Observation photograph of the surface of the bioartificial organ in Example 1 Observation photograph of the surface of the bioartificial organ in Comparative Example 1

  One aspect of the present invention includes a semipermeable membrane for isolating cells from the external environment, and a gel for isolating cells from humoral immunity, the gel being capable of holding cells dispersed in the gel It is a device for transplantation. Another embodiment of the present invention is a bioartificial organ including a transplant device and cells.

  In this specification, a bioartificial organ is an artificial organ comprising a semipermeable membrane, a cell fixing substrate wrapped in the semipermeable membrane, and cells fixed by the cell fixing substrate.

  An example of a bioartificial organ is a bioartificial pancreas. Cells provided in the bioartificial pancreas include insulin-secreting cells. Insulin-secreting cells may be pancreatic islets collected from humans or pigs, or pancreatic islets differentiated from ES cells or iPS cells, pancreatic cells, or pancreatic progenitor cells. In this specification, the bioartificial pancreas may be referred to as a bioartificial islet.

  In this specification, the device for transplantation is a device for transplanting cells to be transplanted in a living body into the living body. The device for transplantation is a device comprising a semipermeable membrane and a cell fixing substrate wrapped in the semipermeable membrane. The transplant device may or may not include cells fixed with a cell fixing substrate. The transplant device can also be provided as a kit for producing a bioartificial organ.

<Cell>
The term “cell” as used herein includes, but is not limited to, adherent cells and suspension cells. Adherent cells refer to cells that proliferate by attaching to a carrier during cell culture. Suspension cells are cells that basically do not require attachment to a carrier for cell growth. Suspended cells include cells that can weakly adhere to a carrier.

  Examples of adherent cells include osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (such as mammary epithelial cells), endothelial cells (such as vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal cells, and cardiomyocytes. , Myogenic cells, smooth muscle cells, living skeletal muscle cells, human tumor cells, fiber cells, EB virus mutant cells, hepatocytes, kidney cells, bone marrow cells, macrophages, liver parenchymal cells, small intestinal cells, mammary cells, salivary gland cells , Thyroid cells, and skin cells, but are not limited to these.

  Examples of suspension cells include, but are not limited to, T cells, B cells, killer cells, lymphocytes, and lymphoblasts.

  The cells may aggregate with each other or may be differentiated. Aggregated cells may have an organ function. The cell may be a cell immediately after being collected from a living body or a cultured cell. Cells collected from a living body may form a tissue or an organ. Examples of the tissue include endocrine tissues such as islets or exocrine tissues. The cell to be transplanted may be a single type of cell or multiple types of cells. Multiple types of cells may coexist in the cell fixing substrate. A single type of cell may be uniformly distributed in the cell fixing substrate. A plurality of types of cells may be dispersed without deviation in the cell fixing substrate. The number of the plurality of types of cells may be equal to each other or different from each other. The plurality of types of cells may be 2, 3 or 4 or more types of cells.

<Semi-permeable membrane>
[resin]
The semipermeable membrane used in the present invention is a material containing a resin. The semipermeable membrane can be produced, for example, by dissolving at least one kind of resin in a solvent and solidifying the dissolved resin. Such resin is not particularly limited. Examples of such resins include resins such as ethylene-vinyl alcohol copolymers, polysulfone polymers, polyacrylonitrile polymers, cellulose polymers such as cellulose acetate, polyamide polymers, and polycarbonate polymers. I can do it. The weight average molecular weight of these resins is preferably 10,000 or more.

  The content of the resin in the semipermeable membrane used in the present invention is preferably 80% by mass or more, more preferably 90% by mass or more. In addition to the resin, additives and solvents used for film formation may remain in the semipermeable membrane, but the content thereof is preferably 20% or less, and more preferably 10% by mass or less.

  As the resin contained in the semipermeable membrane used in the present invention, it is preferable to use an ethylene-vinyl alcohol copolymer or a polysulfone polymer. These resins are suitable for medical use because they are difficult to activate complement and are excellent in biocompatibility and chemical stability. In addition, these resins are difficult to biodegrade in vivo, and the amount of eluate generated is small.

  The ethylene-vinyl alcohol copolymer that can be contained in the semipermeable membrane may be any of a random polymer, a block polymer, and a graft polymer. The ethylene structural unit content in the ethylene-vinyl alcohol copolymer is preferably 10 mol% or more and 90 mol% or less. By making the content rate of an ethylene structural unit 10 mol% or more, it can suppress that a semipermeable membrane will activate a complement. In order to further suppress complement activation, the ethylene structural unit content is more preferably 30 mol% or more. By making the content rate of an ethylene structural unit 90 mol% or less, the fall of the softness | flexibility of a semipermeable membrane can be suppressed. In order to further suppress the decrease in flexibility, the content of the ethylene structural unit is more preferably 60 mol% or less. The saponification degree of the ethylene-vinyl alcohol copolymer is preferably 95 mol% or more.

  The ethylene-vinyl alcohol copolymer is mainly composed of an ethylene monomer and a vinyl alcohol monomer as structural units. The ethylene-vinyl alcohol copolymer may contain other monomers as structural units. Further, the ethylene-vinyl alcohol copolymer may not contain other monomers. Examples of other monomers include methacrylic acid, vinyl chloride, methyl methacrylate, and acrylonitrile. The blending ratio of other monomers in the ethylene-vinyl alcohol copolymer can be appropriately adjusted within a range not impairing the gist of the present invention. Such a blending ratio is preferably in the range of 15 mol% or less.

[Film thickness]
The thickness of the semipermeable membrane used in the present invention is preferably 50 μm or more and 200 μm or less. When the thickness is 50 μm or more, the strength of the semipermeable membrane can be increased. When the thickness is 200 μm or less, it is possible to provide a semipermeable membrane that easily adjusts the permeability of proteins such as albumin and the amount of water permeation to a desired range.

[Pore diameter]
The semipermeable membrane used in the present invention is a porous membrane provided with a plurality of minute holes. The average pore diameter of such holes is preferably 0.1 μm or more and 3 μm or less, more preferably 0.3 μm or more and 3 μm or less, and more preferably 0.5 μm or more and 2 μm or less. By having the lower limit for the average pore diameter, protein permeability and water permeation can be increased. Moreover, permeation | transmission of an external cell etc. and the outflow of an internal cell can be suppressed by having the upper limit which an average pore diameter requires. In the semipermeable membrane, the hole diameter of each hole has a predetermined distribution. At this time, in order to prevent permeation of external cells and the like, it is preferable that the semipermeable membrane surface has no pores having a pore diameter of 20 μm or more.

[Film formation]
There is no restriction | limiting in particular in the film-forming method of the semipermeable membrane used for this invention. For example, a semi-permeable membrane can be obtained by making a non-porous membrane by a known method and making a hole in the non-porous membrane by drilling. For example, a semipermeable membrane can be produced by the following method.

  First, a resin, a solvent, and an additive are mixed and dissolved by heating in a range of 50 ° C. to 120 ° C., and a mixture of the components is used as a film forming stock solution. The film-forming stock solution is preferably kept at a temperature of 20 ° C. to 95 ° C., more preferably 50 to 90 ° C. Next, a film is formed by coagulating the film-forming stock solution at a temperature of 20 ° C. to 80 ° C.

  As an example, a resin, an additive, and a solvent are mixed and dissolved by heating at 50 ° C. to 120 ° C., more preferably 50 ° C. to 80 ° C. to obtain a film forming stock solution. Furthermore, a film-forming stock solution heated to 20 ° C. to 95 ° C., more preferably 20 ° C. to 80 ° C., is applied to a plate-shaped molding tool. The film-forming stock solution on the molding tool is coagulated in a coagulation bath of 20 ° C. to 80 ° C., more preferably 20 ° C. to 60 ° C., and the additive and the solvent diffuse into the coagulation bath, so A permeable membrane is obtained. Thereafter, a purification step and a drying step can be appropriately provided.

  As for the mass ratio in the film-forming stock solution, the resin ratio is preferably 7.5% by mass to 25% by mass, and preferably 10% by mass to 20% by mass. It is preferable that the ratio of a solvent is 55 mass%-87.5 mass%, and it is preferable that it is 60 mass%-80 mass%. It is preferable that the ratio of an additive is 5 mass%-20 mass%. In order to promote the formation of the film, the additive ratio is preferably smaller than the resin ratio.

  Regarding the film formation of the semipermeable membrane used in the present invention, the entire disclosure of Japanese Patent Application Publication No. 2001-314736 of Patent Document 3 is incorporated herein.

[Membrane stock solution]
The viscosity of the film-forming stock solution at 30 ° C. is preferably 1,000 mPa · s or more and 20,000 mPa · s or less, and more preferably 1,000 mPa · s or more and 10,000 mPa · s or less.

  When the viscosity of the film-forming stock solution is less than 1,000 mPa · s or higher than 20,000 mPa · s, it is difficult to adjust the coagulation time of the film-forming stock solution, and film formation may be difficult. In addition, the membrane may be easily damaged after the stock solution is solidified.

[Measurement of viscosity of film forming stock solution]
The viscosity of the film-forming stock solution at 30 ° C. can be measured using a BL-type viscometer or a BH-type viscometer under conditions of a rotor rotational speed of 6 rpm and a temperature of 30 ° C.

[Solvent for film formation]
The solvent constituting the film forming stock solution is not particularly limited as long as it is a good solvent for the resin used for film formation. Examples thereof include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), dimethylacetamide (DMAc) and the like, or a mixed solvent containing these as components. Among them, dimethyl sulfoxide (DMSO) is preferable from the viewpoint of film forming property and low toxicity.

[Additives for film formation]
The additive constituting the film-forming stock solution is preferably a water-soluble polymer. For example, what is generally called a surfactant can be used as an additive. The surfactant is preferably a polymer surfactant. The average pore diameter of the semipermeable membrane can be adjusted by adjusting the type and amount of the additive. Moreover, the viscosity of the film-forming stock solution can be adjusted. Moreover, the phase separation temperature (LST, Land Surface Temperature) of the film-forming stock solution can be adjusted.

  The high molecular surfactant refers to a surfactant having a molecular weight of any one of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, and 9000. The surface active function may be nonionic, anionic, or cationic. Of these, nonionic polymer surfactants are preferred.

  Nonionic polymer surfactants include, for example, the following: polyethylene glycol, polyethylene oxide, polypropylene glycol, and polymers typified by polypropylene oxide; polyethylene glycol-polypropylene glycol, polyethylene glycol-polytetramethylene glycol , Polyethylene glycol-polybutylene glycol, and diblock copolymers represented by polyethylene glycol-polypentylene glycol or random copolymers; polyethylene glycol-polypropylene glycol-polyethylene glycol, polyethylene glycol-polytetramethylene glycol-polyethylene glycol , Polyethylene glycol-polybutylene glycol-polyethylene glycol And a triblock copolymer represented by polyethylene glycol-polypentylene glycol-polyethylene glycol; polypropylene glycol-sulfate sodium salt, polytetramethylene glycol-sulfate sodium salt, polybutylene glycol-sulfate sodium salt, and poly Pentylene glycol-sulfate sodium salt; and polyethylene glycol-polypropylene glycol-alkyl ether.

  In addition to what is generally referred to as a surfactant, water-soluble polymers that can be used as additives are listed. Examples thereof include polysaccharides represented by various known vinyl alcohol polymers, starch, carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and dextran.

[Complement activation]
It is preferable that the surface of the semipermeable membrane used in the present invention hardly activates complement in serum. The fact that the surface of the semipermeable membrane hardly activates complement is expressed based on the following evaluation method.

<Method A: Evaluation Based on Serum Complement Value (CH50) Measurement>
Contact the semipermeable membrane with fresh human serum. At this time, if the rate of decrease in serum complement value (CH50) is 25% or less, the surface of the semipermeable membrane is evaluated to be difficult to activate complement. Serum complement titers are proportional to the total amount of complement C1, C2, C3, C4, C5, C6, C7, C8 and C9 remaining without being activated. The degree of complement activation can be evaluated based on the serum complement value.

<Method B: Evaluation Based on Measurement of Individual Complement Activity>
Complements in the blood usually exist as inactive enzyme precursors. The enzyme precursor is activated by being stimulated by a foreign substance that has entered the body. The enzyme precursor is partially decomposed by activation and becomes a decomposition product. This degradation product forms a complex partly and partly liberates. Therefore, when the semipermeable membrane behaves as a foreign substance, the complement is activated on the surface of the semipermeable membrane, and the decomposition product as described above is generated. It can be evaluated by the following evaluation method.

  Contact the semipermeable membrane with fresh human serum. At this time, if the activation rate of the complement in serum by the semipermeable membrane is 30% or less, it is evaluated that the surface of the semipermeable membrane hardly activates the complement.

  In one embodiment, the increase in activated protein is calculated based on a measurement of the amount of each complement in fresh human serum contacted with the membrane. The complement to be measured is preferably one or more complements of C1, C2, C3, C4, C5, C6, C7, C8 and C9, and one or more complements of C3 and C4. More preferably it is.

  In one embodiment, the increase in activity is calculated based on the measurement of the amount of protein produced by degradation of the complement. The protein produced by decomposing the complement to be measured is preferably a protein that is released from the complement when the complement is activated.

  The protein released from the complement to be measured is preferably one or more of C1r, C1q, C2b, C3a, C4a and C5a, and the protein released from the complement is one or more of C3a and C5a. More preferably, it is a protein. C3a and C5a have an action of attracting inflammatory leukocytes. In particular, C3a can be effectively used as an indicator of initiation of complement system activation.

  In one embodiment, the increase in activity is calculated based on a measurement of the amount of the complement complex. The complex to be measured is preferably formed by binding of complements to each other when the complements are activated. The complex is preferably SC5b-9. SC5b-9 is the final product of the complement system activation reaction.

An example of a technique for quantifying the increase in complement activity is shown below. A semipermeable membrane of a predetermined size is immersed in a predetermined amount of fresh human serum. The volume of serum is 1 mL per cm ² surface area of the semipermeable membrane. Here, the surface area inside the membrane caused by the holes may be ignored. The same applies hereinafter. Incubate the semipermeable membrane and serum for 90 minutes at 37 ° C. with agitation. The inner surface of the incubation container is preferably previously blocked with albumin. As a reference, prepare fresh human serum without soaking the semipermeable membrane. In addition, fresh human serum to which a predetermined amount of zymosan is added instead of a semipermeable membrane is also prepared. These are incubated for 90 minutes at 37 ° C. with agitation.

  After the incubation, disodium ethylenediaminetetraacetate (EDTA) is added to each fresh human serum to 10 mM as an activation reaction terminator. After stopping the reaction, the amount of complement in serum is measured. The measurement can be performed by the ELIZA method. Let X be the amount of complement of serum immersed in a semipermeable membrane. Xmin is the amount of complement of serum that was not immersed in the semipermeable membrane. The amount of complement of serum to which zymozan has been added is defined as Xmax. The activation rate is calculated according to the following formula.

  Activation rate (%) = {(X−Xmin) / (Xmax−Xmin)} × 100

[Adsorbability of protein (albumin)]
It is preferable that the surface of the semipermeable membrane used in the present invention hardly adsorbs protein. The fact that the surface of the semipermeable membrane hardly adsorbs proteins can be evaluated based on the following evaluation method using albumin.

In the evaluation method using albumin, a 1 cm square semipermeable membrane is first immersed in a 0.1% human albumin solution for 90 minutes. At this time, if the amount of albumin adsorbed to the semipermeable membrane is 10 μg / cm ² or less, it is evaluated that the surface of the semipermeable membrane is difficult to adsorb proteins. Furthermore, it is more preferable that the albumin adsorption amount is not more than any one of 9, 8, 7, 6, 5, 4, 3, ² , and 1 μg / cm ² . The albumin adsorption amount can be adjusted by selecting a resin used for producing the semipermeable membrane. When the surface of the semipermeable membrane is difficult to adsorb proteins, it can be evaluated that the material permeability of the semipermeable membrane can be prevented from decreasing with time and the biocompatibility is high.

[Water permeability]
The water permeability of the semipermeable membrane used in the present invention is preferably high. The water permeability of the semipermeable membrane is determined by the amount of water per unit area and per unit time of the semipermeable membrane when distilled water at 25 ° C. is sucked through the semipermeable membrane at a negative pressure of 3 ± 0.2 kPa {L / (M ² · h)}. If the water permeation amount is 1,000 {L / (m ² · hr)} or more, it is evaluated that the water permeability of the semipermeable membrane is high.

[Permeability of substance (albumin)]
The permeability of albumin of the semipermeable membrane used in the present invention is 30% or more. In this embodiment, the albumin permeability is used as an indicator of the substance permeability of the semipermeable membrane. Thereby, the permeability | transmittance of various proteins other than albumin can also be evaluated indirectly. In addition, permeability of various amino acids such as glutamine, glucose, hormones, vitamins and the like can be indirectly evaluated. These substances desirably penetrate the semipermeable membrane of the present invention. The permeability of albumin can be measured by the following method.

  A semipermeable membrane having a predetermined size is folded in half, and the two sides are melt-bonded to produce a bag-like member with one side not bonded. A bag is formed from the bag-shaped member by injecting a predetermined amount of human albumin solution into the bag-shaped member and then melting and bonding one unbonded side of the bag-shaped member to close the seal. . The bag containing the human albumin solution is immersed in a predetermined volume, for example, 10 mL of Hanks buffer solution present in a predetermined volume, for example, a 15 mL tube. Shake the tube at 37 ° C. for 30 minutes. After 30 minutes, collect the Hanks buffer on the outside of the bag. The albumin concentration in the collected Hanks buffer is measured by the ELISA method. The measurement by ELISA method may be carried out using Human Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc.). The transmittance can be calculated by the following equation. Let Xn be the permeation amount of albumin transferred from the bag to the Hanks buffer. Let Xmax be the same amount of aluminum bumine as the amount injected into the bag.

  Transmittance (%) = Xn / Xmax × 100

<Cell fixation substrate>
The cell fixing substrate used in the present invention is to fix cells so as not to be detached from the cell fixing substrate.

  Examples of the cell fixing substrate include a sheet form, a fibrous form such as a nonwoven fabric, and a gel form. As the cell fixing substrate, hydrogel is preferable. In the present specification, the hydrogel refers to a polymer having a three-dimensional network structure insoluble in water and a swollen body of the polymer. Hereinafter, the hydrogel may be simply referred to as a gel.

  In one aspect of this embodiment, the cells are encapsulated in the gel by encapsulating or embedding the cells in the gel. Encapsulation suppresses aggregation between cells and uniformly disperses cells in the gel. Further, the permeation of complement proteins and antibody proteins is suppressed by the substance permeation characteristics of the gel, so that cells in the gel are isolated from humoral immunity.

  The gel is preferably composed of the following polymers. For example, collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptide, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, alginate, agarose, Agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen and derivatives thereof, in addition to fibrin, fibrinogen, thrombin, and polyglutamic acid, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, vinyl alcohol polymer, gellan gum , Xanthan gum, galactomannan, guar gum, locust bean gum and tara gum.

  Among these, collagen, heparin, chitin, chitosan, alginate, agarose, agar, thrombin, vinyl alcohol polymer, and gellan gum are preferable from the viewpoint of affinity with water and low cytotoxicity.

  By changing the concentration of the polymer used when making the gel, the molecular weight of the molecules that can pass through the network structure of the gel can be changed greatly and freely. That is, it is conceivable that the gel network structure has a small mesh when the polymer concentration is high, and a large mesh when the polymer concentration is low. If the mesh of the network structure is too large, antibodies and the like enter the network structure. In this case, rejection of the living cells in the gel is likely to occur. Rejection inhibits the production of necessary substances such as insulin.

  In the present invention, the gel, which is a cell fixing substrate, is wrapped with the above-mentioned semipermeable membrane. It is sufficient that only a part of the gel is wrapped with the semipermeable membrane. Preferably, 50% or more of the surface area of the gel, more preferably 80% or more, and still more preferably 90% or more is covered with a semipermeable membrane. The entire gel may be covered with a semipermeable membrane.

[Porting device]
The transplant device according to the present invention can be used to isolate cells from the external environment. FIG. 1 shows a bioartificial organ 20 which is an embodiment of the use of the transplant device according to the present invention. In a bag made of a semipermeable membrane 10, a cell fixing substrate 11 and desired cells 12 are placed. The cells 12 are sealed with a bag. Here, the bag is a member provided with a region closed by the semipermeable membrane 10. The bag can be produced, for example, by the method described in the column of [Substance permeability of substance (albumin)]. Moreover, a bag may be produced using a plurality of semipermeable membranes, or a bag may be produced by overlapping with a film made of another material as long as the permeability of the semipermeable membrane 10 is not suppressed. Moreover, the tube for inject | pouring a cell in a bag may be connected to the bag. Alternatively, after placing the transplant device in the living body, the bioartificial organ may be completed by injecting the cells into the transplant device. Further, after the bioartificial organ is completed in advance by arranging cells in the transplantation device, the bioartificial organ may be transplanted into the living body.

  The semipermeable membrane 10 permeates nutrients 15 necessary for the activity of the cells 12. The nutrient is, for example, an amino acid or a substance adsorbed on albumin. The nutrient 15 moves from the external environment toward the inside of the bag. The semipermeable membrane 10 permeates the secretory substance 16 that has exited from the cells 12. The secretory substance 16 moves from the inside of the bag toward the external environment. The base material 11 suppresses aggregation of cells and uniformly disperses the cells in the bag. Moreover, the humoral immunity is suppressed by the base material 11. Moreover, you may combine a bag and another raw material in the range which does not impair the effect of this embodiment. For example, the inside or outside of the implantation device may be reinforced with various mesh materials.

  In the transplant device according to the present invention, the albumin permeability in 30 minutes from the inside of the transplant device to the outside of the transplant device is preferably 30% or more. Can be measured based on the amount of albumin permeated from the inside of the bag (device for evaluation) formed of a semipermeable membrane to the outside of the bag. As in the case of the semipermeable membrane, if the albumin permeability is high, the substance permeability is high, and it can be said that it is favorable as a transplant device.

[effect]
The inventors have found that conventional implantable bioartificial organs have the following problems. That is, when cells encapsulated in cells and cells encapsulated in a bag are transplanted into a living body as a bioartificial organ, proteins in the living body adhere to the bag surface. Moreover, cells adhere using the protein as a scaffold, and a cell layer is formed on the bag surface. For this reason, the substance permeability of a bag falls. The decrease in substance permeability not only suppresses the release of substances secreted by the cells in the bag, but also prevents the cells in the bag from receiving sufficient nutrients from the external environment. For this reason, it is difficult to maintain the function of the bioartificial organ for a long time.

  As shown in FIG. 1, the semipermeable membrane 10 constituting the bag has pores large enough to pass through the nutrients 15 and the secretory material 16, while external cells including leukocytes 18 containing phagocytic cells. Does not pass through. For this reason, spreading | diffusion of the nutrient 15 and the secretory substance 16 is accelerated | stimulated, preventing the penetration | invasion of the white blood cell 18 in a bag.

  Further, since the surface of the bag hardly activates complement, external cells such as leukocytes 18 are difficult to adhere to the semipermeable membrane 10. Also, the bag surface is difficult to adsorb proteins. Therefore, a cell layer is hardly formed on the bag surface. For this reason, the time-dependent fall of the substance permeability of the bag in the living body can be suppressed. For this reason, the semipermeable membrane 10 contributes to maintaining the function of the bioartificial organ for a long period of time. As shown in FIG. 1, the cell fixing substrate 11 surrounds the periphery of the cell. For this reason, the base material 11 can prevent the approach of the complement protein or the antibody protein to the cell 12. Or the base material 11 prevents that the thing produced when the cell 12 itself decompose | disassembled flows out of a bag. Therefore, the base material 11 suppresses rejection due to humoral immunity. By using the semipermeable membrane 10 and the base material 11 in combination, it is possible to realize diffusion of nutrients and secretory substances while suppressing a decrease in substance permeability with time and suppressing a rejection reaction due to humoral immunity.

  The transplant device of the present embodiment can be used in various modes such as a tube shape and a hollow fiber shape in addition to the above modes. The transplant device of the present embodiment can be widely used for medical applications including the production of bioartificial organs, drug discovery research applications, and the like. The transplant device of the present invention is characterized in that a semi-permeable membrane having good biocompatibility is used on the device surface. The transplant device of the present invention is characterized by good permeability when substances such as nutrients move in and out of the device. Taking advantage of these characteristics, the transplantation device can be used as a treatment device before transplantation of a bioartificial organ. Further, as proposed in Patent Document 7, a blood vessel inducing factor such as bFGF may be encapsulated inside the device. Thereby, a blood vessel is born at the transplantation site, and a region suitable for cell transplantation can be prepared in advance in the living body. Alternatively, a transplantation device having a collagen sheet or the like containing a blood vessel inducing factor on its surface may be implanted subcutaneously.

[Modification of Embodiment]
Note that the present invention is not limited to the above-described embodiment, and can be changed as appropriate without departing from the spirit of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by this Example. In the following examples and comparative examples, unless otherwise specified, parts and% represent parts by mass and% by mass, respectively.

[Production of semipermeable membrane]
The semipermeable membrane used in each Example and Comparative Example is produced based on the method of Production Example 1 shown below. By changing the composition of the membrane-forming stock solution and the composition of the coagulating solution, semipermeable membranes (porous membranes) having different pore diameters and internal structures can be obtained. In each production example, the points changed from production example 1 will be described later.

1. In order to prepare a film-forming stock solution, all the following materials were put in a pressure-resistant test tube. The total material was 12 g. As the material, an ethylene-vinyl alcohol copolymer (manufactured by Kuraray Co., Ltd., Kuraray Eval (registered trademark) G110B, a content ratio of ethylene structural units of 47 mol%, a saponification degree of 98 mol%, EVOH-1 in this specification) is used. ), Dimethyl sulfoxide (DMSO) as a solvent and polyethylene oxide (PEO, molecular weight of about 60,000) as an additive, and the mass ratio thereof was 15:75:10. The pressure test tube was capped and the material was sealed. The temperature in the test tube was set to 90 ° C. by heating the test tube with a hot air dryer supplying hot air at 100 ° C. While shaking the test tube, the temperature in the test tube was maintained at 90 ° C. for 3 days to obtain a film forming stock solution.
2. The temperature of the film-forming stock solution was adjusted to 80 ° C. by adjusting the hot air dryer.
3. A glass plate was used as a molding tool. A glass plate was placed on the hot plate, and the temperature of the hot plate was adjusted so that the surface of the glass plate was 30 ° C. A tape was affixed to one end and the other end of the glass plate to adjust the thickness during film formation.
4). A film forming stock solution at 80 ° C. was hung on one end of the glass plate. The stock solution on the glass plate was uniformly stretched with a glass rod.
5. The sample was left in the atmosphere at room temperature (25 ° C.) for 1 minute.
6). The glass plate coated with the stock solution was immersed in a coagulation bath containing warm water at 30 ° C.
7). A semipermeable membrane was formed by the addition of the additive and solvent in the membrane-forming stock solution into the coagulation bath and the resin component coagulating. Ten minutes after the start of immersion, the semipermeable membrane was peeled off from the glass plate in water.
8). The four sides of the semipermeable membrane were fixed by sandwiching the semipermeable membrane between two rectangular metal frames in water and fixing two portions on each side.
9. 15 minutes after the start of immersion, the semipermeable membrane was transferred to 30 ° C. warm water and immersed in water overnight.
10. The semipermeable membrane was removed from the water. The semipermeable membrane was transferred to a mixture of acetone and water. The volume ratio of acetone and water was 8/2 and the temperature was 25 ° C. The semipermeable membrane was immersed in the mixed solution for 15 minutes.
11. The semipermeable membrane was removed from the mixed solution. The semipermeable membrane was transferred to acetone and immersed in acetone at 25 ° C. for 15 minutes.
12 The semipermeable membrane was taken out from acetone and allowed to stand in the atmosphere at room temperature (25 ° C.) for 1 hour.
13. The semipermeable membrane E-1 having a long side of 150 mm, a short side of 120 mm, and a thickness of 100 μm was obtained by drying the semipermeable membrane in a dryer set at 40 ° C. for 1 hour.

In Production Example 2, the above 1. Was changed to polyethylene glycol (PEG, molecular weight of about 20,000).
In Production Example 3, the above 1. EVOH-1 represented by formula (1) is an ethylene-vinyl alcohol copolymer (manufactured by Kuraray Co., Ltd., Kuraray Eval (registered trademark) F101B, ethylene structural unit content 32 mol%, saponification degree 98 mol%, in this specification EVOH- Changed to 2). Each composition was changed as shown in Table 1.
In Production Example 4, the above 1. EVOH-1 shown in the following was changed to a polysulfone polymer (PS) (ADEL CO., UDEL P-1700), and the solvent was changed to dimethylacetamide (DMAc). Each composition was changed as shown in Table 1.
In Production Example 5, the above 1. The mass ratio of EVOH-1, DMSO, and PEO shown in Fig. 5 was changed to 15: 85: 0.
In Production Example 6, a semipermeable membrane was produced according to Example 3 of JP-A No. 2001-314736. That is, the above 1. The additives shown in Table 1 were changed to water and lithium chloride. Each composition was changed as shown in Table 1.

  Serum complement values, individual complement activities, and albumin adsorbability were measured using the semipermeable membrane. The measuring method is shown below.

[Method A: Measurement of serum complement value (CH50)]
Serum complement value (CH50) can be measured using a commercially available measuring reagent. Examples of the measuring reagent include Complement Value-HA Test Wako manufactured by Wako Pure Chemical Industries, Ltd. and Immunoturbidimetric Test CH50 Auto (KW) manufactured by Nippon BCG Co., Ltd.

  The reduction rate (complement value reduction rate) of serum complement value (CH50) of fresh human serum by the semipermeable membrane according to Production Examples 1 to 6 was 25% or less. For this reason, it was evaluated that the surface of the semipermeable membrane hardly activates complement.

[Method B: Measurement of individual complement activity]
The measurement of the activity of individual complements was performed using a Human OPtEIA ELISA Set C3a (Cat. No 550499) from BD Biosciences. In such measurement, the measurement target is protein C3a desorbed from complement. C3a can be used effectively as an indicator of initiation of complement system activation.

1. The semipermeable membrane according to each production example was cut and separated into pieces of 10 mm × 10 mm without changing the thickness.
2. The separated semipermeable membrane was immersed in ethanol at 25 ° C. for 30 minutes or more to expel bubbles in the holes of the semipermeable membrane. Subsequently, it was washed with PBS (−). The term PBS (-) represents PBS (phosphate buffered saline) free of magnesium and calcium.
3. A semipermeable membrane separated into a test tube and 2.0 mL of fresh human serum were added. The ratio of the semipermeable membrane to the serum was 1 (cm ² / mL) (the area of the thickness portion is negligible because it is minute). The inner surface of the test tube was previously blocked with albumin before use.
4). For negative control, fresh human serum without an individualized semipermeable membrane was similarly placed in a test tube. For positive control, fresh human serum without singulated semipermeable membrane but containing 20 mg of zymosan (Z4350-1G manufactured by SIGMA) was placed in a test tube. These tubes were warmed with agitation and incubated at 37 ° C. for 90 minutes.
5. The separated semipermeable membrane was pulled up from the test tube. The activity of complement in each serum was measured. The measurement was performed using an ELISA kit of BD Bioscience, product number 550499. Measurement was performed according to the protocol attached to the kit.
6). The activation rate was determined by the following formula. The value of complement activity in fresh human serum containing the singulated semipermeable membrane according to the production example was defined as Xn. The value of the negative control activity was defined as Xmin. The value of the positive control activity was defined as Xmax. The activation rate can be calculated by the following formula.

  Activation rate (%) = {(Xn−Xmin) / (Xmax−Xmin)} × 100

  The activation rate (complement activation rate) of individual complements in serum, that is, C3a, by the semipermeable membranes according to Production Examples 1 to 6 was 30% or less. For this reason, it was evaluated that the surface of the semipermeable membrane hardly activates complement.

[Measurement of albumin adsorption]
1. The semipermeable membrane according to each production example was cut and separated into pieces of 10 mm × 10 mm with the thickness unchanged. The separated semipermeable membrane was immersed in ethanol at 25 ° C. for 30 minutes or more. Subsequently, it was washed with PBS (−).
2. The separated film was immersed in a human albumin (A1653-10G manufactured by SIGMA) solution. The concentration of the albumin solution was 0.1% (1 mg / mL), the temperature was 30 ° C., and the volume of the solution was 2.4 mL. Immersion was performed for 90 minutes.
3. After immersion, the semipermeable membrane separated into pieces from the solution was taken out.
4). By measuring the concentration of albumin in the remaining solution, the amount of albumin adsorbed on the semipermeable membrane separated into pieces was calculated. The measurement was performed by the ELISA method. Measurement by ELISA was performed using a Human Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc.).
The amount of albumin adsorbed to the semipermeable membranes according to Production Examples 1 to 6 was 10 μg / cm ² or less. For this reason, it was evaluated that the surface of the semipermeable membrane hardly adsorbs proteins.

[Measurement of water permeability]
1. By cutting the semipermeable membrane according to each of the production examples, the semi-permeable membrane was separated into pieces having a thickness of 3 cm × 3 cm.
2. The separated film was immersed in ethanol at 25 ° C. for 30 minutes or more.
3. Thereafter, the separated film was immersed in distilled water at 30 ° C. for 30 minutes or more.
4). The membrane was mounted on a calibrated filter holder (FILTER HOLDER: ADVANTEC 25 mm, filter area 3.14 cm ² ). The filter holder consists of a lower part, a spacer, an upper part and a clip. The upper part is a part that functions as a support for the outer frame and the semipermeable membrane. A spacer was placed on the lower part, a semipermeable membrane was placed on the spacer, and an upper part was placed on the semipermeable membrane. The upper part and the lower part were clamped and fixed. The lower part was connected to a suction bottle.
5. 11 mL of distilled water was placed in the upper part of the calibrated filter holder.
6). After connecting the aspirator to the calibrated filter holder, the aspirator was operated, and the lower part side where the semipermeable membrane was not in contact with distilled water was decompressed. The water temperature at this time was 25 ° C., and the negative pressure was 3 ± 0.2 kPa.
7). The time when the volume of distilled water in the upper part was changed from 10 mL to 5 mL was recorded. When the said time was 10 minutes or more, the amount of water filtration in 10 minutes after the start of pressure reduction was recorded.
8). The water permeability of the semipermeable membrane is represented by the permeation amount of water {L / (m ² · hr)} per effective unit area and unit time of the membrane attached to the filter holder at the above negative pressure. In each production example, four semipermeable membranes were prepared, and the measurement was performed four times. The average value was used as a representative value to determine water permeability.

In the membrane according to Production Example 1, since the average of 4 measurements in the time when the volume of distilled water was changed from 10 mL to 5 mL was 22.9 seconds, the permeation amount of water was 5 mL / (3.14 cm ^2. 22.9 sec), that is, the first digit was rounded off to 2500 L / (m ² · hr).

The water permeability of the semipermeable membrane of each production example is as shown in Table 1, and the water permeability of the semipermeable membrane according to Production Examples 1 to 4 was 1000 L / (m ² · hr) or more. For this reason, the semipermeable membrane was evaluated as having high water permeability.

[Measurement of albumin permeability]
1. By cutting the semipermeable membrane according to each of the production examples, the semi-permeable membrane was separated into pieces having a short side of 25 mm and a long side of 40 mm without changing the thickness. The separated semipermeable membrane was immersed in ethanol at 25 ° C. for 30 minutes or more, and then washed with PBS (−).
2. The long side portion was bent and two sides of the original long side portion were melted and bonded with a heat sealer. Thereby, the bag-shaped member by which the original short side part was open | released was produced.
3. After injecting 200 μL of a 20 mg / L human albumin solution into the member, the original short side was sealed with a heat sealer to prepare a bag.
4). The bag containing the albumin solution was immersed in 10 mL of Hanks buffer in a 15 mL tube and shaken at 37 ° C. for 30 minutes.
5. One hour later, Hanks buffer solution outside the bag was collected.
6). The albumin concentration in the collected Hanks buffer was measured by ELISA. Measurement by ELISA was performed using a Human Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc.).
7). The transmittance was determined by the following formula. The albumin permeation amount transferred from the bag to the Hanks buffer was defined as Xn. The amount of aluminum bumine injected into the bag was taken as Xmax. That is, Xmax is 4000 ng at 20 mg / L × 200 μL.

  Transmittance (%) = Xn / Xmax × 100

  The transmittance of albumin of the semipermeable membrane according to Production Examples 1 to 4 was 30% or more. For this reason, the semipermeable membrane was evaluated as having high permeability to substances such as proteins.

[Measurement of film thickness]
The film thickness was measured with a film thickness meter, and the average of the measured values at the four corners of the formed semipermeable membrane was taken as the film thickness of the semipermeable membrane. Table 1 shows the film thickness of each of the production examples.

[Measurement of average pore size]
The pore diameter of the semipermeable membrane was measured by observing the surface of the semipermeable membrane. One surface of the semipermeable membrane was photographed with a scanning electron microscope at a magnification of 2,000, and after photographing, 30 holes, that is, voids were randomly selected, and the diameter of each void was measured. The average of the measured values was taken as the average pore diameter of the semipermeable membrane.

[Measurement of albumin permeability in implantable devices]
1. As described in [Evaluation of albumin permeability in transplantation device] in Examples and Comparative Examples described later, an albumin permeability evaluation device was obtained. In this evaluation method, gel washing and dipping treatment were all omitted. This is because, as described below, the purpose of carrying out this evaluation method is to measure the albumin inside the device that is permeated when the evaluation device is immersed in the liquid.
2. The bag containing the albumin solution was immersed in 10 mL of Hanks buffer in a 15 mL tube and shaken at 37 ° C. for 30 minutes.
3. One hour later, Hanks buffer solution outside the bag was collected.
4). The albumin concentration in the collected Hanks buffer was measured by ELISA. Measurement by ELISA was performed using a Human Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc.).
5. The transmittance was determined by the following formula. The albumin permeation amount transferred from the bag to the Hanks buffer was defined as Xn. The amount of aluminum bumine injected into the bag was taken as Xmax. That is, Xmax is 1000 ng at 200 mg / L × 5 μL or 20 mg / L × 50 μL.

  Transmittance (%) = Xn / Xmax × 100

  The transmittance of albumin of the evaluation device corresponding to each example was 30% or more. For this reason, the transplantation device according to each example was evaluated as having high permeability to substances such as proteins.

[Evaluation of bioartificial organs]
<Preparation of islets>
Islets were isolated from Wister rats (Shimizu animal Co. Ltd. Kyoto, Japan), 10-14 weeks old, weighing approximately 300 g, by the following collagenase digestion method.
1. 12 mL of collagenase / Hanks solution containing 1000 U / ml of type XI collagenase (Sigma aldrich, C7657-100MG) was injected from the open bile duct of the rat, then the pancreas was excised and transferred to a 50 mL conical tube. .
2. The said tube was moved to the 37 degreeC thermostat, and the enzyme process for 20 minutes was performed.
The enzyme reaction was stopped by adding 35 mL of Hank's solution at 3.4 ° C. The pancreatic tissue was dispersed using a pipette and collected as a tissue pellet by centrifugation. Thereafter, 40 mL of Hanks' solution was further added twice, and the tissue pellet was dispersed again by pipetting, centrifuged and washed. Centrifugation was performed at 400 G for 1 minute.
4). 10 mL of Hank's solution was added and dispersed by pipetting to obtain a tissue suspension.
5. The tissue suspension was filtered using a sieve having a 800 μm size mesh. The filtrate was collected in a 50 mL conical tube, and the tube was centrifuged (400 G, 3 minutes) to obtain an islet-containing pellet.
6). The pellet was suspended in 10 mL of Hank's solution (density 1.094 g / cm ³ ) at 4 ° C. in which dextran (manufactured by Sigma aldrich, MW 70,000, 31390-500G) was dissolved. Thereafter, a density of 1.094 g / ml dextran solution (A) and a density of 1.081 g / ml dextran solution (B) and a density of 1.041 g / ml dextran solution (C) were gently added to form a three-layer structure. . A discontinuous density gradient centrifugation was performed. Discontinuous density gradient centrifugation was performed at 40 G for 4 minutes and further at 800 G for 16 minutes.
7). After centrifugation, the islet-containing fraction was collected from the interface between the uppermost two layers (B) and (C) using a Pasteur pipette.
8). The obtained islets were washed 3 times with Hank's solution at 4 ° C.
9. Suspended in 37 ° C. CMRL-1066 medium (Life technologies / Gibco, product number 11530037) and incubated overnight at 37 ° C., 5% CO ₂ . Approximately 500 islets were obtained per rat.

  Bioartificial organs according to Examples and Comparative Examples are prepared according to the method of Example 1 shown below. In addition, in Examples 2-7 and Comparative Examples 1-4, the point changed from Example 1 is mentioned later.

Example 1.
<Bag making>
1. The semipermeable membrane E-1 produced in Production Example 1 was cut into a size of 20 mm × 30 mm, the long side was bent in two, and the two sides were welded with a heat sealer.
2. It was immersed in 70% ethanol for 3 minutes and then immersed in sterilized distilled water overnight.

<Preparation of PVA-containing cell fixative>
1. 0.48 g of PVA (polymerization degree 18,000, saponification degree 98.5 mol%) was dissolved in 9.52 mL of distilled water, and then subjected to heat dissolution sterilization treatment by autoclave (121 ° C., 15 minutes) twice.
2. 6.3 mL of the obtained sterile PVA solution was taken in a 15 mL conical tube, 2 mL of 5 times concentration ET-Kyoto electrolyte solution (hereinafter abbreviated as ETK solution), DMSO (manufactured by SIGMA aldrich, D4540-100ML) 0.5 mL, FBS ( 1 mL of biological industries, Cat.04-121-1A) and 0.1 mL of 1M nicotinamide (manufactured by SIGMA aldrich, N0636-100G), 0.1 mL of Antibiotics-Antilytic (product of Thermo Fisher Scientific, product number 15240-062) 0.1 mL By aseptically dropping and mixing, 10 mL of a sterile 3% PVA-ETK solution was obtained.

<Preparation of islet dispersion system (Example 1)>
1. 800 pancreatic islets obtained above were collected and suspended in 37 ° C. CMRL-1066 medium. The suspension was placed in a 15 mL conical tube and centrifuged at 800 G for 1 minute to remove the medium.
2. 100 μL of cell banker (CELLBANKER 1, manufactured by Nippon Zenyaku Kogyo Co., Ltd.) was added to the obtained islet deposit, and the islet was suspended and then allowed to stand for 5 minutes in an ice-cooled state.
3. After centrifugation again to remove the cell bunker, the islets were collected as pellets in a 1.5 mL tube.
4). The collected islets were suspended in the PVA-ETK solution by mixing the pellet and 100 μL of the sterilized 3% PVA-ETK solution. As a result, a PVA-ETK islet suspension was obtained.

<Production of bioartificial islets>
1. The whole amount of the PVA-ETK islet suspension obtained above was injected into the bag, and the remaining one side was welded with a heat sealer to seal the PVA-ETK islet suspension.
2. The sealed bag was stored in a special container at −80 ° C. for 24 hours. Thereby, in the PVA-ETK solution in the bag, the swelling of PVA was advanced, and the islet dispersion system was gelled.
3. After 24 hours, thawed rapidly in 37 ° C. CMRL-1066 medium to obtain bioartificial islets.
4). The thawed bioartificial islet was immersed in 5 mL of 4 ° C. ETK solution for 5 minutes. After 5 minutes, the ETK solution was removed, 5 mL of ETK solution was newly added, and the bioartificial islets were immersed. This immersion and removal operation was repeated three times in total to remove DMSO contained in the bioartificial islet.
The bioartificial islet was immersed in an ETK solution at 5.4 ° C. and stored at 4 ° C. for 24 hours.
6. After 24 hours, the cells were transferred to CMRL-1066 medium and cultured for 24 hours (37 ° C., 5% CO ₂ ). The obtained bioartificial islets were used in subsequent tests.

[Evaluation of albumin permeability in implantable devices]
For the evaluation of albumin permeability, do not perform steps 1, 2, and 3 in <Preparation of islet dispersion system> in the above operation when preparing a transplant device, but use 5 μL of a 200 mg / L human albumin solution instead of 4 pellets. It was mixed with 150 μL of 3% PVA-ETK solution and prepared as a substitute for PVA-ETK islet suspension. Thereafter, a device for evaluating albumin permeability was prepared by the operations 1 and 2 in <Preparation of bioartificial islets>.

Example 2
In Example 2, the procedure of <Preparation of islet dispersion system> in Example 1 was changed as follows.

<Preparation of islet dispersion system (Example 2)>
1. 0.5 g of chitosan (417963, Sigma, USA) was dissolved in 20 mL of acetic acid (017-00256, Wako, Japan) solution adjusted to 0.1 M and sterilized by autoclave to obtain a chitosan solution.
2. Dissolve 12 g of β-glycerophosphate disodium hydrate (G6251, Sigma, USA) in 15 mL of pure water, filter sterilize with a 0.22 μm filter (Millex-HA, Millipore, USA), Obtained. This is a GP solution.
3. While stirring the chitosan solution in a clean bench, the GP solution was added dropwise to adjust the pH to 7.4, and the solution was stored at 4 ° C. This is a gel preparation solution.
4). 800 pancreatic islets obtained above were collected and suspended in 37 ° C. CMRL-1066 medium. The suspension was placed in a 15 mL conical tube, centrifuged at 800 G for 1 minute, and the medium was removed to obtain a pellet.
5. The pellet was suspended in 200 μL of gel preparation solution to obtain an islet suspension.
<Production of bioartificial islets>
1. After the islet suspension was sealed in a bag, it was allowed to gel in an incubator at 37 ° C. for 10 minutes to obtain a bioartificial islet.
2. This was immersed in a CMRL-1066 medium, cultured for 24 hours (37 ° C., 5% CO ₂ ), and used for the subsequent experiments.

[Evaluation of albumin permeability in implantable devices]
In the case of evaluation of albumin permeability, during preparation of the transplantation device, 1, 2, and 3 were performed in <Preparation of islet dispersion system> in the above operation, 4 was not performed, 200 mg / L instead of 5 pellets Using 5 μL of human albumin solution, it was mixed with 150 μL of gel preparation solution to prepare instead of islet suspension. Thereafter, an albumin permeability evaluation device was prepared by the operation 1 in <Preparation of bioartificial islets>.

Example 3 FIG.
In Example 3, <Preparation of islet dispersion system> and <Preparation of bioartificial islet> shown above were changed as follows.

<Preparation of islet dispersion system (Example 3)>
1. Sterile CMRL-1066 medium was added to agarose powder (manufactured by Nacalai Tesque, Agarose LGT) in a dry autoclave-resistant bottle to make a 12% suspension, and then sterilized by autoclaving (121 ° C., 20 minutes).
2. 0.1 mL of a sterile CMRL-1066 medium in which 800 islets were dispersed was added to 0.1 mL of an agarose solution kept at 40 ° C. and gently mixed to obtain an islet suspension.

<Production of bioartificial islets>
1. The whole amount of 0.2 mL was injected into the bag in which the above islet suspension was prepared, and was welded and sealed with a heat sealer.
2. The bag was placed in a sterile 20 mm × 15 mm × 10 mm mold.
3. The mold and bag were stored in a refrigerator at 4 ° C. for 10 minutes to gel the agarose to obtain a bioartificial islet.
4). This was immersed in a 37 ° C. CMRL-1066 medium, cultured for 24 hours (37 ° C., 5% CO ₂ ), and used for the subsequent experiments.

[Evaluation of albumin permeability in implantable devices]
In the case of evaluation of albumin permeability, during preparation of the transplant device, 1 was performed in <Preparation of islet dispersion system> in the above operation, and 50 μL of 20 mg / L human albumin solution was used instead of 2 islets, and 50 μL of agarose solution And prepared as an alternative to islet suspension. Thereafter, a device for evaluating albumin permeability was prepared by the operations of 1, 2 and 3 in <Preparation of bioartificial islets>.

Example 4
In Example 4, <Preparation of islet dispersion system> and <Preparation of bioartificial islet> shown above were changed as follows.

<Preparation of islet dispersion system (Example 4)>
1. Sodium alginate (manufactured by Wako Pure Chemical Industries) was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Factory) to obtain a 2.0% sodium alginate solution.
2. The 800 islets obtained above were dispersed in 0.1 mL of sterile CMRL-1066 medium and mixed with 0.1 mL of the above 2.0% sodium alginate solution to obtain an islet suspension.

<Production of bioartificial islets>
1. The whole amount of 0.2 mL was injected into the bag in which the above islet suspension was prepared, and was welded and sealed with a heat sealer.
2. The bag was placed in a sterile 20 mm × 15 mm × 10 mm mold.
3. 20 mL of a 1.5% calcium chloride (Wako Pure Chemical Industries) aqueous solution was poured to gel the islet-containing calcium alginate to obtain a bioartificial islet.
After 4.5 minutes, the supernatant was removed by aspiration.
5. It was immersed in 10 mL of 1.0% calcium chloride solution and allowed to stand for 5 minutes.
6). The calcium chloride solution was removed by aspiration and immersed in 20 mL of Hank's solution for 1 minute.
7). The Hanks solution was removed by aspiration and again immersed in 20 mL of Hanks solution for 1 minute.
8). The bioartificial islets were removed from the mold, immersed in CMRL-1066 medium, cultured for 24 hours (37 ° C., 5% CO ₂ ), and used for the subsequent experiments.

[Evaluation of albumin permeability in implantable devices]
In the case of evaluation of albumin permeability, during preparation of the transplant device, 1 was performed in <Preparation of islet dispersion system> in the above operation, and 50 μL of 20 mg / L human albumin solution was used instead of 2 islets, and 50 μL of agarose solution And prepared as an alternative to islet suspension. Thereafter, an albumin permeability evaluation device was prepared by the operations 1, 2, 3, 4 and 5 in <Preparation of bioartificial islets>.

Examples 5-7.
In Example 5, the semipermeable membrane in <Bag Production> shown above was changed from E-1 to E-2.
In Example 6, the semipermeable membrane in <Bag Production> shown above was changed from E-1 to E-3.
In Example 7, the semipermeable membrane in <Bag Production> shown above was changed from E-1 to P-1.

Comparative Example 1
In Comparative Example 1, the above-described <Bag Production> was not performed, and a bioartificial pancreatic islet (but no semipermeable membrane) was produced by the following method.

<Molding of PVA gel (Comparative Example 1)>
1. Two sterilized 15 mm x 20 mm PET mesh sheets (manufactured by Sunplantech: TN70, 115) were immersed in a sterilized 3% PVA-ETK solution in advance, one of which was sterilized on a glass plate Put it on.
2. The PVA-ETK pancreatic islet suspension obtained by the operation according to Example 1 was uniformly applied on a mesh sheet, and another mesh sheet was covered from above, (PET mesh sheet) / (PVA-ETK pancreatic islet suspension) A three-layer structure of (turbid liquid) / (PET mesh sheet) was adopted. Furthermore, another sterilized glass plate is placed on the top, adjusted to a three-layered sheet with a thickness of about 1 mm, and stored for 24 hours in a freezer at −80 ° C. while sandwiched between the two glass plates. Then, the PVA in the liquid was gelled to obtain a bioartificial islet.
3. After 24 hours, thawed quickly in 37 ° C. CMRL-1066 medium.
4). The thawed bioartificial islet was immersed in 5 mL of 4 ° C. ETK solution for 5 minutes. After 5 minutes, the ETK solution was removed, 5 mL of ETK solution was newly added, and the bioartificial islets were immersed. This operation of immersion and removal was repeated a total of 3 times to remove DMSO contained in the bioartificial islet.
The bioartificial islet was immersed in an ETK solution at 5.4 ° C. and stored at 4 ° C. for 24 hours.
6. After 24 hours, the cells were transferred to CMRL-1066 medium and cultured for 24 hours (37 ° C., 5% CO ₂ ), which was used in the subsequent experiments.

[Evaluation of albumin permeability in implantable devices]
In the case of albumin permeability evaluation, a mixed solution of a PVA-ETK solution and a human albumin solution is prepared according to the method of Example 1, and a semi-permeable membrane is used using a PET mesh sheet according to the above method. The device for evaluation was produced.

Comparative Example 2
In Comparative Example 2, the above-described <Bag Production> was not performed, and a bioartificial islet (however, no semipermeable membrane) was produced by the following method.

<Preparation of islet dispersion system>
1. To agarose powder (manufactured by Nacalai Tesque, Agarose LGT) in a dry autoclave resistant bottle, a sterilized CMRL-1066 medium is added to make a 12% suspension, and then sterilized by autoclaving (121 ° C., 20 minutes), An agarose solution was obtained.
2. 0.1 mL of sterile CMRL-1066 medium in which 800 islets were dispersed was added to 0.1 mL of an agarose solution kept at 40 ° C. and gently mixed to obtain a suspension.

<Production of bioartificial islets>
1. The suspension was placed in a sterilized 20 mm × 15 mm × 10 mm mold.
2. The mold was stored in a refrigerator at 4 ° C. for 10 minutes to gel the agarose to obtain a bioartificial islet.
3. The bioartificial islets were immersed in a 37 ° C. CMRL-1066 medium and cultured for 24 hours (37 ° C., 5% CO ₂ ), and this was used in the subsequent experiments.

[Evaluation of albumin permeability in implantable devices]
In the case of evaluation of albumin permeability, a mixed solution of an agarose solution and a human albumin solution was prepared according to the method of Example 4, and an evaluation device was prepared without using a semipermeable membrane.

Comparative Examples 3-4.
In Comparative Example 3, the semipermeable membrane E-1 in the above <Making bag> was changed to E-4.
In Comparative Example 4, the semipermeable membrane E-1 in the above-described <Bag Production> was changed to E-5.

[Transplantation evaluation of bioartificial organs]
It was confirmed whether or not immune isolation was performed on the islets in the bioartificial islets. Furthermore, it was confirmed whether or not the coating on the bioartificial organ made by the fibrous coating derived from body fluid can be suppressed.

<Transplantation test: in vivo evaluation>
Preparation of diabetes model mice:
1. Streptozotocin (manufactured by Sigma aldrich, S0130-50MG) dissolved in citrate buffer (pH 4.5) was intraperitoneally administered to C57BL / 6 mice (7 weeks old, male) at 200 mg / kg per mouse body weight.
2. One week after the administration, blood glucose was measured three times, and a mouse having a blood glucose of 600 mg / dL or more twice was judged to be diabetic and used in the experiment. In addition, the mouse | mouth which does not perform streptozotocin administration was made into the normal mouse group P, and the mouse | mouth which does not transplant a bioartificial islet after administration of streptozotocin was made into the diabetic mouse group N.

Transplant:
1. Under ether anesthesia, pentobarbital was intraperitoneally administered to a diabetic model mouse (those with diabetes) per mouse body weight.
2. The abdominal wall was closed into two layers after placing the bioartificial islet obtained in Example 1 in the abdominal cavity after a midline incision. This mouse was designated as transplanted mouse group PE1.

  Similarly, mice transplanted with the bioartificial islets obtained in Examples 2 to 7 were designated as transplanted mouse groups PE2 to PE7, respectively. Mice transplanted with the bioartificial islets obtained in Comparative Examples 1 to 4 were designated as transplanted mouse groups CE1 to CE4.

<Evaluation after transplantation>
The blood glucose level was measured as follows. Blood was collected from the tail of the mouse one week and four weeks after transplantation, and the blood glucose level was measured using DRI-CHEMNX500 manufactured by Fuji Film. The average of the values obtained from three mice in each group was determined according to the following criteria.

A: Blood glucose level is less than 200 mg / dL B: Blood glucose level is 200 mg / dL or more and less than 300 mg / dL C: Blood glucose level is 300 mg / dL or more and less than 400 mg / dL D: Blood glucose level is 400 mg / dL or more

The body weight was measured as follows. The mice were weighed one week and four weeks after transplantation. The increase / decrease rate of the body weight was calculated by the following formula. In each group, the average of the increase / decrease rate values obtained from three mice was determined according to the following criteria.
Formula: [Change rate (%)] = ([weight after transplantation]-[weight before transplantation]) / [weight before transplantation]
<Standard>
A: + 5% or more B: + 0% or more and less than + 5% C: −10% or more and less than 0% D: Less than −10%

Tissue reactivity was evaluated as follows. Eight weeks later or after death, bioartificial islets were removed from the mouse body. The extraction method was as follows.
1. Pentobarbital was injected into the left abdominal cavity at 25 mg / kg of mouse body weight.
2. After confirming opacity due to anesthesia, the hair was shaved and the four legs were fixed on their back.
3. An incision was made about 4 cm along the midline.
4). Visual observation of the surface and surrounding tissue of the implanted bioartificial islets was performed.
5. We tried to detach bioartificial islets with tweezers without damaging surrounding tissues.
A typical observation example is shown in FIGS. FIG. 2A relates to Example 1, and FIG. 2B relates to Comparative Example 1. 2A and 2B, the scale of the ruler directed to the bioartificial organs 20 and 21 is in 0.5 mm increments. The observation results were determined according to the following criteria.
<Standard>
The criteria for evaluating the surface of a bioartificial organ are as follows.
A: Although a thin fibrous coating is formed on the surface of the bioartificial organ, the fibrous coating can be peeled off from the surface of the bioartificial organ.
B: A thick fibrous coating is formed on the surface of the bioartificial organ, and the fibrous coating cannot be peeled off from the surface of the bioartificial organ.

The criteria for evaluating the inflammation around the transplant site are as follows.
A: Minor and can be detached from the surrounding tissue of the bioartificial organ itself.
B: Severe inflammation is seen, and peeling from the surrounding tissue of the bioartificial organ itself is impossible.

Table 2 shows main configurations and evaluation results of Examples 1 to 7 and Comparative Examples 1 to 4. Table 2 shows the evaluation results of normal mouse group P and diabetic mouse group N that were not transplanted (Reference Examples 1 and 2).

[Evaluation]
As described above, the surface of the semipermeable membrane according to Example 1 was difficult to adsorb albumin, that is, protein. Therefore, it was suggested that the semipermeable membrane according to Example 1 has good biocompatibility. The surface of the semipermeable membrane according to Example 1 was difficult to activate complement. Therefore, it was suggested that the membrane according to Example 1 suppresses the temporal decrease in the material permeability of the semipermeable membrane in vivo. The same applies to Examples 2 to 7. Moreover, from the evaluation result of the bioartificial islet transplantation test using mice, it was found that the bioartificial organs of Examples 1 to 7 using the semipermeable membrane permeate secretory substances for a long time.
Since Comparative Examples 1 and 2 did not have a semipermeable membrane, the tissue reactivity evaluation was inferior. In Comparative Examples 3 and 4, since the bioartificial pancreatic islet had a low albumin permeability, a favorable course of the mouse was not obtained in the blood glucose level and body weight measurement. Therefore, it was found that the bioartificial organs of Examples 1 to 7 have characteristics that cells in the bioartificial organ can be isolated from the immune system of the recipient.

  From the above results, it was found that the bioartificial organ of the present invention is resistant to a decrease in substance permeability over time in vivo. For this reason, it was found that the bioartificial organ of the present invention can maintain the metabolic function of the transplanted cells over a long period of time.

10 semipermeable membrane, 11 substrate, 12 cells, 15 nutrients, 16 secretory substance, 18 leukocytes, 20, 21 bioartificial organ

Claims (14)

Comprising a semipermeable membrane containing a resin and a cell-fixing base material wrapped in the semipermeable membrane,
A device for transplantation in which the amount of adsorbed albumin is 10 μg / cm ² or less when the 1-cm square semipermeable membrane is immersed in a 0.1% albumin solution for 90 minutes.   The device for transplantation according to claim 1, wherein the cell fixing substrate is a hydrogel.   The implantable device according to claim 1 or 2, wherein the semipermeable membrane has an albumin permeability of 30% or more in 30 minutes.   The transplant device according to any one of claims 1 to 3, wherein in the transplant device, the albumin permeability in 30 minutes is 30% or more. When the semipermeable membrane sucks water through the semipermeable membrane at a negative pressure of about 3 ± 0.2 kPa, the water permeation amount is represented by 1,000 L / (m ² · hr) or more. 5. The implantable device according to any one of 4 above.   The transplant according to any one of claims 1 to 5, wherein when the semipermeable membrane is contacted with serum, a reduction rate of complement value (CH50) in the serum by the semipermeable membrane is 25% or less. Device. When the semipermeable membrane is brought into contact with serum, the rate of increase in the activity of complement protein in the serum by the semipermeable membrane is 30% or less,
The increase in activity is due to the amount of one or more complements of C1, C2, C3, C4, C5, C6, C7, C8 and C9, the amount of protein produced by degradation of the complement, and the complement. The implantable device according to any one of claims 1 to 6, which is calculated based on a measurement amount of one or more body complexes.   The transplant device according to any one of claims 1 to 7, wherein the semipermeable membrane has a thickness of 50 µm or more and 200 µm or less.   The transplant device according to any one of claims 1 to 8, wherein an average surface pore diameter when measuring a semipermeable membrane on the surface of the transplant device is 0.1 µm or more and 3 µm or less.   The implantable device according to any one of claims 1 to 9, wherein the semipermeable membrane contains 50% by mass or more of an ethylene-vinyl alcohol copolymer.   The cell according to any one of claims 1 to 10, wherein the cell is isolated from a living environment outside the transplant device by fixing the cell with a cell fixing substrate and transplanting the cell into the living body together with the transplant device. An implantable device according to any one of the above. A cell-fixing substrate made of at least one polymer selected from the group consisting of collagen, heparin, chitin, chitosan, alginate, agarose, agar, thrombin, vinyl alcohol polymer and gellan gum A method for producing an implantable device by wrapping a membrane bag,
When the 1-cm square semipermeable membrane is immersed in a 0.1% albumin solution for 90 minutes, the amount of albumin adsorbed is 10 μg / cm ² or less, and the permeability of albumin in 30 minutes in the semipermeable membrane is 30% or more. A method for manufacturing an implantable device. A resin containing a dispersion in which at least one polymer and cells are dispersed, selected from the group consisting of collagen, heparin, chitin, chitosan, alginate, agarose, agar, thrombin, vinyl alcohol polymer, gellan gum The bioartificial organ is produced by wrapping in a bag made of a semipermeable membrane and fixing the cells in the gel by gelling the polymer in a bag made of a semipermeable membrane containing a resin. And
When the 1-cm square semipermeable membrane is immersed in a 0.1% albumin solution for 90 minutes, the amount of albumin adsorbed is 10 μg / cm ² or less, and the permeability of albumin in 30 minutes in the semipermeable membrane is 30% or more. A method for producing a bioartificial organ.   The method for producing a bioartificial organ according to claim 13, wherein the cell is an islet.