Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/37—Digestive system
  • A61K35/39—Pancreas; Islets of Langerhans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
  • A61K47/38—Cellulose; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/20—Polysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/52—Hydrogels or hydrocolloids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

• the present invention relates to a biocompatible device for transplanting cells and the like into a living body, a method for producing the same, and a kit for biological transplantation.
• a treatment method in which cells or tissues are transplanted into a living body is known, and devices for use in the treatment have been developed.
• a technology for a bioartificial pancreas also called bioartificial pancreas (BAP), also called bioartificial pancreatic islet
• BAP bioartificial pancreas
• a pancreatic islet is covered (encapsulated) with a polymer gel or semipermeable membrane that can be isolated from the recipient's immune cells and the like but is permeable to nutrients, insulin, and the like, and then transplanted into the body, and various transplantation devices have been reported (Patent Documents 1 to 4).
• Patent Documents 5 to 7 biocompatible implantable devices for use in treating diseases other than type 1 diabetes have also been reported.
• Patent Document 8 an immunoisolation device using a membrane made of polypropylene fibers and an ethylene-vinyl alcohol copolymer has been reported.
• Patent Document 9 and 10 implantable devices using chemically crosslinked alginate have been reported.
• the present invention has the following features (1) to (5).
• (1) It becomes possible to prepare a biocompatible composite membrane including a semipermeable membrane layer containing a cellulose derivative laminated on a nonwoven fabric support, and a nonwoven fabric layer containing a nonwoven fabric having thermoplastic properties.
• (2) By using the composite membrane, it is possible to improve the strength of the membrane when covering a hydrogel that can contain cells, tissues, etc. that secrete biologically active substances.
• the nonwoven fabric surface of the composite membrane can be fused, which allows devices to be easily fabricated by heat sealing.
• (4) When a device made using the composite membrane is transplanted, it is expected that the adhesion rate in the living body will improve and the healing rate of the transplanted subject will improve.
• the hydrogel contained in the internal space of the device and capable of containing cells, tissues, etc. that secrete biologically active substances is fixed to the nonwoven fabric surface of the composite membrane, making it possible to prevent a decrease in the survival rate of the cells, etc.
• the present invention relates to a biocompatible device having an internal space that can contain cells or tissues that secrete a biologically active substance, the internal space being formed by placing a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer on the device, with the semipermeable membrane layer on the outside and the nonwoven fabric layer on the internal space side, the semipermeable membrane layer including a cellulose derivative laminated on the nonwoven fabric using the nonwoven fabric as a support, and the nonwoven fabric layer including a nonwoven fabric having thermoplastic properties, and a method for producing the same.
• Exemplary aspects of the present invention are as follows: [1-1] to [4A-3].
• a biocompatible device having an internal space formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, the semipermeable membrane layer contains a cellulose derivative formed using a nonwoven fabric as a support, the nonwoven fabric layer contains the nonwoven fabric having thermoplastic properties, In the device, the semipermeable membrane layer is disposed on the outer side and the nonwoven fabric layer is disposed on the inner space side, The interior space contains cells or tissues that secrete a biologically active substance; Biocompatible devices.
• [1-2] The device described in [1-1], having immune isolation ability.
• [1-3] The device according to [1-1] or [1-2], wherein the cellulose derivative is cellulose acetate.
• the nonwoven fabric contains one or more resins selected from the group consisting of polyethylene, polypropylene, and polyethylene terephthalate.
• [1-5] The device according to any one of [1-1] to [1-4], wherein the internal space is formed by partial fusion of the nonwoven fabric layer.
• [1-6] The device described in any one of [1-1] to [1-5], wherein the composite membrane has a structure in which the cellulose derivative partially penetrates into a nonwoven fabric.
• [1-7] The device according to any one of [1-1] to [1-6], wherein the cells or tissues are encapsulated in an internal space using a hydrogel as a carrier.
• [1-8] The device according to any one of [1-1] to [1-7], wherein the cells are insulin-secreting cells or pancreatic islets.
• [1-9] The device according to any one of [1-1] to [1-8], wherein the thickness of the composite film is 10 to 1500 ⁇ m.
• [1-10] The device according to any one of [1-1] to [1-9], wherein the strength of the composite film measured by a tensile test in accordance with JIS K 7127 is 1 MPa or more.
• [1-11] The device according to any one of [1-1] to [1-10], which is used for biological transplantation.
• [1-12] A biocompatible composite membrane for producing the device according to any one of [1-1] to [1-11].
• a kit for biological transplantation comprising: a biocompatible device in which an internal space is formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer; and a container containing cells or tissues that secrete a biologically active substance, the kit being used so that the cells or tissues are enclosed in the internal space.
• a method for producing a biocompatible device comprising a step of covering a hydrogel, in which cells or tissues that secrete a biologically active substance are embedded, with a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, in which the semipermeable membrane layer is placed on the outer side and the nonwoven fabric layer is placed on the hydrogel side.
• a method for producing a biocompatible device comprising the steps of injecting a solution containing cells or tissues that secrete a biologically active substance, the cells or tissues being mixed with a hydrogellable solution, into an internal space formed by a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer, and gelling the hydrogellable solution, wherein in the step, the semipermeable membrane layer of the composite membrane is on the outer side, and the nonwoven fabric layer is on the internal space side.
• a biocompatible device having an internal space capable of containing cells or tissues that secrete a biologically active substance,
• the internal space is formed by disposing a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer in the device, with the semipermeable membrane layer on the outer side and the nonwoven fabric layer on the internal space side;
• the semipermeable membrane layer contains a cellulose derivative laminated on a nonwoven fabric using the nonwoven fabric as a support,
• the nonwoven fabric layer includes a nonwoven fabric having thermoplastic properties.
• a biocompatible device having an internal space capable of containing cells or tissues that secrete a biologically active substance,
• the internal space is formed by disposing a biocompatible composite membrane composed of a semipermeable membrane layer and a nonwoven fabric layer in the device, with the semipermeable membrane layer on the outer side and the nonwoven fabric layer on the internal space side,
• the semipermeable membrane layer contains a cellulose derivative laminated on a nonwoven fabric using the nonwoven fabric as a support,
• the nonwoven fabric layer includes a nonwoven fabric having thermoplastic properties.
• a biocompatible device having an internal space capable of containing cells or tissues that secrete a biologically active substance,
• the internal space is formed by disposing a biocompatible composite membrane having a cellulose derivative laminated on one side of a nonwoven fabric, with the surface of the composite membrane on which the cellulose derivative is laminated being on the outer side in the device.
• the biocompatible device is
• [1A-2] The device according to any one of [1A-1-1] to [1A-1-3], which has an immune isolation ability.
• [1A-3] The device according to any one of [1A-1-1] to [1A-2], wherein the cellulose derivative is cellulose acetate.
• [1A-4] The device according to any one of [1A-1-1] to [1A-3], wherein the nonwoven fabric contains one or more resins selected from the group consisting of polyethylene (PE), polypropylene (PP) and polyethylene terephthalate (PET).
• PE polyethylene
• PP polypropylene
• PET polyethylene terephthalate
• [1A-5] The device according to any one of [1A-1] to [1A-4], wherein the internal space is formed by partial fusion of a nonwoven fabric surface in the composite membrane that is not laminated with a cellulose derivative.
• [1A-6] The device described in any one of [1A-1-1] to [1A-5] above, wherein the composite membrane has a structure in which the cellulose derivative partially penetrates into a nonwoven fabric.
• [1A-7] The biocompatible device according to any one of [1A-1] to [1A-6], wherein the cells secreting the biologically active substance are insulin-secreting cells or pancreatic islets.
• [1A-8] The device described in any one of [1A-1-1] to [1A-7] above, in which the cells or tissues secreting the bioactive substance are encapsulated in the internal space using a hydrogel as a carrier.
• the hydrogel is a gel of one or more selected from the group consisting of alginic acid, collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptide, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen and derivatives thereof, fibrin, fibrinogen, thrombin, polyglutamic acid, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, gellan gum, xanthan gum, galactomannan, guar gum, locust bean gum, and tara gum
• the hydrogel is a gel of one or more selected from the group consisting of alginic acid, collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptide, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen, and derivatives thereof.
• hydrogel is a hydrogel obtained by gelling alginic acid or a hydrogel obtained by gelling by forming crosslinks using an alginic acid derivative represented by the following formula (IA) or formula (II-A) (crosslinked alginic acid gel).
• -CH 2 - may be substituted with 1 to 15 groups such as -C( ⁇ O)-, -O-, -NH-, -N(C 1-3 alkyl group)-, -S-, a C 3-8 cycloalkyl ring, a benzene ring, a 5- to 6-membered aromatic heterocycle, or a 5- to 6-membered non-aromatic heterocycle;
• 1 to 10 hydrogen atoms of -CH 2 - may be substituted with groups such as halogen atoms, hydroxyl groups, amino groups, C 1-3 alkyl groups, -O-C 1-3
• -CH 2 - may be substituted with 1 to 15 groups such as -C( ⁇ O)-, -O-, -NH-, -N(C 1-3 alkyl group)-, -S-, a C 3-8 cycloalkyl ring, a benzene ring, a 5- to 6-membered aromatic heterocycle, or a 5- to 6-membered non-aromatic heterocycle;
• the hydrogen atom of -CH 2 - in the formula may be substituted with 1 to 10 groups such as a halogen atom, hydroxyl group, amino group, C 1-3 alkyl group,
• -L 1A - of the chemically modified alginic acid derivative represented by formula (IA) is preferably any of the following groups: A linker selected from the following (wherein the dashed lines at both ends are not included): More preferably, the following table: A linker selected from the following (wherein the dashed lines at both ends are not included): More preferably, the following table: A linker selected from the following (wherein the dashed lines at both ends are not included): Particularly preferably, (In each formula, the areas outside the dashed lines at both ends are not included.)
• -L 2A - of the alginic acid derivative represented by formula (II-A) is preferably any one of the following groups: A linker selected from the following (wherein the dashed lines at both ends are not included): More preferably, the following table: A linker selected from the following (wherein the dashed lines at both ends are not included): More preferably, the following table: A linker selected from the following (wherein the dashed lines at both ends are not included): Particularly preferably, is a linker selected from the following (wherein the formula does not include the areas outside the dashed lines at both ends):
• the alginic acid derivative represented by formula (IA) is preferably represented by the following formulae (A01) to (A15): [In each formula, (ALG) represents alginic acid; and -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid]; More preferably, the compound represented by the following formula (A01) or (A02): In each formula, (ALG) represents alginic acid; -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid.
• the alginic acid derivative represented by formula (II-A) is preferably represented by the following formulas (N01) to (N15): [In each formula, (ALG) represents alginic acid; and -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid]; More preferably, the following formulas (N01) to (N04): In each formula, (ALG) represents alginic acid; -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid.
• [1A-12] The biocompatible device according to any one of [1A-1-1] to [1A-11], wherein the thickness of the composite film is about 10 to about 1500 ⁇ m.
• [1A-13] The biocompatible device according to any one of [1A-1-1] to [1A-12], wherein the strength of the composite film measured by a tensile test in accordance with JIS K 7127 is about 1 MPa or more.
• [1A-14] The biocompatible device according to any one of [1A-1-1] to [1A-13], which is used for biological transplantation.
• [1A-15] The biocompatible device according to any one of [1A-1-1] to [1A-14], wherein the biocompatible composite membrane is formed by laminating a cellulose acetate derivative on one side of a nonwoven fabric by the Robb-Thrilleryan method.
• [1A-16] A biocompatible composite membrane for producing the biocompatible device described in any one of [1A-1-1] to [1A-15] above.
• a method for producing a biocompatible device comprising the step of covering a hydrogel, in which cells or tissues that secrete a biologically active substance are embedded, with a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, in which the semipermeable membrane layer is placed on the outer side and the nonwoven fabric layer is placed on the hydrogel side.
• a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, in which the semipermeable membrane layer is placed on the outer side and the nonwoven fabric layer is placed on the hydrogel side.
• a method for producing a biocompatible device comprising the step of covering a hydrogel, in which cells or tissues that secrete a biologically active substance are embedded, with a biocompatible composite membrane having a cellulose derivative laminated on one side of a nonwoven fabric, in which the surface of the biocompatible composite membrane on which the cellulose derivative is laminated is placed on the outside.
• [1A-18-3] The method for producing a biocompatible device described in [1A-18-1] or [1A-18-2] above, wherein the hydrogel is a hydrogel obtained by gelling one or more of the gellable substances described in any one of [1A-9] to [1A-11] above.
• a method for producing a biocompatible device comprising the steps of injecting a solution containing cells or tissues that secrete a biologically active substance, the cells or tissues being mixed with a hydrogellable solution, into an internal space formed by a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer, and gelling the hydrogellable solution, wherein in the step, the semipermeable membrane layer of the composite membrane is on the outer side, and the nonwoven fabric layer is on the internal space side.
• a method for producing a biocompatible device comprising the steps of injecting a mixed solution containing cells or tissues that secrete a biologically active substance, the cells or tissues being mixed in a hydrogelable solution, into an internal space formed by a biocompatible composite membrane having a cellulose derivative laminated on one side of a nonwoven fabric, and gelling the mixed solution, wherein in the step, the surface of the biocompatible composite membrane on which the cellulose derivative is laminated is positioned on the outside.
• [1A-19-3] The method for producing a biocompatible device described in [1A-19-1] or [1A-19-2] above, wherein the hydrogelable solution is a solution containing one or more of the gellable substances described in any one of [1A-9] to [1A-11] above.
• a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer, wherein the semipermeable membrane layer comprises a cellulose derivative formed using a nonwoven fabric as a support, and the nonwoven fabric layer comprises the nonwoven fabric having thermoplastic properties, the composite membrane being used to encapsulate cells or tissues.
• the composite membrane described in [2-1] which has immune isolation ability.
• the nonwoven fabric contains one or more resins selected from the group consisting of polyethylene, polypropylene, and polyethylene terephthalate.
• [2-5] The composite membrane according to any one of [2-1] to [2-4], wherein an internal space can be formed by partial fusion of the nonwoven fabric layer.
• [2-6] The composite membrane according to any one of [2-1] to [2-5], wherein the composite membrane has a structure in which the cellulose derivative partially penetrates into a nonwoven fabric.
• [2-7] The composite membrane according to any one of [2-1] to [2-6], wherein the cells or tissues are encapsulated in an internal space using a hydrogel as a carrier.
• [2-8] The composite membrane according to any one of [2-1] to [2-7], wherein the cell or tissue is a cell or tissue that secretes a biologically active substance.
• a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer, the semipermeable membrane layer comprising a cellulose derivative laminated on a nonwoven fabric using the nonwoven fabric as a support, and the nonwoven fabric layer comprising a nonwoven fabric having thermoplastic properties, the composite membrane being used for encapsulating cells or tissues.
• a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer, the semipermeable membrane layer comprising a cellulose derivative laminated on the nonwoven fabric using the nonwoven fabric as a support, and the nonwoven fabric layer comprising a nonwoven fabric having thermoplastic properties, the composite membrane being used for encapsulating cells or tissues.
• [2A-2] The composite membrane according to any one of [2A-1-1] to [2A-1-3] above, which has immune isolation ability.
• [2A-3] The composite membrane according to any one of [2A-1] to [2A-2] above, wherein the cellulose derivative is cellulose acetate.
• [2A-4] The composite membrane according to any one of [2A-1] to [2A-3], wherein the nonwoven fabric contains one or more resins selected from the group consisting of polyethylene (PE), polypropylene (PP), and polyethylene terephthalate (PET).
• PE polyethylene
• PP polypropylene
• PET polyethylene terephthalate
• [2A-5] The composite membrane according to any one of [2A-1-1] to [2A-4], wherein an internal space can be formed by partial fusion of the nonwoven fabric surface on which the cellulose derivative of the composite membrane is not laminated.
• [2A-6] The composite membrane according to any one of [2A-1-1] to [2A-5], wherein the composite membrane has a structure in which the cellulose derivative partially penetrates into a nonwoven fabric.
• [2A-7] The composite membrane according to any one of [2A-1-1] to [2A-6], wherein cells or tissues are encapsulated using a hydrogel as a carrier in an internal space formed by partial fusion of the nonwoven fabric surface on which the cellulose derivative of the composite membrane is not laminated.
• [2A-7-1] The composite membrane described in [2A-7] above, wherein the hydrogel is a hydrogel in which one or more of the gellable substances described in any one of [1A-9] to [1A-11] above are gelled.
• [2A-8] The composite membrane according to [2A-7] or [2A-7-1] above, wherein the cell or tissue is a cell or tissue that secretes a biologically active substance.
• [2A-9] The composite membrane according to any one of [2A-1-1] to [2A-8], wherein the cell is an insulin-secreting cell or a pancreatic islet.
• [2A-10] The composite membrane according to any one of [2A-1-1] to [2A-9] above, having a thickness of about 10 to about 1500 ⁇ m.
• [2A-11] The composite membrane according to any one of [2A-1-1] to [2A-10] above, wherein the strength of the membrane measured by a tensile test in accordance with JIS K 7127 is about 1 MPa or more.
• [2A-12] The composite membrane according to any one of [2A-1-1] to [2A-11], which is used for producing a bioimplant device.
• [2A-13] The composite membrane according to any one of [2A-1-1] to [2A-12], wherein the composite membrane is formed by laminating a cellulose acetate derivative on one side of a nonwoven fabric by the Robb-Srirayan method.
• the hydrogel comprises a gel of one or more selected from the group consisting of alginic acid, collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptide, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen and derivatives thereof, fibrin, fibrinogen, thrombin, polyglutamic acid, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, gellan gum, xanthan
• the hydrogel comprises a gel of one or more selected from the group consisting of alginic acid, collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptide, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen, and derivatives thereof.
• alginic acid derivative comprises an alginic acid derivative represented by the following formula (HI) and formula (H-II): [Alginic acid derivative represented by formula (HI)]
• -L 1 - is -(CH 2 ) n1 - (wherein n1 is 1 to 50, -CH 2 - in the group may be replaced by 1 to 10 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, and the hydrogen atom of the -CH 2 - may be substituted by a C 1-3 alkyl group or a C 1-3 alkyl group substituted with a phenyl group),
• Akn is a compound which is an 8-membered cyclic alkyne group (wherein the cyclic alkyne group may be further condensed with 1 to 2 benzene rings, cyclopropane rings or 1,2,3-triazole rings and may be bonded to -L 1 - at the condensed ring, or may be an 8-membered
• -L 1 - is -(CH 2 ) n1 - (wherein n1 is 2 to 15, -CH 2 - in the group may be replaced by 1 to 5 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, and the hydrogen atom of the -CH 2 - may be substituted by a C 1-3 alkyl group or a C 1-3 alkyl group substituted with a phenyl group),
• Akn is a compound that is an 8-membered cyclic alkyne group (wherein the cyclic alkyne group may further be condensed with 1 or 2 benzene rings, or may be an 8-membered cyclic alkyne group in which -CH 2 - is replaced with -NH-),
• [Alginic acid derivative represented by formula (HI)] The following formula (H-I): [In formula (HI), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 1 - represents the following partial structural formula (in each formula, the areas outside the wavy lines at both ends are not included)]: A represents a divalent linker selected from the group consisting of: A represents a divalent linker selected from the group consisting of the following partial structural formulas (in each formula, the right side of the wavy line is not included): wherein the asterisk represents a chiral center; [Alginic acid derivative represented by formula (H-II)] The following formula (H-II): [In formula (H-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 - represents the following partial structural formula [in each
• the alginic acid derivative represented by formula (H-I) is the following formula (A01) or (A02), and the alginic acid derivative represented by formula (H-II) is the following formula (N01), (N02), (N03) or (N04).
• (ALG) represents alginic acid; -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid]
• the chemically crosslinked alginic acid derivative is characterized in that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (H-III-L): [In formula (H-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L 1 - is as defined in any one of [3-5] to [3-8]; -L 2 - is as defined in any one of [3-5] to [3-8]; X is the following partial structural formula: (In each formula, the areas outside the dashed lines at both ends are not included, and an asterisk represents a chiral center.)
• -X A - is preferably a group represented by the following partial structural formula: (In each formula, the areas outside the dashed lines at both ends are not included); More preferably, the compound has the following partial structural formula: (In each formula, the areas outside the dashed lines at both ends are not included); More preferably, the partial structural formula: (In each formula, the areas outside the dashed lines at both ends are not included.)
• the hydrogel comprises an alginic acid derivative or crosslinked alginic acid described in any one of [1A-9] to [1A-11] and [3A] to [3A-5].
• the biological transplant kit described in [1-13] and [1A-17].
• [4A-3] The manufacturing method according to [1-15], [1A-19-1] or [1A-19-2], wherein the solution contains an alginic acid solution containing the alginic acid derivative or crosslinked alginic acid according to any one of [1A-9] to [1A-11] and [3A] to [3A-5].
• One aspect of the present invention provides a novel and practical biocompatible device and a biocompatible composite membrane that can be used as a component of the device. Furthermore, one aspect of the present invention provides a method for producing the device and a kit for transplantation into a living body.
• FIG. 1 is a schematic diagram of a biocompatible device according to one embodiment of the present invention.
• FIG. 2 is a schematic diagram of a cross section of a biocompatible composite membrane according to one embodiment of the present invention.
• FIG. 3 is a schematic diagram of a cross-section of a biocompatible device according to one embodiment of the present invention.
• FIG. 4 is a photograph of a biocompatible device according to one embodiment of the present invention.
• Biocompatible Device in which an internal space is formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, the semipermeable membrane layer includes a cellulose derivative formed using a nonwoven fabric as a support, the nonwoven fabric layer includes the nonwoven fabric having thermoplastic properties, the semipermeable membrane layer is disposed on the outside and the nonwoven fabric layer is disposed on the internal space side, and the internal space includes cells or tissues.
• a biocompatible device having an internal space that can contain cells or tissues that secrete a biologically active substance, the internal space being formed by placing a biocompatible composite membrane including/consisting of a semipermeable membrane layer and a nonwoven fabric layer, with the semipermeable membrane layer on the outside and the nonwoven fabric layer on the internal space side in the device, the semipermeable membrane layer comprising a cellulose derivative laminated on the nonwoven fabric using the nonwoven fabric as a support, and the nonwoven fabric layer comprising a nonwoven fabric having thermoplastic properties.
• a biocompatible device having an internal space capable of containing cells or tissues that secrete a biologically active substance, the internal space being formed by placing a biocompatible composite membrane having a cellulose derivative laminated on one side of a nonwoven fabric, with the surface of the composite membrane laminated with the cellulose derivative on the outside of the device.
• a biocompatible device refers to a device in which an internal space is formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, regardless of whether cells or tissues are enclosed therein.
• a device in which an internal space is formed by a biocompatible composite membrane in which a cellulose derivative is laminated on one side of a nonwoven fabric.
• biocompatible device and “biocompatible composite membrane” may be simply referred to as “device” and “composite membrane”.
• biocompatibility means a property that is considered to pose no problems in terms of biological safety when retained in a living body, and examples of this include the property of not inducing interactions between the various biological materials that make up the biocompatible device and the living body, local reactions in tissues adjacent to the biological materials, or systemic reactions, or of reducing such reactions. Possessing these properties is said to be biocompatible.
• biocompatible device and “biocompatible composite membrane” mean “a device having biocompatibility” and "a composite membrane having biocompatibility", respectively.
• a preferred embodiment of the biocompatible device of the present invention has not only a semipermeable membrane layer but also a nonwoven fabric layer, and therefore has appropriate substance permeability while also having good flexibility and strength, and is a device with physical properties suitable for long-term in vivo use, including transplantation into a living body.
• the internal space of the biocompatible device according to a preferred embodiment of the present invention for example, when cells or tissues are encapsulated in a hydrogel as a carrier, is excellent in maintaining the shape of the hydrogel, and therefore the internal space of the biocompatible device according to a preferred embodiment of the present invention can provide an environment suitable for the survival and functional maintenance of the cells or tissues, and the cells or tissues encapsulated in the internal space can exert their functions (for example, blood glucose lowering effect due to glucose-responsive insulin secretion in the case of using insulin-producing cells) for a long period of time.
• functions for example, blood glucose lowering effect due to glucose-responsive insulin secretion in the case of using insulin-producing cells
• the biocompatible device of the present invention by arranging the semipermeable membrane layer and the nonwoven fabric layer (a biocompatible composite membrane in which a cellulose derivative is laminated on one side of a nonwoven fabric) as described above, a highly safe device with less adhesion, inflammation, etc. can be obtained.
• the device has immunoisolation capabilities.
• immunoisolation ability refers to the property of isolating cells or tissues from the recipient's immune system. By providing this property, it is possible to prevent immune rejection (e.g., those due to cellular immune response and humoral immune response) in the recipient during transplantation.
• the immune isolation ability can be adjusted by selecting the permeability of the semipermeable membrane described below so as to block the entry of antibodies, immune cells, and the like.
• crosslinking means “ionic crosslinking (reaction)” or “chemical crosslinking (reaction)”.
• ionic crosslinking (reaction) means that an ionic crosslink (ionic bond) is formed between an alginic acid or alginic acid derivative of a certain embodiment via a bond with a divalent or higher metal ion.
• chemical crosslinking (reaction) means “crosslinking (reaction) by chemical bond” and means that a chemical crosslink (chemical bond) is formed between the alginic acid derivative of a certain embodiment by performing a chemical reaction (e.g., Huisgen reaction) using the alginic acid derivative of the certain embodiment.
• a chemical reaction e.g., Huisgen reaction
• Biocompatible composite membrane in this specification, includes a semipermeable membrane layer and a nonwoven fabric layer.
• the semipermeable membrane layer includes a cellulose derivative formed using a nonwoven fabric as a support, and the nonwoven fabric layer includes the nonwoven fabric having thermoplastic properties.
• the semipermeable membrane layer includes a cellulose derivative laminated on the nonwoven fabric using the nonwoven fabric as a support, and the nonwoven fabric layer includes a nonwoven fabric having thermoplastic properties.
• the biocompatible composite membrane is a membrane in which a cellulose derivative is laminated on one side of a nonwoven fabric, and the nonwoven fabric is thermoplastic.
• the semipermeable membrane layer includes a cellulose derivative formed using a nonwoven fabric as a support.
• the semipermeable membrane layer includes a cellulose derivative laminated on a nonwoven fabric using a nonwoven fabric as a support.
• the semipermeable membrane layer includes a semipermeable membrane including the cellulose derivative.
• the semipermeable membrane layer is made of a semipermeable membrane including the cellulose derivative.
• the term "semipermeable membrane” refers to a membrane that allows only molecules or ions of a certain size or less to pass through.
• a semipermeable membrane is a system of a solute that does not pass through the semipermeable membrane and a solvent that shows permeability, and when solutions of two concentrations are brought into contact with each other via the semipermeable membrane, osmotic pressure is generated across the membrane, and only the solvent passes through.
• cellulose derivatives is a general term for compounds in which different substituents have been introduced into the hydroxyl groups contained in cellulose molecules, and are classified into cellulose ethers formed by ether bonds (e.g., methyl cellulose, ethyl cellulose, ethyl methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, benzyl cellulose, trityl cellulose, cyanoethyl cellulose, aminoethyl cellulose, etc.) and cellulose esters formed by ester bonds (e.g., cellulose acetate, diacetyl cellulose, triacetyl cellulose, cellulose propionate, cellulose butyrate, etc.).
• ether bonds e.g., methyl cellulose, ethyl cellulose, ethyl methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, hydroxyethyl cellulose,
• the cellulose derivative includes a cellulose ester.
• the cellulose ester includes a cellulose acetate.
• the semipermeable membrane layer may contain a resin other than the cellulose derivative as a component of the semipermeable membrane.
• a resin is not particularly limited, but examples thereof include polysulfone-based polymers, polyacrylonitrile-based polymers, polyamide-based polymers, polycarbonate-based polymers, and the like.
• the content of the cellulose derivative in the components of the semipermeable membrane is about 50 to about 100 mass %, in another embodiment about 60 to about 100 mass %, and in yet another embodiment about 70 to about 100 mass %, based on the total amount of the components of the semipermeable membrane.
• the content of the resin other than the cellulose derivative in the components of the semipermeable membrane is about 1 to about 50 mass %, about 1 to about 40 mass %, and about 1 to about 30 mass %, based on the total amount of the components of the semipermeable membrane.
• the semipermeable membrane is made of a cellulose derivative, i.e., in this embodiment, the semipermeable membrane does not substantially contain any resin other than the cellulose derivative.
• the term "substantially free” does not exclude the case where a certain component is inevitably mixed in during the manufacturing process, but it is preferable that such unavoidable mixed components are kept to a minimum.
• the content of resins other than cellulose derivatives is specifically less than about 0.1 mass%, less than about 0.05 mass% in another embodiment, and less than about 0.01 mass% in still another embodiment, based on the total amount of the constituent components of the semipermeable membrane.
• the semipermeable membrane of one embodiment of the present invention is substantially free of an ethylene-vinyl alcohol-based copolymer.
• the content of the ethylene-vinyl alcohol-based copolymer in the components of the semipermeable membrane is, specifically, less than about 0.1% by mass, less than about 0.05% by mass in another embodiment, and less than about 0.01% by mass in yet another embodiment, based on the total amount of the components of the semipermeable membrane.
• Semipermeable membranes that form the semipermeable membrane layer include, for example, those that have the same characteristics and functions as membranes or tubes used in dialysis, and examples of product names include Cellu-Sep T Tubular Membrane (Membrane Filtration Products), Spectra Biotec Membrane (REPLIGEN), Spectra/Por CE Dialysis Tube (REPLIGEN), and the like.
• the semipermeable membrane can be produced, for example, by dissolving a resin containing a cellulose derivative in a solvent and solidifying the dissolved resin.
• the semipermeable membrane used herein has a "molecular weight cutoff".
• the “molecular weight cutoff” refers to the size of the maximum molecular weight that is not substantially blocked. Molecules having a molecular weight exceeding the molecular weight cutoff are substantially prevented from entering and exiting the semipermeable membrane.
• the "molecular weight cutoff" of the semipermeable membrane is 100 kDa, and in another embodiment, 300 kDa.
• a membrane having a molecular weight cutoff value of the above value it is possible to obtain a semipermeable membrane layer that has a permeation inhibitory function for macromolecules such as antibodies and complements or immune cells, while maintaining a constant rate of permeation of low molecular weight components such as glucose, insulin, cell nutrients, and oxygen.
• the semipermeable membrane is a cellulose ester dialysis membrane, such as Spectra/Por CE dialysis tube (Repligen), which is sold with the cutoff value as "MWCO" in specifications of 100 to 500 Da, 0.5 to 1 kDa, 3.5 to 5 kDa, 8 to 10 kDa, 20 kDa, 50 kDa, 100 kDa, 300 kDa, 1000 kDa, etc.
• the unit Dalton symbol is Da, and 1,000 Da means 1 kDa.
• the thickness of the semipermeable membrane layer (semipermeable membrane) can be calculated, for example, by cutting the composite membrane to an arbitrary size from the biocompatible device, observing the cross section of the composite membrane under a microscope, etc., and subtracting the thickness of the nonwoven fabric from the thickness of the entire composite membrane.
• the thickness (SLT) of the semipermeable membrane layer (semipermeable membrane) is not particularly limited, but examples include 0 ⁇ m or more, about 0.1 ⁇ m or more, about 1.0 ⁇ m or more, about 5.0 ⁇ m or more, about 10.0 ⁇ m or more, etc.
• the upper limit of the thickness of the semipermeable membrane layer (semipermeable membrane) is not particularly limited, but examples include about 500 ⁇ m or less, about 400 ⁇ m or less, about 300 ⁇ m or less, about 200 ⁇ m or less, about 100 ⁇ m, etc. However, SLT does not equal 0 ⁇ m.
• the composite membrane of one embodiment of the present invention has a structure in which the semipermeable membrane component (e.g., cellulose derivative) partially penetrates the nonwoven fabric, but as described below, the thickness of the portion where the semipermeable membrane component partially penetrates the nonwoven fabric (212 in Figure 2) is not included in the thickness of the semipermeable membrane layer (semipermeable membrane).
• the semipermeable membrane component e.g., cellulose derivative
• the semipermeable membrane layer can be formed, for example, by applying a solution containing a cellulose acetate derivative to one side of a nonwoven fabric and laminating it using the Robb-Thrilleryan method, as described in the Examples below.
• penetration and “penetrated” refer to partial penetration of the cellulose derivative (semipermeable membrane) from the surface of the nonwoven fabric into the interior.
• cellulose derivative semipermeable membrane
• the nonwoven fabric forming the nonwoven fabric layer is not particularly limited as long as it can be a support for the semipermeable membrane.
• Conventional semipermeable membranes tend to have difficulty in obtaining sufficient strength against external forces such as tearing.
• the strength of the composite membrane can be improved by forming a composite membrane by laminating the semipermeable membrane on the nonwoven fabric using the nonwoven fabric as a support for the semipermeable membrane.
• the heat fusion property of the composite membrane can be improved, and the leakage of cells or tissues from the device can be suppressed or prevented, and the immune isolation ability of the device can be maintained.
• the unevenness of the nonwoven fabric surface also functions as an anchor for the hydrogel that is enclosed in the internal space of the biocompatible device described below. This, for example, maintains the shape of the hydrogel in the internal space of the biocompatible device, and prevents the gel from becoming biased, folding, or collapsing in the internal space of the device. Without being bound by theory, it is believed that this results in improved utilization of the cells or tissues embedded in the hydrogel, and improved therapeutic effects.
• the nonwoven fabric layer includes a portion where the semipermeable membrane component (e.g., a cellulose derivative) has partially penetrated into the nonwoven fabric. That is, the nonwoven fabric layer of one embodiment of the present invention includes a portion where the semipermeable membrane component has not penetrated into the nonwoven fabric, and a portion where the semipermeable membrane component has partially penetrated into the nonwoven fabric.
• the semipermeable membrane component e.g., a cellulose derivative
• the portion where the semipermeable membrane component has partially penetrated into the nonwoven fabric is a portion formed by immersing the nonwoven fabric in a solution of a cellulose derivative or the like that forms a semipermeable membrane layer, thereby allowing the semipermeable membrane component (e.g., a cellulose derivative) to penetrate into the mesh structure of the nonwoven fabric.
• a semipermeable membrane component e.g., a cellulose derivative
• the composite membrane of one embodiment of the present invention does not have a structure in which a semipermeable membrane and a nonwoven fabric are simply bonded together (for example, a laminate structure), but has a structure in which the components of the semipermeable membrane partially penetrate into the mesh structure of the nonwoven fabric, and the components of the semipermeable membrane solidify, resulting in an integrated structure of the semipermeable membrane and the nonwoven fabric. Therefore, the device of one embodiment of the present invention does not include a laminate membrane formed by lamination processing as the composite membrane. This makes it possible to prevent peeling, loss, and defects of the semipermeable membrane, and to obtain a composite membrane that is stable over time.
• the nonwoven fabric layer may have a semipermeable membrane component (e.g., a cellulose derivative) permeated throughout the entire nonwoven fabric.
• the nonwoven fabric layer does not have any portion (211 in FIG. 2) that is not permeated by the semipermeable membrane component.
• the nonwoven fabric layer includes a portion (211 in FIG. 2) in which the components of the semipermeable membrane have not penetrated and a portion (212 in FIG. 2) in which the components of the semipermeable membrane have partially penetrated into the nonwoven fabric.
• fusion bonded refers to a state in which fibers are melted and bonded to each other.
• sealing refers to, for example, a process in which two biocompatible composite membranes are overlapped with the semipermeable membrane layer disposed on the outer surface, and then a portion several mm to several cm from the edge of the membrane is heated to fuse the thermoplastic nonwoven fabric layer, thereby forming a seal (sealing).
• the nonwoven fabric can be made from a thermoplastic resin.
• resins include polyolefin resins such as polyethylene (PE) and polypropylene (PP), polyester resins such as polyethylene terephthalate (PET), fluorine resins such as polytetrafluoroethylene (PTFE), polyvinyl resins such as polystyrene (PS), and polyamide resins such as nylon.
• PE polyethylene
• PP polypropylene
• PET polyethylene terephthalate
• fluorine resins such as polytetrafluoroethylene
• PS polystyrene
• polyamide resins such as nylon.
• the nonwoven fabric contains a resin having a melting point in the range of about 90 to about 180°C.
• the nonwoven fabric contains at least a polyolefin resin or polyester resin having a melting point in the range of about 90 to about 180°C, and it is preferable that the nonwoven fabric contains at least polyethylene, for example.
• the nonwoven fabric of one embodiment of the present invention may be formed from fibers containing two components with different melting points.
• the nonwoven fabric of one embodiment of the present invention may be formed from, for example, core-sheath type composite fibers.
• the nonwoven fabric may be formed from heat-fusible core-sheath fibers from the viewpoint of enhancing heat fusion properties.
• such nonwoven fabrics include, for example, nonwoven fabrics formed from core-sheath fibers with polypropylene or polyethylene terephthalate as the core material and polyethylene as the sheath material.
• the nonwoven fabric does not use a biodegradable or bioabsorbable resin, such as polylactic acid, polyglycolic acid, poly-p-dioxane, etc.
• the nonwoven fabric of one embodiment of the present invention is substantially free of an ethylene-vinyl alcohol copolymer.
• the content of the ethylene-vinyl alcohol copolymer in the components of the nonwoven fabric is less than about 0.1 mass %, less than about 0.05 mass %, and less than about 0.01 mass %, based on the total amount of the components of the nonwoven fabric.
• the thickness of the nonwoven fabric layer refers to the thickness of the nonwoven fabric itself, regardless of the degree of penetration of the components of the semipermeable membrane.
• the thickness of the nonwoven fabric layer (nonwoven fabric) is specifically about 10 to about 1000 ⁇ m, in another embodiment about 50 to about 750 ⁇ m, and in yet another embodiment about 100 to about 500 ⁇ m.
• the basis weight (grammage) of the nonwoven fabric is about 5 to about 100 g/m 2 , in another embodiment about 10 to about 75 g/m 2 , and in yet another embodiment about 15 to about 50 g/m 2 .
• the thickness of the biocompatible composite membrane can be the sum of the thickness of the nonwoven fabric itself and the thickness of the semipermeable membrane layer, regardless of the degree of penetration of the semipermeable membrane components into the nonwoven fabric.
• the thickness of the biocompatible composite membrane is specifically about 10 to about 1500 ⁇ m in one embodiment, about 25 to about 800 ⁇ m in another embodiment, about 25 to about 400 ⁇ m in yet another embodiment, and about 100 to about 300 ⁇ m in yet another embodiment.
• the biocompatible composite membrane of the present invention is produced, for example, by laminating a cellulose derivative on one side of a nonwoven fabric using the Robb-Srirayan method, as described in the Examples below, and is sometimes called an asymmetric porous membrane.
• the biocompatible device of the present invention has an internal space formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer.
• the semipermeable membrane layer is disposed on the outside and the nonwoven fabric layer is disposed on the internal space side, and the internal space contains cells or tissues that secrete a biologically active substance.
• the biocompatible device of the present invention has an internal space formed by a biocompatible composite membrane having a cellulose derivative laminated on one side of a nonwoven fabric, the surface of the biocompatible composite membrane on which the cellulose derivative is laminated being disposed on the outside, and the internal space contains cells or tissues that secrete a biologically active substance.
• FIG. 1 is a conceptual diagram of a biocompatible device according to one embodiment of the present invention.
• the biocompatible device 1 has an internal space 3 formed by a biocompatible composite membrane 2.
• the internal space 3 is hollow so that cells or tissues 4 can be enclosed therein.
• the shape, volume, etc. of the internal space 3 are not particularly limited, and can be designed appropriately according to the application of the device 1.
• the internal space 3 may be formed by partial fusion of the nonwoven fabric layer (nonwoven fabric) 21.
• the cells or tissues 4 may be enclosed in the internal space 3 using the hydrogel 5 as a carrier.
• the cells or tissues 4 may not use the hydrogel 5 as a carrier. In this case, the cells or tissues 4 can be enclosed in the internal space 3 in a state where they are mixed with a culture medium or the like.
• FIG. 2 is a conceptual diagram showing a cross section of a biocompatible composite membrane 2 according to one embodiment of the present invention.
• the semipermeable membrane layer (semipermeable membrane) 22 constituting the biocompatible composite membrane 2 is disposed on the outside of the biocompatible device 1
• the nonwoven fabric layer (nonwoven fabric) 21 is disposed on the side of the internal space 3 of the biocompatible device 1.
• the nonwoven fabric layer (nonwoven fabric) 21 may have a portion 211 where the semipermeable membrane components have not penetrated, and a portion 212 where the semipermeable membrane components have partially penetrated the nonwoven fabric.
• the shape of the biocompatible device is not limited to the shape shown in FIG. 1, as long as an internal space is formed by the biocompatible composite membrane.
• the method of forming an internal space by a biocompatible composite membrane is not particularly limited, but for example, two biocompatible composite membranes (e.g., square, rectangular, etc.) are prepared, overlapped so that the semipermeable membrane layer is on the outside and the nonwoven fabric layer is on the internal space side, and the ends are fused and sealed (6 in Fig. 3 is the fusion site).
• one biocompatible composite membrane e.g., square, rectangular, etc.
• the device of one embodiment may have one internal space or may have multiple internal spaces. Examples of a method for forming multiple internal spaces include separating one internal space into multiple spaces, or connecting multiple devices each having one internal space to form a single device having multiple internal spaces.
• the shape of the device can be designed to a shape and size according to the site to be implanted.
• Specific examples of the device shape include a flat plate.
• a flat plate means a flat plate having a nearly constant thickness and a wide area.
• Examples of the plate shape include flat plate shapes such as polygonal shapes such as triangles, squares, and pentagons, and circles.
• the thickness of the flat device is about 0.1 to about 100 mm, in another embodiment, about 0.2 to about 50 mm, in yet another embodiment, about 0.5 to about 20 mm, and in yet another embodiment, about 1 to about 20 mm.
• the flat device has the above thickness and is a nearly constant thickness throughout the entire plate. In a plate-shaped device, the thickness variation is preferably within ⁇ 10%, more preferably within ⁇ 5%.
• the thickness of a flat device is the thickness of the thickest part of the device.
• the device may have a shape that at first glance looks like a rugby ball, with both ends being slightly thinner and the center being thicker than the ends.
• the thickness of the device means the thickness near the center, which is the thickest part of the device.
• the flat device has a length of about 1 to about 200 mm, a width of about 1 to about 200 mm, and a thickness of about 0.2 to about 50 mm, for example, when expressed in terms of length x width x thickness.
• the flat device has an area, for example, in terms of length x width, of about 1 to about 40,000 mm 2 and a thickness of about 0.2 to about 50 mm, about 0.5 to about 50 mm, or about 1 to about 50 mm.
• the device can have a shape such as a circle, a square, a hexagon, or an octagon.
• the shape of the device is not particularly limited as long as it is tubular or fibrous and elongated.
• a tubular shape is not limited to a circular cross-sectional shape perpendicular to the central axis, but may be an asymmetrical structure or a deformed shape, for example, a polygonal cross-section such as a triangle, square, or pentagon, or an elliptical cross-section.
• the cross-sectional shape is circular.
• the cross-sectional diameter (hereinafter, in the case of a non-circular shape, the long diameter or maximum diameter) is, for example, about 0.1 to about 100 mm in one embodiment, about 0.2 to about 50 mm in another embodiment, about 0.5 to about 20 mm in yet another embodiment, and about 1 to about 10 mm in yet another embodiment.
• the length is not limited, but for example, in one embodiment, it is from about 10 mm to about 5 m, in another embodiment, it is from about 10 mm to less than about 30 cm, and in yet another embodiment, it is from about 30 cm to about 5 m.
• Cells or tissues to be encapsulated in a biocompatible device are not particularly limited, and examples thereof include cells or tissues that secrete a biologically active substance (a substance having pharmacological activity or biological activity).
• the bioactive substance is not particularly limited, but is preferably one used as a pharmaceutical, and examples thereof include low molecular weight pharmaceuticals, medium molecular weight pharmaceuticals such as peptides and oligonucleic acids, biopharmaceuticals such as proteins, and physiologically active substances.
• the bioactive substance includes not only physiologically active substances inherent in living organisms (biologically active natural products), but also substances obtained by modifying or altering physiologically active substances, and substances that activate or inhibit physiological activity, and includes naturally occurring substances as well as substances produced by genetic engineering or chemically synthesized, and also includes prodrugs thereof.
• Specific examples of the bioactive substance include vitamins, coenzymes, hormones, antibiotics, neurotransmitters, cytokines, enzymes, growth factors, antibodies, and other biological factors, and more specific examples of biological factors include insulin, dopamine, factor VIII, factor IX, and the like.
• the cells secreting the bioactive substance include natural cells, artificially modified cells, and cell masses consisting of multiple cells.
• the cells secreting the bioactive substance may be cells for transplantation.
• Examples of the cells for transplantation include insulin-secreting cells, pancreatic islets, and pancreatic islet cells.
• examples of cells capable of secreting other biologically active substances include dopamine-secreting cells, pituitary cells, growth hormone-secreting cells, parathyroid cells, nerve growth factor-secreting cells, blood coagulation factor-secreting cells, hepatic cells, parathyroid cells, erythropoietin-secreting cells, and norepinephrine-secreting cells.
• Insulin-secreting cells refers to cells that have the function of secreting insulin, and for example, in the case of cells that constitute pancreatic islets, it refers to ⁇ cells that secrete insulin.
• insulin-secreting cells may be cells that have acquired insulin secretion function through differentiation, maturation, modification, etc., and may include, for example, cells with insulin secretion function obtained by differentiating stem cells such as iPS cells, ES cells, or somatic stem cells (e.g., mesenchymal stem cells), cells with insulin secretion function obtained by maturing immature cells or precursor cells, and cells that have been given insulin secretion function through genetic recombination.
• stem cells such as iPS cells, ES cells, or somatic stem cells (e.g., mesenchymal stem cells)
• differentiating or maturing the cells includes culturing the cells, and in other words, cells obtained by differentiating or maturing may include cells obtained by culture.
• a pancreatic islet also known as an islet of Langerhans, is a cell mass made up of an average of about 2,000 islet cells.
• a pancreatic islet is made up of five types of cells: alpha cells that secrete glucagon, beta cells that secrete insulin, delta cells that secrete somatostatin, epsilon cells that secrete ghrelin, and PP (pancreatic polypeptide) cells that secrete pancreatic polypeptide.
• islet cells may refer to cells that contain at least one of the five types of cells that make up the above-mentioned pancreatic islets, but preferably contain at least ⁇ cells.
• the islet cells may be a mixture that contains all of ⁇ cells, ⁇ cells, ⁇ cells, ⁇ cells, and PP cells, or may be cells that are contained in a pancreatic islet.
• pancreatic islet cells may be cells that have become pancreatic islet cells through differentiation, maturation, modification, etc.
• pancreatic islet cells may also include, for example, pancreatic islet cells obtained by differentiating stem cells such as iPS cells, ES cells, and somatic stem cells (e.g., mesenchymal stem cells), as well as pancreatic islet cells obtained by maturing immature cells or precursor cells.
• insulin-secreting cells or “pancreatic islets (including pancreatic islet cells)" preferably have a degree of viability and function that allows them to recover from a pathological condition when transplanted into a patient.
• Functions of insulin-secreting cells, pancreatic islets, or pancreatic islet cells include, for example, the secretion of insulin, and it is preferable that glucose responsiveness is maintained even after transplantation.
• the donor of the "insulin-secreting cells", “pancreatic islets” or “pancreatic islet cells” is an animal in one embodiment, a vertebrate in another embodiment, or a mammal in yet another embodiment, specifically a human, pig, monkey, rat, or mouse, and particularly a human or pig.
• the donor of the "insulin-secreting cells", “pancreatic islets” or “pancreatic islet cells” is a pig in order to alleviate the donor shortage.
• the "insulin-secreting cells”, “pancreatic islets” or “pancreatic islet cells” may be either a pancreatic islet or islet cell obtained from a donor animal, or an insulin-secreting cell or islet cell obtained from a donor-derived cell, and may be, for example, an insulin-secreting cell or islet cell differentiated from a human-derived ES cell or iPS cell.
• the “insulin-secreting cells,” “pancreatic islets,” or “islet cells” are derived from pigs
• examples of such cells include adult porcine islets, or fetal, neonatal, or perinatal porcine islets, or insulin-secreting cells or islet cells obtained from such islets.
• the islets may be appropriately cultured before use, or islets obtained by maturing fetal, neonatal, or perinatal porcine islets may be used.
• blood coagulation factor-secreting cells examples include factor VIII-secreting cells and factor IX-secreting cells.
• Other cells for encapsulation include animal cells in one embodiment, vertebrate-derived cells in another embodiment, and mammalian-derived cells in yet another embodiment, particularly human-derived cells or porcine-derived cells.
• the type of vertebrate-derived cells may be any of stem cells (e.g., pluripotent stem cells (multipotent cells) or somatic stem cells), progenitor cells, or mature cells.
• pluripotent stem cells for example, embryonic stem (ES) cells, germline stem (GS) cells, or induced pluripotent stem (iPS) cells can be used.
• somatic stem cells for example, mesenchymal stem cells (MSCs), hematopoietic stem cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, cardiac muscle stem cells, adipose-derived stem cells, or neural stem cells can be used. These stem cells can also be induced to differentiate and used.
• the precursor cells and mature cells may be derived from, for example, skin, dermis, epidermis, muscle, cardiac muscle, nerve, bone, cartilage, endothelium, brain, epithelium, heart, kidney, liver, spleen, oral cavity, cornea, bone marrow, umbilical cord blood, amniotic membrane, or hair.
• the human-derived cells may be derived from, for example, ES cells, iPS cells, MSCs, chondrocytes, osteoblasts, osteoblast precursor cells, mesenchymal cells, myoblasts, cardiac muscle cells, cardiac muscle blasts, nerve cells, hepatocytes, fibroblasts, corneal endothelial cells, vascular endothelial cells, corneal epithelial cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, or hematopoietic stem cells.
• the origin of the cells may be either autologous or allogeneic.
• the biocompatible device may contain substances for maintaining the viability and function of cells or tissues, or substances (such as hydrogels) that uniformly disperse cells or tissues that secrete biologically active substances.
• the device of the present invention includes both embodiments that contain these and embodiments that do not.
• the cells or tissues are encapsulated in the internal space using a hydrogel as a carrier.
• hydrogel refers to a water-insoluble polymer with a three-dimensional mesh structure and its water-swollen form. In this specification, hydrogel may be simply referred to as "gel.”
• materials used to form hydrogels include alginic acid (including alginic acid esters and alginates, as described below), collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptides, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen and derivatives thereof, as well as fibrin, fibrinogen, thrombin, and polyglutamic acid, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymers, gellan gum, xanthan gum, galactomannan, guar gum, locust bean gum, and
• the material used to form the hydrogel may be alginic acid or a derivative thereof.
• alginic acid or a derivative thereof it is preferable to use alginic acid or a derivative thereof that has been modified so that it can be gelled by chemical crosslinking.
• chemically modified alginic acid derivatives include those described in the above aspects [3-5] to [3-9] and [1A-9-2] to [1A-11].
• alginic acid with chemical crosslinking examples include those described in the above aspects [3-10] to [3-13] and [3A] to [3A-5].
• alginic acid as the hydrogel, or in some aspects, chemically crosslinked alginic acid, the network structure of alginic acid or chemically crosslinked alginic acid is more firmly bound to the unevenness of the nonwoven fabric surface, and the anchoring effect of the nonwoven fabric can be further improved.
• the hydrogel of one embodiment of the present invention is substantially free of vinyl alcohol-based polymers.
• the content of vinyl alcohol-based polymers in the hydrogel is less than about 0.1% by mass, less than about 0.05% by mass in another embodiment, and less than about 0.01% by mass in yet another embodiment, based on the total amount of the components of the hydrogel.
• alginic acid and alginic acid derivatives which are the raw materials for the chemically crosslinked alginic acid that constitutes the hydrogel in one embodiment of the device.
• alginic acid refers to at least one type of alginic acid (sometimes referred to as “alginic acid compounds”) selected from the group consisting of alginic acid, alginic acid esters, and salts thereof (e.g., sodium alginate).
• alginic acid used may be of natural origin or synthetic origin, but is preferably of natural origin.
• alginic acid may be expressed as (ALG)-COOH, with alginic acid being (ALG) and one of the carboxyl groups of alginic acid being -COOH.
• the alginic acid is sodium alginate.
• Commercially available sodium alginate can be used as the sodium alginate.
• sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 (manufactured by Mochida Pharmaceutical Co., Ltd.) shown in the table below is used as the sodium alginate.
• the viscosity, weight average molecular weight, and M/G ratio of a 1 w/w% aqueous solution of each sodium alginate are shown in the table below.
• alginic acid ester and "alginate salt” are not particularly limited, but must not have a functional group that inhibits the crosslinking reaction.
• Preferred examples of alginic acid esters include propylene glycol alginate.
• examples of alginates include monovalent salts of alginic acid and divalent salts of alginic acid.
• Preferred examples of monovalent salts of alginic acid include sodium alginate, potassium alginate, ammonium alginate, etc., more preferably sodium alginate or potassium alginate, and particularly preferably sodium alginate.
• Preferred examples of divalent salts of alginic acid include calcium alginate, magnesium alginate, barium alginate, strontium alginate, etc.
• alginic acid please refer to the descriptions in PCT/JP2019/023478 (filed June 13, 2019), PCT/JP2020/047100 (filed December 17, 2020), and PCT/JP2019/007655 (filed February 27, 2019).
• the alginic acid derivative in the present specification is an alginic acid derivative in which a reactive group in the Huisgen reaction or a complementary reactive group to the reactive group is introduced to any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker.
• the divalent linker (-L 1 - or -L 2 -) may be any straight-chain group as long as it does not inhibit the reaction between a reactive group and a reactive group complementary to the reactive group.
• the hydrogen atom of the imino group (-NH-) can be replaced with a methyl group to form an -N(Me)-CO- group.
• the bonding mode between the linker (-L 1 -, -L 2 -) and alginic acid in the chemically modified alginic acid derivative represented by formula (HI) or (H-II), or the bonding mode between the linker (-L 1A -, -L 2A -) and alginic acid in the chemically modified alginic acid derivative represented by formula (IA) or (II-A), is an -NH-CO- bond or an -N(Me)-CO- bond; preferably, an -NH-CO- bond.
• the -CO- in the -NH-CO- bond or the -N(Me)-CO- bond is derived from a carboxyl group of alginic acid.
• cyclic alkyne group means a "5- to 9-membered cycloalkyne group” and also includes a cycloalkyne group in which 1 to 4 -CH 2 - of the "5- to 9-membered cycloalkyne group” are substituted with 1 to 4 groups selected from the group consisting of -NH-, -S-, -O-, or ⁇ C( ⁇ O).
• a "5- to 9-membered cycloalkyne group” means a group in which -CH 2 -CH 2 - of a monocyclic saturated cycloalkyl group having 5 to 9 carbon atoms is replaced with -C ⁇ C-, and examples include a cyclopentyne group, a cyclohexyne group, a cycloheptyne group, a cyclooctyne group, a cyclononyne group, etc.
• -L 1A - or -L 1 - is -(CH 2 ) n1 - (wherein n1 is 1 to 50, -CH 2 - in the group may be replaced by 1 to 10 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, and the hydrogen atom of the -CH 2 - may be substituted by a C 1-3 alkyl group or a C 1-3 alkyl group substituted with a phenyl group),
• Aky or Akn is an 8-membered cyclic alkyne group (wherein the cyclic alkyne group may be further condensed with 1 to 2 benzene rings, cyclopropane rings or 1,2,3-triazole rings, and -L 1 -,
• n1 is 1 to 20, and a more preferred embodiment is 1 to 15 (for example, 2 to 15).
• a preferred embodiment of -L 1A - or -L 1 - is a group in which -CH 2 - in -(CH 2 ) n1 - may be replaced by 1 to 5, more preferably 1 to 3, groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-.
• a preferred embodiment of Aky or Akn is an 8-membered cyclic alkyne group in which 1 to 2 benzene rings may be condensed and in which -CH 2 - in the 8-membered cyclic alkyne group may be replaced by -NH-.
• a preferred embodiment of n2 is 1 to 20, and a more preferred embodiment is 1 to 15 (for example, 2 to 15).
• a preferred embodiment of -L 2A - or -L 2 - is a group in which -(CH 2 ) n2 - may be replaced by 1 to 5, more preferably 1 to 3, groups selected from -CONH-, -NHCO-, -O- and -NH-.
• halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
• examples of the " C1-3 alkyl group” include methyl, ethyl, propyl and isopropyl groups.
• C2-4 alkanoyl group means a " C1-3 alkylcarbonyl group” in which a carbonyl group is bonded to the above-mentioned " C1-3 alkyl group", and examples thereof include acetyl, propionyl, butyryl and the like.
• C 3-8 cycloalkyl ring includes a monocyclic or polycyclic saturated or unsaturated cycloalkyl ring having 3 to 8 carbon atoms, such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, or cyclooctane ring.
• C 5-9 cycloalkene ring includes a monocyclic cycloalkene ring having 5 to 9 carbon atoms, such as cyclopentene, cyclohexene, cycloheptene, cyclooctene, and cyclononene rings.
• 5- to 6-membered aromatic heterocycle means a 5- to 6-membered unsaturated ring containing 1 to 4 heteroatoms selected from the group consisting of nitrogen atoms, sulfur atoms, and oxygen atoms.
• examples of the "5- to 6-membered aromatic heterocycle” include pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, triazole, oxadiazole, furazan, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, thiadiazine, and other groups.
• the term "5- to 6-membered non-aromatic heterocycle” means a 5- to 6-membered saturated heterocycle containing 1 to 4 heteroatoms selected from an oxygen atom, a sulfur atom, and a nitrogen atom.
• examples of the "5- to 6-membered non-aromatic heterocycle” include rings such as pyrrolidine, tetrahydrofuran, thiolane, piperidine, dihydropyran, tetrahydropyran, tetrahydrothiopyran, piperazine, dioxane, morpholine, thiomorpholine, and quinuclidine.
• alginic acid derivatives represented by the formula (I-A), formula (II-A), formula (H-I) or formula (H-II) can be produced, for example, by the synthesis method of alginic acid derivatives described below.
• alginic acid derivative is a compound in which the definitions of -L 1 - and Akn in the above formula (HI) are the same as those in the above embodiment [3-8]. Moreover, one embodiment of the alginic acid derivative is a compound in which the definition of -L 2 - in the above formula (H-II) is the same as that in the above embodiment [3-8].
• An embodiment of the alginic acid derivative is a compound in which the definitions of -L 1A - and Aky in the formula (IA) are the same as those in the above embodiment [1A-9-2]. Also, an embodiment of the alginic acid derivative is a compound in which the definition of -L 2A - in the formula (II-A) is the same as those in the above embodiment [1A-9-2].
• alginic acid derivatives represented by the above formula (IA) or formula (HI) include those represented by the following formula:
• alginic acid derivatives include, but are not limited to, those represented by the following formula:
• alginic acid derivatives represented by the formula (II-A) or formula (H-II) include, for example, the following formula:
• alginic acid derivatives include, but are not limited to, those represented by the following formula:
• variable substituent when a variable substituent is substituted on a cyclic group, it means that the variable substituent is not bonded to a specific carbon atom of the cyclic group.
• the variable substituent Rs in the following formula A means that it can be substituted on any of the carbon atoms i, ii, iii, iv, or v in the formula A.
• the weight average molecular weight of the alginic acid derivative represented by formula (I-A) or formula (II-A) and the alginic acid derivative represented by formula (H-I) or formula (H-II) in this specification is about 100,000 Da to about 3,000,000 Da, preferably about 300,000 Da to about 2,500,000 Da, and more preferably about 500,000 Da to about 2,000,000 Da.
• the molecular weight of the alginic acid derivative can be determined by the methods described in PCT/JP2019/023478 (filed June 13, 2019) and PCT/JP2020/047100 (filed December 17, 2020).
• the Aky-L 1A -NH- group or the Akn-L 1 -NH- group of formula (IA) or formula (HI) does not need to be bonded to all of the carboxyl groups of the alginic acid constitutional unit
• the N 3 -L 2A -NH- group or the N 3 -L 2 -NH- group of formula (II-A) or formula (H-II) does not need to be bonded to all of the carboxyl groups of the alginic acid constitutional unit.
• the Aky-L 1A -NH- group or the Akn-L 1 -NH- group of formula (IA) or formula (HI) is referred to as a reactive group
• the N 3 -L 2A -NH- group or the N 3 -L 2 -NH- group of formula (II-A) or formula (H-II) is the complementary reactive group.
• the N 3 -L 2A -NH- group or the N 3 -L 2 -NH- group of formula ( II -A) or formula (H-II) is referred to as a reactive group
• the Aky- L 1A -NH- group or the Akn-L 1 -NH- group of formula (IA) or formula (HI) is the complementary reactive group.
• the introduction rate of the reactive group or the complementary reactive group is about 0.1% to about 30% or about 1% to about 30%, preferably about 2% to about 20%, and more preferably about 3% to about 10%.
• the introduction rate of the reactive group or complementary reactive group is the number of uronic acid monosaccharide units into which each reactive group has been introduced, expressed as a percentage, among the uronic acid monosaccharide units, which are the repeating units of alginic acids.
• the percentage used for the introduction rate of the reactive group or complementary reactive group in the alginic acid derivative means mol%.
• the introduction rate of each reactive group or complementary reactive group can be determined by the methods described in PCT/JP2019/023478 (filed June 13, 2019) and PCT/JP2020/047100 (filed December 17, 2020).
• the cyclic alkyne group (Akn) in formula (H-I) and the azide group in formula (H-II) form a triazole ring by a Huisgen reaction, thereby forming a crosslink.
• the cyclic alkyne group (Aky) in formula (I-A) and the azide group in formula (II-A) form a triazole ring by a Huisgen reaction, thereby forming a crosslink.
• Huisgen reaction please refer to the descriptions in PCT/JP2019/023478 (filed June 13, 2019) and PCT/JP2020/047100 (filed December 17, 2020).
• alginic acid derivatives represented by formula (IA), formula (II-A), formula (HI), and formula (H-II) can be produced, for example, by the production method shown below (see above for the definitions of Aky, -L 1A -, -L 2A -, (ALG), etc. in each formula; it is also possible to replace Aky with Akn, -L 1A - with -L 1 -, and -L 2A - with -L 2 -).
• Crosslinked alginic acid can be (i) via a divalent metal ion bond, (ii) via a chemical bond, or (iii) via both a divalent metal ion bond and a chemical bond. All crosslinked alginic acids have the property of forming a gel to a semi-solid, or in some cases a sponge-like form.
• the divalent metal ion is not particularly limited, but examples thereof include calcium ion, magnesium ion, barium ion, strontium ion, zinc ion, etc., and in one embodiment, it is calcium ion or barium ion.
• crosslinked alginic acid via divalent metal ion bonds the reaction proceeds at an ultrahigh speed and is reversible, whereas in crosslinked alginic acid via chemical bonds, the reaction proceeds slowly under relatively mild conditions and is irreversible.
• the physical properties of crosslinked alginic acid can be adjusted, for example, by changing the concentration of the aqueous solution containing the divalent metal ion (e.g., calcium chloride aqueous solution, barium chloride aqueous solution, etc.) used, or by changing the introduction rate of the reactive group introduced into alginic acid.
• the divalent metal ion concentration (e.g., calcium ion or barium ion concentration) of an aqueous solution containing a divalent metal ion is not particularly limited, and is, for example, in the range of about 1 mmol/L to about 1 mol/L in one embodiment, in the range of about 2 mmol/L to about 500 mmol/L in another embodiment, and in yet another embodiment, about 5 mmol/L to about 100 mmol/L.
• an aqueous solution containing a divalent metal ion may be simply referred to as a divalent metal ion solution, and unless otherwise specified, the divalent metal ion solution refers to an aqueous solution.
• crosslinking reaction By utilizing the crosslinking reaction, it is possible to prepare various alginate structures (e.g., hydrogels). For example, a specific structure can be instantly created from an alginate solution by an ionic crosslinking reaction, and a crosslinking reaction by chemical bonds can be used to strengthen the structure (e.g., to obtain long-term stability, etc.). Also, for example, in a crosslinked alginate structure via both divalent metal ion bonds and chemical bonds, it is possible to create a structure in which the divalent metal ions incorporated by ionic crosslinking are reversibly released and only crosslinks by chemical bonds remain.
• a preferred embodiment of crosslinked alginate includes chemically crosslinked alginate obtained by utilizing a crosslinking reaction by chemical bonds.
• crosslinked alginate includes the chemically crosslinked alginate and ionic crosslinked alginate by divalent metal ion bonds.
• Yet another preferred embodiment of crosslinked alginate includes the chemically crosslinked alginate and, optionally, the ionic crosslinked alginate.
• the cross-linked alginic acid can be obtained by mixing the alginic acid derivatives of the formula (H-I) and the formula (H-II) and carrying out the Huisgen reaction.
• the cross-linked alginic acid can be obtained by mixing the alginic acid derivatives of the formula (I-A) and the formula (II-A) and carrying out the Huisgen reaction.
• the cross-linked alginic acid forms a three-dimensional mesh structure through chemical cross-linking (cross-linking by triazole rings formed from alkyne groups and azide groups).
• the crosslinked alginic acid has a bond between any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid represented by the following formula (H-III-L): [In formula (H-III-L), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; -L 1 -, -L 2 -, and X are as defined in any one of the above embodiments [3-10] to [3-13]].
• the mixing ratio of the alginic acid derivative of formula (H-I) and the alginic acid derivative of formula (H-II) when preparing the crosslinked alginic acid is, for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1, in terms of the weight ratio of the derivative of formula (H-I) to the derivative of formula (H-II).
• the mixing ratio of the alginic acid derivative of formula (H-II) and the alginic acid derivative of formula (H-I) when preparing the crosslinked alginic acid is, for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1, in terms of the weight ratio of the derivative of formula (H-II) to the derivative of formula (H-I).
• the mixing ratio of the alginic acid derivative of formula (H-I) to the alginic acid derivative of formula (H-II) when preparing the crosslinked alginic acid is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of formula (H-I) to the alginic acid derivative of formula (H-II) is, for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.
• the mixing ratio of the alginic acid derivative of formula (H-II) to the alginic acid derivative of formula (H-I) when preparing the crosslinked alginic acid is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of formula (H-II) to the alginic acid derivative of formula (H-I), for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.
• the alginic acid derivative of formula (H-I) can be replaced with the alginic acid derivative of formula (H-II), and the alginic acid derivative of formula (H-II) can be replaced with the alginic acid derivative of formula (H-I).
• crosslinked alginic acid it is not necessary that all carboxyl groups in the constituent units of alginic acid have crosslinks of the above formula (H-III-L).
• the introduction rate of crosslinks represented by the above formula (H-III-L) in crosslinked alginic acid is, for example, in the range of about 0.1 to about 80%, about 0.3 to about 60%, about 0.5 to about 30%, or about 1.0 to about 10%.
• the concentration of the alginic acid derivative of formula (H-I) or formula (H-II) in the Huisgen reaction to obtain crosslinked alginic acid is usually in the range of about 1 to about 500 mg/mL, preferably about 5 to about 100 mg/mL.
• the crosslinked alginic acid has a bond between any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid represented by the following formula (III-Lx):
• formula (III-Lx) [In formula (III-Lx), -NHCO- on the right end represents an amide bond derived from an alginic acid derivative of formula (IA), and -CONH- on the left end represents an amide bond derived from an alginic acid derivative of formula (II-A);
• -L 1A - and -L 2A - are as defined above in [1A-9-2];
• -X A - is a cross-linked alginic acid bonded via an amide bond, which is the same as defined in the above [3A].
• the mixing ratio of the alginic acid derivative of formula (I-A) and the alginic acid derivative of formula (II-A) when preparing the crosslinked alginic acid is, for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1, in terms of the weight ratio of the derivative of formula (I-A) to the derivative of formula (II-A).
• the mixing ratio of the alginic acid derivative of formula (II-A) and the alginic acid derivative of formula (I-A) when preparing the crosslinked alginic acid is, for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1, in terms of the weight ratio of the derivative of formula (II-A) to the derivative of formula (I-A).
• the mixing ratio of the alginic acid derivative of formula (I-A) to the alginic acid derivative of formula (II-A) when preparing the crosslinked alginic acid is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of formula (I-A) to the alginic acid derivative of formula (II-A), for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.
• the mixing ratio of the alginic acid derivative of formula (II-A) to the alginic acid derivative of formula (I-A) when preparing the crosslinked alginic acid is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of formula (II-A) to the alginic acid derivative of formula (I-A), for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.
• the alginic acid derivative of formula (I-A) can be replaced with the alginic acid derivative of formula (II-A), and the alginic acid derivative of formula (II-A) can be replaced with the alginic acid derivative of formula (I-A).
• crosslinked alginic acid it is not necessary that all carboxyl groups in the constituent units of alginic acid have crosslinks of the above formula (III-Lx).
• the introduction rate of crosslinks represented by the above formula (III-Lx) in crosslinked alginic acid is, for example, in the range of about 0.1 to about 80%, about 0.3 to about 60%, about 0.5 to about 30%, or about 1.0 to about 10%.
• the concentration of the alginic acid derivative of formula (I-A) or formula (II-A) in the Huisgen reaction to obtain crosslinked alginic acid is usually in the range of about 1 to about 500 mg/mL, preferably about 5 to about 100 mg/mL.
• the reaction temperature for the Huisgen reaction is usually an external temperature of about 4 to about 60°C, preferably about 15 to about 40°C.
• the stirring time for forming cross-linked alginic acid is, for example, from a few seconds to about 24 hours, from a few seconds to about 12 hours, from a few seconds to about 30 minutes, or from a few seconds to about 10 minutes.
• the reaction solvent or reaction solution used in the Huisgen reaction is not particularly limited, but examples include tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), and saline, with ultrapure water being preferred.
• tap water pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), and saline, with ultrapure water being preferred.
• pure water e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.
• ultrapure water cell culture medium
• PBS phosphate buffered saline
• saline phosphate buffered saline
• cross-linked alginate are cross-linked alginates that include chemical cross-links formed by triazole rings via the Huisgen reaction, and ionic cross-links formed in part by calcium ions or barium ions.
• the method for producing a biocompatible device according to one embodiment of the present invention may be, for example, the method described in "6-1" or "6-2" below.
• Step (d) A step of covering the cell- or tissue-embedded hydrogel obtained in step (c) with the biocompatible composite membrane obtained in step (a), comprising a semipermeable membrane layer and a nonwoven fabric layer.
• the semipermeable membrane layer is placed on the outside, and the nonwoven fabric layer is placed on the hydrogel side.
• step (a) can be performed before step (b), between step (b) and step (c), or after step (c).
• multiple steps may be performed simultaneously as appropriate.
• step (a) can be replaced with the following step (A1) or step (A2).
• Step (A1) A step of forming a biocompatible composite membrane including/consisting of a semipermeable membrane layer formed by laminating a cellulose derivative on a nonwoven fabric using the nonwoven fabric as a support, and a nonwoven fabric layer having a structure in which the cellulose derivative partially penetrates the nonwoven fabric.
• Step (A2) A step of forming a biocompatible composite membrane by laminating a cellulose derivative on one side of a nonwoven fabric using a nonwoven fabric as a support, and the cellulose derivative partially penetrates the nonwoven fabric.
• a semipermeable membrane layer is formed from a cellulose derivative using a nonwoven fabric as a support, and a biocompatible composite membrane having a structure in which the cellulose derivative partially penetrates the nonwoven fabric is formed.
• A1) is a biocompatible material that includes/consists of a semipermeable membrane layer formed by laminating a cellulose derivative on a nonwoven fabric as a support, and a nonwoven fabric layer having a structure in which the cellulose derivative partially penetrates the nonwoven fabric.
• step (A2) a cellulose derivative is laminated on one side of a nonwoven fabric used as a support, and a biocompatible composite membrane is formed in which the cellulose derivative partially penetrates the nonwoven fabric.
• step (a) a cellulose derivative (e.g., cellulose acetate) is first mixed with a solvent to prepare a solution containing the cellulose derivative (also referred to as a cellulose derivative-containing solution).
• the solvent to be mixed with the cellulose derivative is not particularly limited, but examples thereof include dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, dioxane, acetone, etc.
• the content of the cellulose derivative in the cellulose derivative-containing solution is about 1 to about 30 mass %, about 5 to about 20 mass %, and about 7 to about 15 mass %, based on the total amount of the cellulose derivative-containing solution, in another embodiment.
• the cellulose derivative-containing solution is brought into contact with the nonwoven fabric, and the cellulose derivative-containing solution is permeated into the nonwoven fabric. Examples of methods for permeating the nonwoven fabric with the cellulose derivative-containing solution include pouring or applying the cellulose derivative-containing solution onto the surface of the nonwoven fabric, spraying the cellulose derivative-containing solution onto the nonwoven fabric, and immersing the nonwoven fabric in the cellulose derivative-containing solution.
• the Robb-Srirayan method described in the examples below can also be used. Thereafter, the nonwoven fabric permeated with the cellulose derivative-containing solution is immersed in a water bath or the like kept at about 40 to about 80° C. to solidify the cellulose derivative.
• This provides one embodiment of a biocompatible composite membrane.
• This method also allows the formation of a biocompatible composite membrane having a structure in which the cellulose derivative partially penetrates the nonwoven fabric layer. The composite membrane thus obtained can be appropriately washed, cut, and then stored, preferably in a refrigerator.
• step (b) cells or tissues are mixed with a solution capable of hydrogelling (e.g., an alginic acid solution, the gelling solutions described in [3-1], [3-2], and [1A-9] to [1A-11] above, etc.).
• a solution capable of hydrogelling e.g., an alginic acid solution, the gelling solutions described in [3-1], [3-2], and [1A-9] to [1A-11] above, etc.
• alginic acid solutions that can be hydrogelled include aqueous sodium alginate solutions, and the alginic acid derivatives represented by the above-mentioned formulas (HI), (H-II), (IA) and (II-A).
• step (b) for example, an aqueous solution or physiological saline solution of the alginic acid derivative having a concentration of about 0.1 to about 5% by weight is prepared, and a required amount of the cells or tissues described above is suspended in the solution.
• the "hydrogelable solution” refers to, for example, two types of solutions, a solution of an alginic acid derivative represented by the above formula (H-I) and a solution of an alginic acid derivative represented by the above formula (H-II).
• two types of solutions a solution of an alginic acid derivative represented by the above formula (IA) and a solution of an alginic acid derivative represented by the above formula (II-A).
• these two types of solutions are prepared separately without being mixed.
• the cells or tissues may be mixed into only one of the two types of solutions, or may be mixed into both.
• materials other than alginic acid derivatives such as the alginic acid derivatives represented by the above-mentioned formulas (H-I) and (H-II), and the alginic acid derivatives represented by formulas (I-A) and (II-A), may be added to the alginic acid solution that can be hydrogelled.
• Such materials include, for example, alginic acid (including alginic acid esters and alginates), collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptides, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen and derivatives thereof, as well as fibrin, fibrinogen, thrombin, polyglutamic acid, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, gellan gum, xanthan gum, galactomannan, guar gum, locust bean gum, and tara gum.
• alginic acid including alg
• step (c) the solution obtained in step (b) is gelled.
• the alginic acid derivatives represented by the above formulae (HI) and (H-II) or the alginic acid derivatives represented by the formulae (IA) and (II-A) are used as raw materials for the alginic acid solution capable of hydrogelling, the two solutions obtained in step (b) are mixed to cause chemical crosslinking by the Huisgen reaction, thereby gelling the solution.
• a hydrogel can be produced via bonds due to a crosslinking reaction (e.g., divalent metal ion bonds, chemical bonds).
• a crosslinking reaction e.g., divalent metal ion bonds, chemical bonds.
• the two solutions obtained in step (b) are mixed and then contacted with a solution containing a divalent metal ion, whereby chemical crosslinking and ionic crosslinking proceed simultaneously, and the solution can be gelled.
• a solution containing a divalent metal ion whereby chemical crosslinking and ionic crosslinking proceed simultaneously, and the solution can be gelled.
• step (d) the cell- or tissue-embedded hydrogel obtained in step (c) is covered with a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer obtained in step (a), step (A1) or step (A2).
• the hydrogel is covered so that the semipermeable membrane layer of the biocompatible composite membrane is disposed on the outside and the nonwoven fabric layer is disposed on the hydrogel side (the hydrogel is covered so that the surface of the biocompatible composite membrane where the cellulose derivative is laminated on the nonwoven fabric is disposed on the outside).
• the hydrogel As a method for covering the hydrogel with the biocompatible composite membrane, as described above, the hydrogel is placed between two biocompatible composite membranes cut to an appropriate size, and the ends of the two composite membranes are heat-sealed.
• the hydrogel can be covered with the composite membrane by heat-sealing four or less times, three or less times, two or less times, or once.
• the order of the above steps is not particularly limited. For example, steps (i) and (ii) may be performed before step (iii), or between step (iii) and step (iv). In addition, a plurality of steps may be performed simultaneously in parallel as appropriate.
• step (i) can be replaced with the following step (i-1) or step (i-2).
• Step (i-1) A step of forming a biocompatible composite membrane including/consisting of a semipermeable membrane layer formed by laminating a cellulose derivative on a nonwoven fabric using the nonwoven fabric as a support, and a nonwoven fabric layer having a structure in which the cellulose derivative partially penetrates the nonwoven fabric.
• Step (i-2) A step of forming a biocompatible composite membrane by laminating a cellulose derivative on one side of a nonwoven fabric using the nonwoven fabric as a support, and the cellulose derivative partially penetrates the nonwoven fabric.
• step (i) a semipermeable membrane layer is formed from a cellulose derivative using a nonwoven fabric as a support, and a biocompatible composite membrane having a structure in which the cellulose derivative partially penetrates the nonwoven fabric is formed.
• the specific method of i) is the same as step (a) of the above-mentioned method (1) for producing a biocompatible device.
• step (i-1) a semipermeable membrane layer is formed by laminating a cellulose derivative on a nonwoven fabric using a nonwoven fabric as a support, and the nonwoven fabric layer has a structure in which the cellulose derivative partially penetrates the nonwoven fabric;
• a specific method for the step (i-1) is the same as that for the step (A1) in the above-mentioned method (1) for producing a biocompatible device.
• step (i-2) a cellulose derivative is laminated on one side of a nonwoven fabric as a support, and a biocompatible composite membrane is formed in which the cellulose derivative partially penetrates the nonwoven fabric.
• the specific method is the same as step (A2) of the above-mentioned method (1) for producing a biocompatible device.
• step (ii) an internal space is formed with the biocompatible composite membrane obtained in step (i), step (i-1) or step (i-2).
• the semipermeable membrane layer of the biocompatible composite membrane is arranged on the outside, and the nonwoven fabric layer is arranged on the internal space side.
• the biocompatible composite membrane is arranged so that the surface of the nonwoven fabric on which the cellulose derivative is laminated is arranged on the outside.
• a method for forming an internal space with the biocompatible composite membrane includes placing two biocompatible composite membranes cut to an appropriate size and heat sealing the ends of the two composite membranes.
• the internal space can be formed with the composite membrane by heat sealing four or less times, three or less times, two or less times, or once.
• step (iii) cells or tissues are mixed with the solution capable of hydrogelation.
• the specific method of step (iii) is the same as step (b) of the above-mentioned method (1) for producing a biocompatible device.
• step (iv) the solution obtained in step (iii) is injected into the internal space formed by the biocompatible composite membrane in step (ii).
• the method for injecting the solution into the internal space is not particularly limited, and the solution may be injected into the internal space using a pipette or the like in an amount that does not leak out of the composite membrane.
• step (v) the solution injected in step (iv) is gelled.
• the specific method for step (v) is the same as step (c) in the above-mentioned method for producing a biocompatible device (1). Through these steps, a biocompatible device according to one embodiment of the present invention can be manufactured.
• the physical properties of the biocompatible device can be evaluated based on the physical properties of the biocompatible composite membrane.
• D/P system drug oral absorption evaluation system
• the glucose permeability at 24 hours is about 80% or more, in another embodiment about 85% or more, and in yet another embodiment about 90% or more, as measured by the method described in the Examples below.
• the biocompatible composite membrane of one embodiment of the present invention has an insulin permeability at about 24 hours, as measured by the method described below, of about 30% or more, in another embodiment of about 40% or more, and in yet another embodiment of about 50% or more.
• biocompatible composite membrane of one embodiment of the present invention has a permeability to dextran (molecular weight: about 150 kD) at about 24 hours, as measured by the method described below, of about 30% or less, in another embodiment about 20% or less, and in yet another embodiment about 10% or less.
• the strength of the biocompatible composite membrane of one embodiment of the present invention can be measured by a tensile test, a tear test, a burst test, or the like.
• a tensile test in accordance with JIS K 7127 or ASTM D882.
• the strength of the composite membrane measured by the tensile test is, for example, 1 MPa or more, preferably about 1 to about 100 MPa, and more preferably about 5 to about 100 MPa.
• the tear test and the burst test can be measured by a general test method.
• the heat fusion integrity of the biocompatible composite membrane according to one embodiment of the present invention can be measured by the method described in the Examples below. It can also be measured by a method that applies a filter integrity test, for example, a bubble point value can be measured using an integrity test kit for small volume devices (Merck, SLTEST000) and used as an index of heat fusion integrity.
• a filter integrity test for example, a bubble point value can be measured using an integrity test kit for small volume devices (Merck, SLTEST000) and used as an index of heat fusion integrity.
• the device and composite membrane provided herein are biocompatible. It has been confirmed that the alginic acid derivatives and crosslinked alginic acid constituting the hydrogel in the present invention have good biocompatibility, similar to alginic acid (see, for example, PCT/JP2019/023478, PCT/JP2020/047100, and PCT/JP2019/007655).
• the biocompatible device provided herein can be used as a device for biological transplantation.
• a biocompatible device of a certain embodiment can be suitably used for the treatment of diabetes.
• the device can be used to treat any of type 1 diabetes, type 2 diabetes, and diabetes caused by other mechanisms, and is preferably used for the treatment of type 1 diabetes.
• a preferred embodiment of the device is not only biocompatible, but also has excellent stability, low cytotoxicity, almost no adhesion or inflammation at the implantation site, little gel dissolution, and can maintain a blood glucose lowering effect for a long period of time and regulate blood glucose.
• the implantation site of the device is not particularly limited, and may be subcutaneous, intramuscular, intraperitoneal, intrahepatic, intraomental, or subrenal capsule, but implantation is preferably subcutaneous, intramuscular, intraperitoneal, or intraomental.
• Methods for implanting the device in the living body include incision and placement, endoscopy, and laparoscopy.
• kits for biological transplantation includes a biocompatible device in which an internal space is formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, and a container containing cells or tissues that secrete a biologically active substance, and is used so that the cells or tissues are enclosed in the internal space.
• biocompatible device in which an internal space is formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer
• a container containing cells or tissues that secrete a biologically active substance and is used so that the cells or tissues are enclosed in the internal space.
• a certain embodiment of a biological transplant kit may include, in one package, (1) a pack in which a biocompatible device is enclosed, and (2) a container (e.g., a vial, etc.) in which cells or tissues that secrete a biologically active substance are enclosed in a frozen state.
• the biocompatible device of (1) above may be packed in a state in which a part of the biocompatible composite membrane (e.g., one of the four sides in the case of a flat shape) is not fused so that the cells or tissues are enclosed in the internal space before use.
• the internal space can be sealed by enclosing the cells or tissues in the internal space of the biocompatible device before use and heat-fusing the unfused part (e.g., the remaining side in the case of a flat shape).
• a container such as a vial in which the alginic acid derivative that is gelled by chemical crosslinking is sealed in a freeze-dried state, (4) a container such as a vial in which physiological saline for dissolution is sealed, etc., are included in one package.
• the cells or tissues of (2) and the alginic acid derivative of (3) are dissolved in advance in a container such as a vial.
• the cells or tissues of (2) are mixed with a solution of the alginic acid derivative of (3), gelled, and sealed in a pack in advance.
• the kit of the present invention may further include accessories such as a syringe, pipette, injection needle, dedicated filling device, instruction manual, etc., as appropriate.
• the chemically modified alginic acid derivatives used in the following examples were the alginic acid derivatives (A01) or (A02) having a cyclic alkyne group, and the alginic acid derivative (N01) having an azide group, which were synthesized according to the method described in the examples of WO 2020/262642.
• a Huisgen reaction occurs, and a chemically crosslinked alginic acid containing the crosslinked structures of the formulae (LZ-8) and (LZ-8-r), or a chemically crosslinked alginic acid containing the crosslinked structures of the formulae (LY-2) and (LY-2-r), is formed, forming a hydrogel.
• a hydrogel containing the crosslinked structures of the formulae (LY-2) and (LY-2-r
• Example A Example of preparation of composite membrane (1) Preparation of membrane forming solution (dope) Cellulose acetate (LT-75) (manufactured by Daicel Corporation) was 11% by weight, PEG200 (manufactured by Hayashi Pure Chemical Industries, Ltd.) was 10% by weight, and dimethyl sulfoxide (DMSO) (manufactured by Nacalai Tesque, Ltd.) was 79% by weight, and a mixed solution of PEG200 and DMSO was prepared by weighing each component. Next, the cellulose acetate was gradually added to the mixed solution while stirring it at 70 to 80°C, to obtain a dope to be used for membrane formation.
• LT-75 membrane forming solution
• PEG200 manufactured by Hayashi Pure Chemical Industries, Ltd.
• DMSO dimethyl sulfoxide
• the composite membrane was prepared by the Loeb-Sourirajan method, which is a method for preparing asymmetric porous membranes (for example, S. Loeb and S. Sourirajan, ibid No. 59-28 (1959) and the like are known in the art).
• a nonwoven fabric HOP-30H (manufactured by Hirose Paper Co., Ltd.) was cut to the same size as the glass plate and attached to the glass plate with tape.
• a film applicator was placed on one end of the nonwoven fabric on the glass plate, and the dope kept at 60°C was evenly dripped from one end of the applicator to an area of about 1 cm, and the applicator was pressed against the nonwoven fabric and slid to cast the dope on the nonwoven fabric.
• the nonwoven fabric after casting was submerged together with the glass plate in a water bath at 60°C for a few minutes to solidify the dope, and a composite membrane in which cellulose acetate was laminated on the nonwoven fabric was obtained.
• the composite membrane obtained together with the glass plate was washed with water, and then the composite membrane was peeled off from the glass plate and immersed in water overnight to thoroughly remove the solvent contained in the membrane.
• the composite membrane of the present invention was confirmed to have sufficient heat fusion properties, with no liquid seeping out to the outside of the sealed portion.
• Strength It was confirmed by visual inspection and by touch that the film was stronger than commercially available films.
• Example B Evaluation of substance permeability of composite membrane
• the substance permeability of the composite membrane was evaluated using a horizontal chamber (Osaka Rika) for the drug oral absorption evaluation system (D/P system).
• the evaluation membrane cut to a size of 3 cm x 3 cm was immersed in a 0.1% Tween 20 solution (dialysis solution) prepared with physiological saline, and then set in the chamber.
• 9 mL of the dialysis solution was placed in the tank on the addition side of the chamber, and 7.5 mL was placed in the tank on the opposite side (recovery side) separated by the membrane. Then, with both tanks stirred (200 rpm) at room temperature, 1 mL of the following test solution was added to the addition side to start the substance permeability test.
• test was started when each solution was added, and samples were taken from the recovery side 2 hours and 24 hours after the start of the test. After 24 hours, samples were also taken from the addition side.
• the test substance concentration in each sampled solution was calculated based on the quantitative value using the measurement kit for each test substance or the quantitative value by fluorescence measurement of the label, and the substance permeability was calculated using the following formula.
• Example: Composite membrane prepared in Example A Reference Example: Dialysis tube "Spectra/Pore CE (molecular weight cutoff: 100,000)" manufactured by Repligen (Hereinafter referred to as commercially available membrane) (Test solution) ⁇ 100 mg/dL glucose solution (Otsuka Pharmaceutical) ⁇ 2000 ng/mL insulin solution (Eli Lilly) ⁇ 500 ⁇ g/mL FITC-albumin solution (Sigma) ⁇ 2000 ⁇ g/mL TRITC-dextran150 solution (manufactured by TdB Labs) (Formula for calculating material permeability) Permeability (%) 100 ⁇ test substance concentration in sampled solution / test substance concentration at permeability equilibrium
• the mass permeabilities of the composite membranes of the examples and the commercially available membranes are shown in Table 16.
• the orientation of the evaluation membrane is as follows: for the composite membrane, the semipermeable membrane layer side is the outside and the nonwoven fabric layer side is the inside, and for the commercially available membrane, the outside of the cylindrical dialysis tube is the outside and the inside is the inside, and the direction of substance permeation in the evaluation system is indicated by an arrow. That is, for example, "composite membrane (outside ⁇ inside)" means that the composite membrane was evaluated with the semipermeable membrane layer side facing the addition side and the nonwoven fabric layer side facing the recovery side. As shown in Table 16, the composite membranes of the examples exhibited material permeability equivalent to that of the commercially available membranes, and it was confirmed that they functioned as semipermeable membranes.
• Example C Evaluation of Complement Sequestration Ability
• the device containing a sensitized sheep red blood cell solution was shaken in a human serum mixed buffer solution, and the hemolysis rate was measured to evaluate the complement sequestration ability of the device.
• the above alginic acid derivatives (A02) and (N01) were dissolved in CH50 buffer (buffer solution included with One Point CH50 "Seiken" (Denka Co., Ltd.)) to give a final concentration of 0.5% alginic acid derivative (a mixture of equal amounts of alginic acid derivative (A02) with a final concentration of 0.25% and alginic acid derivative (N01) with a final concentration of 0.25%), and sensitized sheep red blood cells were added to the solution in a volume equivalent to one-third of the solution (red blood cell-containing alginic acid solution).
• Example A Two sheets of the composite membrane prepared in Example A cut into rectangles measuring approximately 1.5 cm x approximately 2.5 cm were prepared, and the nonwoven fabric layer was placed on top and bottom of the gel, and the four sides were heat-pressed to form an example device. (Reference device)
• the hydrogel wrapped in the commercially available membrane prepared above was used as a reference device.
• the hemolysis rate of each sample was calculated by assuming that the hemolysis rate was 100% when 100 ⁇ L of the red blood cell solution was added to 3.01 mL of water.
• the hemolysis rate in the complement hemolysis control group was about 70%, but in both the example device using the composite membrane of the example and the reference example device using the commercially available membrane, the hemolysis rate was less than 1%, and almost no hemolysis was observed. Therefore, it was confirmed that the device made using the composite membrane of the example has complement sequestration ability.
• Example D Glucose-responsive insulin secretion test Glucose-responsive insulin secretion ability was evaluated using a device containing insulin-secreting cells.
• Device Preparation MIN6 cells 2.5 ⁇ 10 6 cells derived from pancreatic ⁇ cells were suspended in 100 ⁇ L of a solution in which 0.5% aqueous solutions of alginic acid derivatives (A01) and (N01) were mixed in equal amounts.
• Two composite membranes prepared in Example A cut into rectangles of about 2 cm x about 1 cm were prepared, and the two were stacked so that the nonwoven fabric layers faced each other on the inside, and the three sides except for one side of 1 cm were heat-pressed to form an envelope-like structure.
• Example C a commercially available membrane was molded into an envelope-like structure of the same size as in Example C.
• the above cell suspension was injected into each internal space generated by molding. After the remaining side of each structure was heat-pressed, it was immersed in a 50 mmol/L CaCl 2 solution for 10 minutes to promote the formation of a hydrogel, and used as an example device and a reference example device for evaluation.
• the device was cultured at 37° C. for one day using a culture medium (Optimized DMEM (AddexBio) containing 15% FBS), and then glucose concentration-dependent insulin secretion was evaluated. Specifically, the device was immersed in a 2 mmol/L glucose solution for 1 hour or in a 20 mmol/L glucose solution for 2 hours, and the insulin concentration secreted into the solution under each condition was measured, allowing the insulin secretion ability of cells in response to glucose concentration to be evaluated.
• Optimized DMEM Optimized DMEM (AddexBio) containing 15% FBS
• Example E Biocompatibility test of composite membrane (rats) (1) Preparation of Membranes The following four types of membranes for implantation were prepared.
• ⁇ The composite membrane prepared in Example A was sterilized in advance by UV irradiation, cut into a rectangle of approximately 2.8 cm x approximately 1.8 cm, two of which were stacked with the nonwoven fabric layers facing each other on the inside, and the four sides were heat-pressed to form a membrane.
• ⁇ A membrane made by cutting polyurethane containing 0.75% zinc diethyldithiocarbamate (Food and Drug Safety Center, Hadano Research Institute) into a size of approximately 2 cm x approximately 1 cm and sterilizing it in an autoclave.
• Non-effect control A high-density polyethylene sheet (Food and Drug Safety Center, Hadano Research Institute) cut into approximately 2 cm x 1 cm pieces and sterilized in an autoclave.
• the composite membranes of the Examples were suitable for transplantation, with no adhesions, changes in the membrane, or adhesions between organs.
• the commercially available membrane of the Reference Example showed slight adhesions between organs. All animals experienced a temporary weight loss, which was thought to be due to the treatment, and recovered afterwards. No abnormalities were noted in their general condition.
• Example F Device biocompatibility test (dog, miniature pig) (1) Fabrication of Devices The following two types of devices were fabricated for implantation.
• Example device The composite membrane prepared in Example A was cut into a square of about 2.4 cm x about 2.4 cm, and two sheets were stacked so that the nonwoven fabric layers faced each other on the inside, and three sides were heat-pressed to form an envelope-like structure.
• the structure was immersed in a 10 mmol/L BaCl2 solution for 10 minutes to promote the formation of a hydrogel, and was used as the example device.
• (Reference device) Two commercially available membranes cut into a square of approximately 2.4 cm x approximately 2.4 cm were stacked, and three sides were heat-pressed to form an envelope-like structure. 156 ⁇ L of an equal mixture of 1% aqueous solutions of alginic acid derivatives (A02) and (N01) was injected into the internal space created by the molding. After the remaining side was heat-pressed, the structure was immersed in a 10 mmol/L BaCl2 solution for 10 minutes to promote the formation of a hydrogel, and used as a reference device.
• Example G Fabrication of devices using a 3D printer and biocompatibility testing (dogs, minipigs)
• aqueous solution containing alginic acid derivatives (A02) and (N01) (final concentration 0.5% each), alginic acid (A-2: Mochida Pharmaceutical Co., Ltd., sodium alginate listed in Table 1) (final concentration 1%), and BaCl 2 (final concentration 2.5 mmol/L) was prepared, and the aqueous solution was ejected from a 410 ⁇ m diameter nozzle using a 3D printer (BIO X TM , CELLINK Co., Ltd.) to form a rectangular shape of approximately 6.5 cm x approximately 3 cm.
• Example A a 10 mmol/L BaCl 2 solution was layered on the ejected material and left to stand for 10 minutes to form a hydrogel.
• Two sheets of the composite membrane produced in Example A were cut into rectangles measuring approximately 7 cm x approximately 3.5 cm. These were placed on top and bottom of the hydrogel so that the nonwoven fabric layer was on the gel side, and the four sides were heat-pressed to form a device for implantation.
• Example H Examination of implantation site in biocompatibility test (dog, mini pig) (1) Fabrication of device Two composite membranes prepared in Example A were cut into a rectangle of about 7 cm x about 3.5 cm in size, stacked so that the nonwoven fabric layers faced each other on the inside, and the three sides except one short side were heat-pressed to form an envelope-like structure. 700 ⁇ L of a mixture of equal amounts of 1% aqueous solutions of alginic acid derivatives (A02) and (N01) was injected into the internal space generated by molding. After the remaining side was heat-pressed, the structure was immersed in a 10 mmol/L BaCl2 solution for 10 minutes to promote the formation of a hydrogel, and was used as a device for implantation.
• A02 alginic acid derivatives
• N01 alginic acid derivatives
• Example F Implantation Method and Evaluation Method According to the method of Example F (Device biocompatibility test (dogs, minipigs)), the above-mentioned devices were implanted in the following three implantation sites using beagle dogs and minipigs (Goettingen minipigs).
• Implantation site 1 intraperitoneal implantation was performed under anesthesia and the wound was closed.
• Implantation site 2 In the omental pocket After laparotomy under anesthesia, the omentum was folded in half and sewn to create a pocket. The device was implanted in the pocket, and the wound was closed.
• Implantation site 3 abdominal wall After laparotomy under anesthesia, the device was sutured to the abdominal wall off the midline, and the wound was closed.
• Example I Drug efficacy evaluation of device (mice)
• Device Preparation MIN6 cells (2.2 x 106 cells) similar to those in Example D were suspended in 50 ⁇ L of a solution obtained by mixing equal amounts of 0.5% aqueous solutions of alginic acid derivatives (A01) and (N01).
• Two composite membranes prepared in Example A cut into rectangles measuring approximately 2cm x approximately 1cm were prepared, and the two were stacked so that the nonwoven fabric layers faced each other on the inside, and the three sides except for one side measuring 1cm were heat-pressed to form an envelope-like structure.
• a commercially available membrane was molded into an envelope-like structure of the same size as in Example C.
• the above cell suspension was injected into each internal space generated by molding. After the remaining side of each structure was heat-pressed, it was immersed in a 50 mmol/L CaCl2 solution for 10 minutes to promote the formation of a hydrogel, and used as an example device and a reference example device for evaluation.
• the pharmacological effects of the example device and the reference example device are shown in Table 5.
• a patient was deemed cured if the blood glucose level at each measurement was 200 mg/dL or more lower than the blood glucose level before implantation.
• Healing was determined based on the blood glucose levels of each animal 5 and 25 days after implantation, and the percentage of cured animals out of all animals was calculated as the cure rate.
• the example device had an effect equal to or greater than that of the reference example device using a commercially available film.
• the healing rate 25 days after implantation was 33% for the reference device, whereas the healing rate was as high as 80% for the example device.
• the unevenness of the nonwoven fabric surface may have acted as an anchor for the hydrogel, preventing the gel from becoming displaced, folding, or collapsing within the device.
• the utilization rate of the cells embedded in the hydrogel was improved, leading to a greater therapeutic effect.
• Biocompatible device Biocompatible composite membrane 21
• Nonwoven fabric layer (nonwoven fabric) 211: Portion where the semipermeable membrane component has not penetrated
• 212 Portion where the semipermeable membrane component has partially penetrated into the nonwoven fabric
• 22 Semipermeable membrane layer (semipermeable membrane) 3 Internal space 4 Cells or tissue 5 Hydrogel 6 Fusion site
• the present invention may include the following aspects.
• a biocompatible device having an internal space capable of containing cells or tissues that secrete a biologically active substance, the internal space being formed by disposing a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer in the device, the semipermeable membrane layer being on the outer side and the nonwoven fabric layer being on the internal space side, the semipermeable membrane layer comprising a cellulose derivative laminated on the nonwoven fabric using the nonwoven fabric as a support, and the nonwoven fabric layer comprising a nonwoven fabric having thermoplastic properties.
• [3] The device according to [1] above, wherein the cellulose derivative is cellulose acetate.
• the nonwoven fabric contains one or more resins selected from the group consisting of polyethylene, polypropylene, and polyethylene terephthalate.
• the internal space is formed by partial fusion of the nonwoven fabric surface in the composite membrane that is not laminated with the cellulose derivative.
• the composite membrane has a structure in which the cellulose derivative partially penetrates into a nonwoven fabric.
• the cells secreting a biologically active substance are insulin-secreting cells or pancreatic islets.
• a kit for biological transplantation comprising a biocompatible device in which an internal space is formed by a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, and a container containing cells or tissues that secrete a biologically active substance, the kit being used so that the cells or tissues are sealed in the internal space.
• a method for producing a biocompatible device comprising a step of covering a hydrogel in which cells or tissues that secrete a biologically active substance are embedded, with a biocompatible composite membrane including a semipermeable membrane layer and a nonwoven fabric layer, wherein in the step, the semipermeable membrane layer is positioned on the outer side and the nonwoven fabric layer is positioned on the hydrogel side.
• a method for producing a biocompatible device comprising the steps of injecting a solution containing cells or tissues secreting a biologically active substance, the cells or tissues being mixed with a hydrogellable solution, into an internal space formed by a biocompatible composite membrane comprising a semipermeable membrane layer and a nonwoven fabric layer, and gelling the hydrogellable solution, wherein in the step, the semipermeable membrane layer of the composite membrane is on the outer side and the nonwoven fabric layer is on the internal space side.

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