WO2023127872A1 - 移植材料の製造方法、及び移植材料 - Google Patents

移植材料の製造方法、及び移植材料 Download PDF

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Publication number
WO2023127872A1
WO2023127872A1 PCT/JP2022/048145 JP2022048145W WO2023127872A1 WO 2023127872 A1 WO2023127872 A1 WO 2023127872A1 JP 2022048145 W JP2022048145 W JP 2022048145W WO 2023127872 A1 WO2023127872 A1 WO 2023127872A1
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Prior art keywords
organ
foreign substance
injection
injected
cells
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English (en)
French (fr)
Japanese (ja)
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比呂志 長嶋
ひとみ 松成
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Pormedtec Co Ltd
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Pormedtec Co Ltd
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Priority to US18/725,479 priority Critical patent/US20250154453A1/en
Priority to JP2023550541A priority patent/JP7440987B2/ja
Publication of WO2023127872A1 publication Critical patent/WO2023127872A1/ja
Priority to JP2024017975A priority patent/JP2024036657A/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • 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
    • A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
    • 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
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/04—Alginic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
    • C08L89/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin, e.g. gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/56—Physical preservation processes for animal cells or human cells
    • C12N5/562—Temperature processes, e.g. following predefined temperature changes over time
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions

Definitions

  • the present invention relates to a method for producing a transplant material containing an organ into which a foreign substance has been injected, which is suitable as a regenerative medicine product, and the transplant material.
  • it relates to a method for producing a xenograft material containing an organ into which a foreign substance has been injected, and the xenograft material.
  • Organ regeneration is actively developing in various organs.
  • kidney which has been considered complex and difficult to regenerate, foreign substances such as human mesenchymal stem cells are injected into the renal developmental region of animals in the developing stage, and a heterogeneous developmental program is borrowed for "embryonic organ complementation.” method” is being developed (see, for example, Non-Patent Document 1).
  • a foreign substance e.g., human mesenchymal stem cells
  • an organ e.g., metanephro
  • the organ such as metanephro
  • it is difficult to puncture a needle-like member such as a glass capillary tube. Therefore, the animal body (fetal body, etc.) is dissected open to some extent, and the needle-like member is pierced into the organ (metnephros, etc.) attached to the animal body (fetal body, etc.) to inject the foreign substance.
  • a needle-like member such as a glass capillary tube
  • the above method has the following problems. 1) Since the injection work can only be performed in a state of being attached to an animal body such as a fetal body, the location and time of the injection work are greatly restricted. 2) Organs such as metanephros excised from animal bodies such as fetal bodies are extremely small (approximately 2 mm in major diameter x 1 mm in minor diameter) and flexible organs, so the needle-shaped member is gently grasped so as not to damage the organ. is difficult to puncture.
  • the injection target area in the organ such as the metanephros is on the unexposed side of the cut open animal body (fetal body, etc.)
  • a needle-like member such as a capillary is punctured from the exposed side of the organ. Then, the foreign matter must be injected by penetrating to the injection target region. This leaves the graft material with a large amount of damage in the injection pathway.
  • the present invention has been made in view of the problems of the prior art as described above, and it is an object of the present invention to provide a method for producing a transplant material that allows a foreign substance to be easily injected into an organ removed from an animal body.
  • the first purpose A second object of the present invention is to provide a graft material in which damage is reduced even though a foreign substance has been injected.
  • the present inventors have found that by positioning at least a part of an organ extracted from the body of an animal in contact with a plastic member, a foreign object can be transported to a desired portion of the organ by the shortest route, and in a complicated manner. It was found that the injection can be easily performed without requiring any special work.
  • the present invention has been completed based on the above findings. That is, the present invention is as follows.
  • ⁇ 1> The organ into which the foreign substance has been injected, comprising the steps of positioning at least a part of an organ extracted from the body of an animal in contact with a plastic member, and injecting the foreign substance into the organ after positioning.
  • a method of making a graft material comprising: ⁇ 2> The method according to ⁇ 1>, wherein the injection is performed from the surface directly above or near the injection target region in the organ.
  • ⁇ 3> The method according to ⁇ 1> or ⁇ 2>, wherein the animal body is a fetal body.
  • ⁇ 4> The method according to any one of ⁇ 1> to ⁇ 3>, wherein the organ is an organ for xenotransplantation.
  • ⁇ 5> The method according to any one of ⁇ 1> to ⁇ 4>, wherein the organ is vitrified and frozen after the excision.
  • the plastic member is a plastic member covering the organ during vitrification and freezing.
  • ⁇ 7> The method according to any one of ⁇ 1> to ⁇ 6>, wherein at least part of the organ is embedded in the plastic material.
  • ⁇ 8> The method according to any one of ⁇ 1> to ⁇ 7>, wherein the plastic member is gel.
  • ⁇ 9> The method according to any one of ⁇ 1> to ⁇ 8>, wherein a needle-like member is used for the injection.
  • ⁇ 10> The method according to ⁇ 9>, wherein the path through which the needle-like member passes has a dense structure impermeable to liquid.
  • the foreign substance is a non-animal-derived cell of the animal body.
  • the organ is the kidney, and the injection target region is the renal cortical formation region of metanephros.
  • Transplantation comprising an organ removed from an animal and a foreign substance injected into the organ, wherein an injection scar is formed in the organ directly above or near the region into which the foreign substance was injected material.
  • ⁇ 14> The material according to ⁇ 13>, wherein the foreign substance is a cell derived from a site other than the organ.
  • INDUSTRIAL APPLICABILITY it is possible to provide a method for producing a transplant material that allows a foreign substance to be easily injected into an organ removed from an animal body. Moreover, according to the present invention, it is possible to provide a transplant material with reduced damage despite the fact that a foreign substance has been injected. Moreover, according to the present invention, it is possible to reduce restrictions on the location and time of the injection work.
  • FIG. 1 is a schematic diagram illustrating one preferred embodiment of the present invention
  • FIG. FIG. 2 shows photomicrographs of metanephros positioned by semi-embedding in gelatin gel.
  • FIG. 3 is a micrograph showing the results of injecting a foreign substance (GFP-expressing cells) into one of the renal cortex-forming regions under the renal capsule.
  • FIG. 10 is a diagram showing micrographs showing the results of injecting foreign substances (GFP-expressing cells) into two locations in the renal cortex-forming region under the renal capsule.
  • FIG. 10 is a diagram showing micrographs showing the results of extensive injection of a foreign substance (GFP-expressing cells) into the metanephric mesenchymal region.
  • FIG. 10 is a micrograph showing the injection results of Example 3; 4 is a diagram showing a micrograph showing the injection results of Comparative Example 1.
  • FIG. 10 shows the pancreatic tissue of a pig deficient in ⁇ cells due to the expression of the Pdx1-Hes1 gene in Example 4.
  • FIG. 9 shows a human iPS cell-derived pancreatic progenitor cell cluster injected into one of the pancreatic tissue fragments shown in FIG.
  • a first aspect of the present invention is the outpatient, comprising the steps of positioning at least a portion of an organ extracted from an animal body by contacting a plastic member, and injecting a foreign substance into the organ after positioning.
  • a method for producing a graft material containing an organ into which an object has been injected in the first aspect, by positioning at least a part of the organ extracted from the animal body in contact with the plastic member, the foreign substance can be easily injected into the organ extracted from the animal body without complicated work. can do.
  • the injection target region 3 of the organ 1 can be easily injected with a foreign substance by any means ( Preferably, it can be injected accurately).
  • the plastic member 2 is preferably brought into contact with (placed, fixed, etc.) on the substrate 4 .
  • animals that provide the animal body include pigs, cows, horses, sheep, goats, primates (e.g., humans, apes (chimpanzees, monkeys, etc.)), rodents (e.g., mice, rats), and the like. mammals.
  • the animals are preferably mammals whose physique and other characteristics are closer to humans than rodents, more preferably pigs, sheep, goats, and primates (e.g., humans, apes), and pigs (that is, the organs are organ of origin) is more preferred.
  • the animal body may be a fetal body, a juvenile body, or an adult body, but from the viewpoint of low immunogenicity, the animal body is preferably a fetal body.
  • the organ is an organ for xenotransplantation, it is preferably a fetal body.
  • the organ is preferably an organ for transplantation, more preferably an organ for xenotransplantation.
  • organs include internal organs (e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestine, etc.), reproductive organs (e.g., ovary, testis), fertilized eggs, embryos, fetuses, bone marrow (e.g., , hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, or tissues derived therefrom, or artificial tissues (cell sheets such as chondrocyte sheets, organoids, etc.).
  • internal organs e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestine, etc.
  • reproductive organs e.g., ovary, testis
  • fertilized eggs embryos, fetuses
  • bone marrow e.g., , hematopoietic organs
  • brain eyes, nose
  • the above organs are preferably internal organs, reproductive organs, fertilized eggs, embryos, and fetuses, more preferably internal organs and reproductive organs, and include pancreas (e.g., pancreatic islets), kidneys (e.g., metanephros, particularly metanephros, ureters, and bladder). Urology) is more preferred.
  • pancreas e.g., pancreatic islets
  • kidneys e.g., metanephros, particularly metanephros, ureters, and bladder.
  • Urology is more preferred.
  • the organ may or may not be a genetically modified organ (recombinant organ or genome edited organ).
  • preparation of genetically modified organs include (1) preparation of genetically modified animal cells, and (2) preparation of the organ from the cells prepared above by somatic cell cloning technology.
  • the preparation of the cells may or may not be introduction of the desired transgene by gene editing technology such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas nuclease.
  • the somatic cell cloning technology involves the use of cells of an animal to be cloned (preferably genetically modified animal cells) is cultured as a donor cell, and the donor cell is nuclear-transplanted into a recipient enucleated egg (oocyte) by a fusion method such as cell fusion.
  • oocyte recipient enucleated egg
  • somatic cell cloning technique genetically modified animals can be cloned, and the genetically modified organ can be prepared by extracting the target organ from the animal.
  • the organ may or may not be a secondary organ.
  • the term "secondary organ” refers to an organ-like tissue formed by nurturing and growing an animal in a part of the body where an organ whose formation is inhibited (e.g. pancreas) is originally formed (Japanese Unexamined Patent Application Publication No. 2019-62929).
  • the secondary organ may or may not be the genetically modified organ. That is, the above-described inhibition of organogenesis can be achieved by genetic modification that modifies the genes of the organ-bearing animal.
  • the Pdx1-Hes1 gene in which the Hes1 gene is linked to the promoter of the Pdx1 gene, can be used, that is, the Hes1 gene can be overexpressed under the control of the Pdx1 promoter (Matsunari et al. , PNAS 110: 4557-4562 (2013); Japanese Patent Application Laid-Open No. 2019-62929).
  • the secondary organ may be formed by inhibiting organogenesis by administering a drug.
  • the secondary organ may have 100% of the function as the original organ, or may have only a part of it.
  • Preferred secondary organs include secondary organs lacking specific functional cells.
  • the term "functional cells" refers to cells that are contained in an organ and have some function.
  • Examples include ⁇ -cells, ⁇ -cells, and ⁇ -cells in the pancreas, and nephron progenitor cells, metanephric mesenchymal cells, ureteroblasts, and the like in the kidney.
  • Preferred functional cells include beta cells in the pancreas and nephron progenitor cells in the kidney.
  • Preferred organs deficient in specific functional cells include pancreas deficient in ⁇ cells, kidney deficient in nephron progenitor cells, kidney deficient in metanephric mesenchymal cells, and the like.
  • the kidney e.g., metanephros, particularly urinary organs including metanephros, ureters, and bladder
  • the kidney after formation of metanephros (renal primordium) at the tip of the ureter is preferable.
  • the kidneys eg metanephros, particularly the urinary system including metanephros, ureters and bladder
  • the kidneys which correspond to the elongation phase of the ureteric buds that are the main components of nephron formation.
  • kidneys of 25 days to 45 days of gestational age corresponding to the elongation period are preferred, and kidneys of 30 days to 40 days of gestational age.
  • kidneys of 30 days to 40 days of gestational age are more preferred.
  • the organ As a mode for bringing at least a portion of the organ into contact with the plastic member, the organ is placed on the plastic member, and at least a portion of the organ does not slide under, sideways, or above the plastic adhesive member. Adhesion by embedding or the like can be mentioned. In any of the above aspects, it is preferable that at least part of the organ is buried in the plastic member. A part of the organ may be buried in the plastic member, and a part of the organ may be exposed, or the organ may be completely buried in the plastic member (completely covered with the plastic member). may be used.).
  • the plastic member when the plastic member is a gel described later, in a liquid (preferably hydrosol) containing a sol (the sol before gelation of the gel) , gelation after immersing the organ, gelation after applying the liquid containing the sol to the organ, or the like.
  • Gelation of the sol can be performed by any method such as addition of polyvalent metal ions such as calcium ions and barium ions, cooling, and the like.
  • positioning refers to maintaining the positional relationship of the organ with respect to the plastic member by suppressing movement of the organ due to slippage or the like.
  • the plastic member is not particularly limited as long as it has plasticity (deformability or flexibility) that can adhere to the organ to the extent that movement of the organ due to slipping or the like can be suppressed, and the object of the present invention can be achieved.
  • a member containing any plastic material can be mentioned.
  • a member containing a plastic material having a coefficient of friction to the extent that the movement of the organ can be suppressed is preferable.
  • the member may or may not contain a plastic adhesive material or the like. Any pharmacologically acceptable plasticity from the viewpoint that there is no actual harm even if the plastic material is not completely removed and a small amount remains, and that the plastic material can be implanted while being coated without removing the plastic material.
  • a member comprising material is preferred.
  • the gel has plasticity (deformability or flexibility) that allows the organ to adhere to the extent that the movement of the organ can be suppressed, and has an appropriate coefficient of friction due to hydrogen bonding, intermolecular force, etc.
  • Preferred are any pharmacologically acceptable gels (non-toxic pharmaceutically acceptable gels), more preferred are hydrogels.
  • the gel is a gel in which the sol-gel phase transition occurs due to ionic cross-linking, or a temperature (for example, a phase transition temperature of 5 to 40 ° C., preferably 5 ⁇ 10 ° C, 10 ⁇ 15 ° C, 15 ⁇ 20 ° C, 20 ⁇ 25 ° C, 25 ° C ⁇ 30 ° C, 30 ° C ⁇ 35 ° C or 35 ° C ⁇ 40 ° C phase transition temperature) by the sol-gel phase transition Temperature-sensitive gels in which .
  • a temperature for example, a phase transition temperature of 5 to 40 ° C., preferably 5 ⁇ 10 ° C, 10 ⁇ 15 ° C, 15 ⁇ 20 ° C, 20 ⁇ 25 ° C, 25 ° C ⁇ 30 ° C, 30 ° C ⁇ 35 ° C or 35 ° C ⁇ 40 ° C phase transition temperature
  • a gel that undergoes a sol-gel phase transition due to the above ionic cross-linking is, for example, ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetic acid disodium ethylenediaminetetraacetic acid (EDTA.2Na).
  • EDTA ethylenediaminetetraacetic acid
  • EDTA.2Na ethylenediaminetetraacetic acid disodium ethylenediaminetetraacetic acid
  • hydrogel examples include alginic acid and salts thereof (e.g., polyvalent metal salts such as calcium alginate and barium alginate, and monovalent metal salts such as sodium alginate and potassium alginate. From the viewpoint of achieving gelation more reliably. , calcium alginate, and polyvalent metal salts of barium alginate are preferred.), gelatin, carrageenan, agar (agarose gel), pectin, chitosan, silicone hydrogel, konjac, and other polysaccharides.
  • a hydrogel in which a sol-gel phase transition occurs due to temperature or a temperature-sensitive hydrogel in which a sol-gel phase transition occurs due to temperature is preferred.
  • Alginic acid or its salts are examples of hydrogels in which the sol-gel phase transition occurs due to ionic cross-linking.
  • Gelatin, carrageenan, agar (agarose gel), pectin and the like are examples of temperature-sensitive hydrogels in which a sol-gel phase transition occurs due to temperature.
  • Gelatin is preferred because it can be sol-formed at a relatively low temperature (for example, 35° C. or lower) and causes less damage to the above-mentioned organs and the like.
  • 5-25% (w/v) gelatin solutions can be used.
  • a 7-20% (w/v) gelatin solution is preferred, and a 10-15% (w/v) gelatin solution is more preferred.
  • the size of the plastic member can be appropriately set by those skilled in the art according to the size of the organ.
  • a sheet-like member having long sides and short sides that are 0.25 to 3 times as large as that of the sheet can be formed.
  • a sheet-like member having long and short sides 0.35 to 2 times the long and short axes of the organ is preferable, and 0.5 to 1.5 times the long and short axes of the organ.
  • a sheet-like member having five times the long side and the short side is more preferable. More specifically, a sheet-shaped member having a long side of 10 to 200 mm and a short side of 5 to 100 mm can be used.
  • the thickness can be, for example, 0.1 mm to 5 mm (preferably 0.5 mm to 2 mm).
  • the method of injecting the foreign substance is not particularly limited as long as the foreign substance can be injected into the organ.
  • a method of injecting by any method and the like can be mentioned.
  • a method of puncturing the surface of the organ with a hollow needle-like member and injecting the foreign matter by any method (eg, capillary action, injection) is preferred.
  • the pore diameter (eg, average diameter) of the pores is not particularly limited, and is, for example, 0.05 mm to 3 mm, preferably 0.1 mm to 2 mm, and more preferably 0.5 mm to 1.5 mm.
  • Examples of the needle-like member include capillaries with sharp tips.
  • the work of injecting a foreign substance may or may not be done freehand (for example, freehand under a microscope by an expert), but from the viewpoint of more accurate execution, it should be done using a micromanipulator. is preferred.
  • the above-mentioned foreign substance means a substance derived from other than the above-mentioned organ, and the above-mentioned foreign substances include substances derived from the same animal species as the recipient (transplantation recipient individual), and the same animal as the donor (transplantation source individual). Substances derived from an individual whose species is different from the donor, substances derived from an animal species different from both the recipient and the donor, drugs (eg, pharmaceuticals), and the like.
  • a substance derived from an animal species a substance derived from the same animal species as the recipient is preferable from the viewpoint of low immunogenicity.
  • Substances include, for example, cells, growth factors, hormones, cytokines, etc. Among them, cells are preferred.
  • the cells may or may not be artificial cells (eg, genetically modified cells, ES cells, iPS cells).
  • the above-mentioned “cells derived from the same animal species as the recipient” include “human-derived cells”.
  • the injection is performed from a surface directly above or near the injection target area in the organ.
  • the “near surface” refers to the surface of the organ that exists on the same side as the injection target area with respect to the center of the organ. A surface of the organ comprising a point on an extension line connecting the center and any point in the injection target area is preferred.
  • the “directly above surface” refers to the surface of the organ that is closest to the injection target region.
  • said “near surface” is the region of the surface that includes said "immediately overlying surface”.
  • the injection target region includes any region within the organ.
  • the injection target region includes the nephrogenic zone of the metanephros, the mesenchymal region of the metanephros, and the like.
  • the substrate 4 is not particularly limited, but includes plates (made of plastic, metal, etc.), wells, glass substrates, plates (for example, dishes), and the like. At least part of the injection scar formed in the organ may or may not be covered with the plastic member. "Injection marks" will be described in detail later.
  • the organ may or may not have undergone vitrification freezing after the extraction.
  • Organs that have been vitrified and frozen include organs that have been vitrified and cryopreserved (for example, stored for a long period of time).
  • the organ may be covered with a plastic member during the vitrification freezing.
  • the plastic member that covers the organ during the vitrification freezing may be the same as or different from the plastic member (member that functions as a positioning jig) in the first aspect. From the viewpoint of process simplification, it is preferable to use the plastic member that covers the organ during vitrification and freezing as it is as the plastic member (member that functions as a positioning jig) in the first aspect.
  • the animal body (fetal body, etc.) is cut open to some extent and disassembled, and the needle-like member is pierced into the organ (metanephrine, etc.) attached to the animal body (fetal body, etc.).
  • Injection of a foreign substance has been performed by using an injection method (see, for example, Non-Patent Document 2).
  • the renal parenchyma is penetrated (by puncturing the needle member) from important vessels such as the renal artery, renal vein, and ureter, and the renal hilum, which has many spaces such as the renal pelvis. I had to inject a foreign substance.
  • the renal hilum has a large number of vessels and spaces, there is a problem that the above-mentioned foreign substances after injection leak out.
  • the renal hilum is an important tissue where important vessels such as the renal artery, renal vein, and ureter converge during kidney development. Although damage to the renal tissue should be avoided as much as possible, the conventional method of injecting a foreign substance from the renal hilum has the problem of being highly invasive to the renal tissue.
  • the injection scar formed in the organ may or may not be covered with the plastic member. Even without coating as described above, according to the present invention, it is possible to avoid injection of a foreign substance from the direction of the above-mentioned vessels such as the renal hilum and the direction with many cavities. Therefore, it is possible to suppress the leakage of the foreign matter in the vitrification freezing. In addition, according to the present invention, damage to the renal hilum can be avoided, and thus the invasiveness is lower than that of the conventional method.
  • the path through which the needle-shaped member passes through the organ has a dense structure.
  • the dense structure includes a structure with fewer tubes, voids, etc. than the lumen of an organ (e.g., a hollow organ), an orifice, etc., or a structure without tubes, voids, etc. (e.g., a solid portion of an organ (solid organ , solid organs, etc.).
  • a dense structure that is impermeable to a liquid is preferable to a lumen of an organ (e.g., a hollow organ), a portal, etc., and a tissue (e.g., connective tissue) or More preferred is a densely packed structure of cells (eg, mesenchymal cells).
  • organ e.g., a hollow organ
  • tissue e.g., connective tissue
  • a densely packed structure of cells eg, mesenchymal cells.
  • metanephric mesenchymal cells such as the metanephric mesenchymal region, the renal cortex-forming region, and the cap mesenchyme, have a substantial structure.
  • vitrification freezing means that the above organs are brought into contact with a refrigerant for vitrification freezing such as liquid nitrogen (boiling point -196°C), liquid helium (boiling point -269°C), or liquid ethane (boiling point -175°C).
  • a refrigerant for vitrification freezing such as liquid nitrogen (boiling point -196°C), liquid helium (boiling point -269°C), or liquid ethane (boiling point -175°C).
  • the method of contact with the vitrification-freezing refrigerant is not particularly limited, but includes immersion or application (flash) of liquid, spraying of vapor, and the like.
  • Contact with the vitrification cryocoolant is vitrification such as a support for vitrification freezing, a cryotop, a straw (for example, a straw for transplantation, a straw with a sharp tip, etc.), a capillary pipette, etc., which can support a plurality of organs at the same time. It may or may not be carried out using a freezing support, but it is preferably carried out using the above support.
  • the support for vitrification and freezing which can support a plurality of organs at the same time, is more preferable from the viewpoint that a plurality of organs can be frozen at once and the production efficiency of the transplant material can be improved.
  • the support for vitrification and freezing that can simultaneously support a plurality of organs has, for example, a plurality of depressions on a glass plate, a metal plate, a plastic plate, etc., and can simultaneously support the plurality of organs in the depressions. chips or plates, A mesh net, a non-woven fabric, or the like may be used as a supporting tool capable of simultaneously supporting a plurality of organs.
  • CRYOTOP registered trademark; manufactured by Kitasato Biopharma
  • CRYOTOP registered trademark; manufactured by Kitasato Biopharma
  • the organs Prior to the vitrification and freezing, the organs are preferably brought into contact (immersion, coating, etc.) with any vitrification solution.
  • the vitrification solution is not particularly limited, but cell-permeable cryoprotectants such as dimethylsulfoxide (DMSO), ethylene glycol (EG), propanediol, and glycerin, and sucrose (sucrose), trehalose, sorbitol, and dextran saccharides, carboxylated polylysine, polyvinyl alcohol, polyvinylpyrrolidone, antifreeze protein and other cell impermeant cryoprotective agents (preferably aqueous solutions).
  • DMSO dimethylsulfoxide
  • EG ethylene glycol
  • propanediol propanediol
  • glycerin sucrose
  • sucrose sucrose
  • trehalose trehalose
  • sorbitol and dextran saccharides
  • carboxylated polylysine carboxylated polylysine
  • the organ after vitrification freezing (preferably after vitrification cryopreservation) may or may not be brought into contact (immersion, coating, etc.) with an arbitrary thawing solution. After contacting with the melt, it may or may not be contacted with any diluent (immersion, coating, etc.).
  • the above-treated organ may or may not be cultured in the above basal medium or basal medium solution before transplantation (preferably xenotransplantation).
  • the organ when used as a regenerative medicine product, it is required that the foreign substance (e.g., cells derived from the same animal species as the recipient) be injected into the organ and then vitrified and frozen for storage. there may be cases.
  • the foreign substance e.g., cells derived from the same animal species as the recipient
  • the foreign matter is removed from the foreign matter by the osmotic pressure gradient between the inside of the organ and the external solution (for example, the vitrification solution, the equilibrium solution, etc.).
  • the external solution for example, the vitrification solution, the equilibrium solution, etc.
  • the organ may or may not be covered with a plastic member during vitrification freezing.
  • the plastic member the member used as the positioning jig in the first aspect may be used as it is. Regardless of whether the injection scar is covered or not, according to the present invention, it is possible to avoid injection of the foreign substance from the direction of the above-mentioned vessels such as the renal hilum and the direction with many voids. leakage can be suppressed.
  • the path through which the needle-like member passes through the organ is a liquid-impermeable path.
  • the path in which a communicating hole described later is formed is a liquid-impermeable path.
  • the organ has a foreign substance injected therein and an injection scar
  • the plastic member covers at least a part of the injection scar.
  • the “injection scar” means a hole formed for artificially injecting the foreign substance into the organ. It means a communicating hole leading to.
  • the "injection scar” may be formed by, for example, a needle-like member or the like.
  • at least a part of the injection marks made up of the holes and the communication holes on the surface and the holes on the surface may be covered with the gel.
  • the method may or may not include a removing step of removing the plastic member covering the surface of the organ.
  • the removal step can be included in the coated plastic member.
  • the gel can be removed by heating the plastic member to a sol-forming temperature, arbitrary sol-forming such as the chelate treatment, or physical pressure. Furthermore, it may or may not be washed with any washing liquid such as any aqueous solution or phosphate buffer (running water).
  • the removal step may or may not be performed multiple times (eg, stepwise).
  • a second aspect of the present invention includes an organ isolated from an animal and a foreign substance injected into the organ, wherein the organ has an injection scar immediately above or near the region into which the foreign substance was injected. is the graft material in which is formed.
  • the organ has a foreign substance injected therein and an injection scar, and the gel covers at least a part of the injection scar, and the gel covers the entire injection scar. More preferably, the gel covers the entire surface of the organ.
  • Specific examples and preferred examples of the foreign matter are as described above.
  • the injection marks are as described above.
  • a capillary tube with a sharply polished tip that expresses green fluorescent protein (GFP) and into which GFP-expressing human mesenchymal stem cells that emit green fluorescence were aspirated was prepared.
  • GFP green fluorescent protein
  • gelatin powder manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • HEPES-buffered TCM199 medium of pH 7.2 was dissolved in HEPES-buffered TCM199 medium of pH 7.2 to prepare a gelatin solution with a concentration of 10-15% (w/v).
  • Example 1 Metanephros excised from pig fetuses obtained by caesarean section from DPF-controlled wild-type sows are placed in the gelatin solution to expose the surface immediately above and near the nephrogenic zone.
  • the metanephros were semi-embedded in the gelatin gel as described above (that is, there were portions of the metanephros that were not in contact with the gel, while portions of the metanephros that were in contact with the gel were embedded).
  • FIG. 2 shows photomicrographs of metanephros positioned by semi-embedding in gelatin gel.
  • the metanephroi semi-embedded in the gelatin gel was placed on a 35 mm dish (manufactured by Iwaki) so that the surface immediately above and near the renal cortical formation area was exposed (the above positioned to face the top side away from the dish).
  • GFP-expressing human mesenchymal stem cells were aspirated into one or two locations on the surface of the semi-embedded metanephros subcapsular renal corticogenesis region directly above or near the renal corticogenesis region; Puncture was performed to the shortest distance, and the human mesenchymal stem cells were injected as a foreign object into the renal cortical formation region.
  • the capillaries are widely spread over the mesenchymal region.
  • the human mesenchymal stem cells were injected into a wide range of the mesenchymal region by puncturing the shortest distance to the tip of the capillary tube and moving the tip of the capillary tube.
  • FIG. 3 is a photomicrograph showing the results of injecting GFP-expressing cells into one of the renal corticogenic regions under the capsule of the semi-embedded metanephros.
  • FIG. 4 shows photomicrographs showing the results of injection of GFP-expressing cells into two sites in the subcapsular renal corticogenic region of the semi-embedded metanephros.
  • FIG. 5 is a photomicrograph showing the results of extensive injection of GFP-expressing cells into the metanephric mesenchymal region. As is clear from the results shown in FIGS.
  • the foreign substance (GFP-expressing cells) could be easily injected into the desired injection target region of the organ excised from the animal body.
  • it can be said that it is possible to provide a transplant material into which a foreign substance is injected, in which damage is reduced by injecting the foreign substance at the shortest distance from the surface immediately above and near the injection target region.
  • Example 2 Metanephros excised from pig fetuses obtained by caesarean section from DPF-controlled wild-type sows were put into the above-mentioned gelatin solution, and the whole metanephros were coated with gelatin gel. The metanephros completely covered with the gelatin gel were immersed in 4.5 ml of an equilibrium solution containing 7.5% by weight EG and 7.5% by weight DMSO as cryoprotectants at room temperature (25 to 27°C) for 25 minutes. processed.
  • the metanephros after the pretreatment were immersed in a vitrification solution containing 15% by mass EG, 15% by mass DMSO, and 0.5M sucrose, which is twice the amount of the above, for 30 minutes at room temperature.
  • the metanephros were placed on a support "Cryotop" (manufactured by Kitasato Biopharma), placed in liquid nitrogen, cooled very rapidly, and vitrified and frozen.
  • the metanephros after vitrification freezing were transferred to a vitrified cryopreservation container, frozen in liquid nitrogen for 10 days, and then thawed by immersion in a 37° C. thawing solution containing 1M sucrose for 1 minute.
  • the plate was washed twice with a diluent containing 0.5 M sucrose for 3 minutes at room temperature and twice with a washing solution for 5 minutes to dilute and remove the cryoprotectant.
  • the capillaries into which GFP-expressing human mesenchymal stem cells were aspirated at one or two points on the surface directly above or near the subcapsular renal cortical formation area of the fully covered metanephros were inserted into the renal cortical formation area.
  • the human mesenchymal stem cells were injected into the renal cortical formation region as foreign substances.
  • the capillaries are widely spread to the mesenchymal region with the shortest distance.
  • the human mesenchymal stem cells were injected into a wide range of the mesenchymal region by moving the tip of the capillary tube.
  • Example 3 Metanephros excised from 35-day-old fetal pigs obtained by caesarean section from DPF-controlled wild-type sows were put into the gelatin solution, and in the same manner as in Example 1, the nephrogenic zone was formed.
  • the metanephros are semi-embedded in the gelatin gel so that the surfaces immediately above and near the metanephros are exposed (i.e., there is a portion of the metanephros that is not in contact with the gel, while the metanephros is in contact with the gel. The part where it is embedded).
  • the metanephroi semi-embedded in the gelatin gel was placed on a 35 mm dish (manufactured by Iwaki) so that the surface immediately above and near the renal cortical formation area was exposed (the dish positioned so that it faces the upper side opposite to the 0.1 ⁇ L of the edible green dye solution was aspirated to one point on the surface of the semi-embedded metanephroid subcapsular renal corticogenesis area directly above or near the renal corticogenesis area. 0.1 ⁇ L of the edible green dye solution was injected as a foreign substance into the renal cortex formation region.
  • Example 4 The present inventors have previously demonstrated that embryos (host embryos) derived from pigs that have the Pdx1-Hes1 gene and in which pancreatic formation is inhibited (deficiency of ⁇ cells in the pancreas) were transformed into embryonic cells derived from normal pigs (donor embryonic cells). ) and allowed to grow in foster mothers to create chimeric pigs with donor-derived pancreases (Matsunari et al., PNAS 110:4557-4562 (2013)). By naturally mating male chimeric pigs with wild-type female pigs, it is possible to obtain neonates in which pancreatic formation is inhibited ( ⁇ cells are deficient in the pancreas) at a constant rate.
  • Newborn pigs with inhibited pancreas formation ( ⁇ -cell deficiency in the pancreas) used in Example 4 were obtained by natural mating with such chimeric pigs (Japanese Patent Application Laid-Open No. 2019-62929).
  • FIG. 8 is a diagram showing pancreatic tissue after laparotomy of the above-mentioned neonatal swine in which pancreatic formation was inhibited ( ⁇ cells are absent in the pancreas).
  • the scale bar is 1 cm.
  • a plurality of fragmentary pancreatic tissues are formed.
  • the pancreatic tissue is very small, it is difficult to inject a foreign substance through a capillary tube while attached to the pig body.
  • pancreatic tissue fragments (arrowheads) were cut out, semi-embedded in gelatin gel in the same manner as in Example 1, and positioned on a 35 mm dish (manufactured by Iwaki).
  • Each of the plurality of semi-embedded pancreatic tissues was punctured with a capillary tube into which human iPS cell-derived pancreatic progenitor cells were aspirated, and the human iPS cell-derived pancreatic progenitor cells were injected as a foreign substance.
  • FIG. 9 is a diagram showing a human iPS cell-derived pancreatic progenitor cell mass injected into one of the plurality of pancreatic tissue fragments shown in FIG.
  • the scale bar is 50 ⁇ m.
  • FIG. 9a is a diagram showing the positions of cell nuclei in the entire tissue and the results of fluorescent immunostaining of amylase by DAPI (4',6-diamidino-2-phenylindole) staining.
  • DAPI 4',6-diamidino-2-phenylindole
  • FIG. 9b shows the results of fluorescence immunostaining of a PDX1-expressing green fluorescence-labeled secondary antibody.
  • FIG. 9c is a figure which shows the result of fluorescent immunostaining of human cell nuclei expressing HNA (human nuclear antigen).
  • HNA human nuclear antigen
  • FIG. 9e is a diagram showing the confirmation results of pancreatic cell structure and pancreatic tissue structure by HE (hematoxylin and eosin) staining.
  • HE hematoxylin and eosin

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JP2013526297A (ja) * 2010-05-07 2013-06-24 ユニバーシティー オブ ノース カロライナ アット チャペル ヒル 実質組織からの細胞の移植方法
JP2016515835A (ja) * 2013-04-12 2016-06-02 サベリオ ラフランチェスカ， 移植のための臓器の改良
WO2020262642A1 (ja) * 2019-06-28 2020-12-30 持田製薬株式会社 化学架橋アルギン酸を用いた移植用デバイス

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* Cited by examiner, † Cited by third party
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JP2013526297A (ja) * 2010-05-07 2013-06-24 ユニバーシティー オブ ノース カロライナ アット チャペル ヒル 実質組織からの細胞の移植方法
JP2016515835A (ja) * 2013-04-12 2016-06-02 サベリオ ラフランチェスカ， 移植のための臓器の改良
WO2020262642A1 (ja) * 2019-06-28 2020-12-30 持田製薬株式会社 化学架橋アルギン酸を用いた移植用デバイス

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