WO2013176239A1 - Substance de type gel réticulé avec un oligosaccharide oxydé - Google Patents

Substance de type gel réticulé avec un oligosaccharide oxydé Download PDF

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WO2013176239A1
WO2013176239A1 PCT/JP2013/064433 JP2013064433W WO2013176239A1 WO 2013176239 A1 WO2013176239 A1 WO 2013176239A1 JP 2013064433 W JP2013064433 W JP 2013064433W WO 2013176239 A1 WO2013176239 A1 WO 2013176239A1
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crosslinkable composition
oligosaccharide
oxide
chitosan
cells
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Japanese (ja)
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渉 上村
博之 小山
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University of Tokyo NUC
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University of Tokyo NUC
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    • 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
    • A61L27/14—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
    • A61L27/50—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
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    • 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
    • A61L27/58—Materials at least partially resorbable by the body
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    • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
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    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
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    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
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    • C08H1/00—Macromolecular products derived from proteins
    • C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
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    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
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    • C08L3/00—Compositions of starch, amylose or amylopectin or of their derivatives or degradation products
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    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
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    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
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    • C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
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    • C12N2533/78—Cellulose

Definitions

  • the present invention relates to a gel material cross-linked with an oligosaccharide oxide, a cross-linking agent composed of an oligosaccharide oxide used for the preparation of the gel material, a cell culture system and a transplant system using the gel material.
  • Non-patent Document 1 a technique for culturing and stacking cells on a cell sheet.
  • Non-patent Document 1 a technique for culturing and stacking cells on a cell sheet.
  • Non-patent Document 1 a technique for culturing and stacking cells on a cell sheet.
  • the exchange of oxygen and substances by passive diffusion is limited to several hundred microns, so even if a large regenerated tissue can be created, if the vascular system is not introduced, the regenerated tissue Even if transplanted to the host, the center is necrotic.
  • Spheroids have attracted attention as microregenerative tissue units.
  • Spheroids are those in which a large number of cells aggregate to form a spherical mass, and spheroid culture can maintain the function of cells for a long period of time compared to conventional monolayer culture, and can be said to be a culture method closer to a living body. Therefore, a technique for transplanting spheroids to a host, inducing (organizing) blood vessels to the spheroids, and constructing the organized spheroids three-dimensionally (Patent Document 1, Non-Patent Documents 4 to 5) has been studied.
  • angiogenesis cannot be successfully induced because there is no support for maintaining the stretch and structure of blood vessels around the spheroids. Therefore, a scaffold material that induces angiogenesis is required.
  • the present invention is a scaffold for constructing a large regenerative tissue. (1) It has a high cell affinity, (2) it has a stable structure even in a physiological environment, and (3) it is rapidly produced after transplantation. (4) can be stably carried inside without leaking the blood vessel inducing factor, and (5) has transparency enough to observe the state of cells cultured in the gel over time, ( 6) It aims at providing the material which has the characteristics which can adjust the intensity
  • the present inventors have conceived that collagen is prevented from forming an aggregate by forming a chemical crosslink in order to stably maintain a collagen gel under conditions suitable for cell culture.
  • water-soluble carbodiimide, glutaraldehyde, and the like are known as water-soluble cross-linking agents that form chemical cross-links.
  • it is not appropriate to use these because of problems of irritation to cells and toxicity.
  • a colorless and transparent gel can be obtained when an oligosaccharide oxide is used as a crosslinking agent.
  • an oxidation reaction in an aqueous system is preferable because the solubility of oligosaccharides in an organic solvent is low, and in the oxidation reaction using lead tetraacetate, the toxicity of residual lead has been a concern. Accordingly, the present inventors have established a technique that allows an iodine compound to be easily removed by selecting an oxidation reaction with periodic acid and adding an alcohol after the oxidation reaction.
  • the present invention (1) a crosslinkable composition comprising an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain; (2) The crosslinkable composition according to (1), wherein the oligosaccharide oxide is generated from periodate oxidation of an oligosaccharide, (3) The crosslinkable composition according to (1) or (2), wherein the polypeptide is selected from collagen, atelocollagen, gelatin, derivatives thereof or mixtures thereof, (4) The polysaccharide is deacetylated chitin or chitosan, succinylated chitosan, glycerylated chitosan, carboxymethylated chitosan, hydroxybutyl-hydroxypropylated chitosan, aminated dextran, aminated starch, aminoalkylated pullulan, amino The crosslinkable composition according to (1) or (2), which is selected from hydrogenated hyaluronic
  • (6) A crosslinked product obtained from the crosslinkable composition according to any one of (1) to (5), (7) The crosslinked product according to (6), wherein an aldehyde group of the oligosaccharide oxide and an amino group of the polypeptide or polysaccharide are covalently bonded, (8) The crosslinked product according to (6) or (7), which is in a gelled state, (9) The cross-linked product according to any one of (6) to (8), which contains an angiogenic factor, (10) The crosslinked body according to any one of (6) to (9), comprising a cell or a cell tissue, (11) The crosslinked product according to (10), which contains a cell culture medium, (12) A cell culture system using the cross-linked product according to any one of (6) to (11), (13) A system for transplanting the crosslinked body according to any one of (6) to (11) into the body of a subject, (14) A system for transplanting cultured cells obtained by the cell culture system according to (12) into the body of a subject, (15) (
  • a polysaccharide having an amino group in the side chain is deacetylated chitin or chitosan, succinylated chitosan, glycerylated chitosan, carboxymethylated chitosan, hydroxybutyl-hydroxypropylated chitosan, aminated dextran, aminated starch,
  • Polysaccharides with amino groups in the side chains such as collagen or polysaccharides with amino groups such as chitosan derivatives are chemically cross-linked with oligosaccharide oxides to suppress the formation of aggregates and prepare transparent and uniform gels It became possible to do.
  • a transparent and uniform gel allows easy observation of cells and progresses promptly with no degradation in vivo.
  • chemical cross-linking limits and suppresses structural changes such as gel shrinkage, swelling, and dissolution under physiological conditions, and maintains an appropriate cross-link density that provides sufficient strength and decomposition rate as a cell scaffolding material. be able to.
  • oligosaccharide oxides are less irritating and toxic than lower aldehydes, and are safe crosslinking agents for cells and as a transplant material.
  • the aldehyde group of the oligosaccharide oxide can carry angiogenic factors and the like by covalent bonds.
  • a carboxylic acid generated by oxidation of an aldehyde can also carry a basic factor such as bFGF by electrostatic interaction. Therefore, since the gel material of the present invention hardly releases angiogenic factors or the like outside in a living body, it becomes possible to induce angiogenesis efficiently.
  • Crosslinking agent One form of this invention is a crosslinking agent which consists of an oligosaccharide oxide which has an aldehyde group.
  • oligosaccharide refers to a compound having a molecular weight of about 300 to 3,000 among compounds in which monosaccharides are bonded by a glycosidic bond.
  • examples of oligosaccharides that can be used in the present invention include disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose; trisaccharides such as raffinose, panose, maltotriose, melezitose, and gentianose; Cyclic dextrins such as cyclodextrin and cycloawaodoline, etc .; fructooligosaccharides and galactooligosaccharides that are monosaccharide polymers; mannan oligosaccharides and xylooligosaccharides as degradation products of polysaccharides; Extract; selected from the group consisting of these derivatives and mixtures of two or more thereof.
  • the oligosaccharide oxide having an aldehyde group refers to one in which the vicinal diol group of the oligosaccharide is partially dissociated and oxidized to an aldehyde group.
  • an oligosaccharide having a vicinal diol group oxidized to an aldehyde group can be obtained by various methods.
  • an oligosaccharide oxide having an aldehyde group an oligosaccharide periodate is used as an oligosaccharide oxide having an aldehyde group.
  • an oxide generated from oxidation is particularly preferred.
  • a method for producing oligosaccharide by periodate oxidation will be described.
  • Oligosaccharide is dissolved in a basic aqueous solution such as water or sodium hydroxide solution, and periodate oxidation reaction of oligosaccharide is performed by adding sodium periodate under ice cooling.
  • the pH of the reaction solution is preferably 7 or less, particularly preferably around 5-6.
  • the ratio of sodium periodate to oligosaccharide sugar units is preferably 1: 2 or less.
  • the reaction solvent for example, various solvents such as water, water / ethanol mixed solvent, water / DMF mixed solvent and the like can be used, but water is preferably used.
  • the reaction time is appropriately determined depending on the type of sugar, but it can usually be carried out in 5 minutes to 96 hours, particularly preferably 1 to 60 hours.
  • a polar solvent is added to precipitate and remove the iodine compound.
  • the polar solvent is preferably a low boiling point solvent because it must be removed under reduced pressure during the preparation of the oligosaccharide oxide.
  • the cross-linking agent comprising an oligosaccharide oxide having an aldehyde group of the present invention can be suitably used for a cross-linking reaction with a polypeptide or polysaccharide having an amino group in the side chain as described later.
  • Crosslinkable composition Another form of the present invention is a crosslinkable composition comprising a crosslinking agent comprising an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain.
  • Non-limiting examples of polypeptides having an amino group in the side chain include naturally occurring polypeptides, artificially synthesized polypeptides that mimic or improve their functions, genetically modified proteins, and the like.
  • Naturally-derived polypeptides include silkworm-derived fibroin, sericin, and the like, and naturally-derived extracellular matrix components such as collagen, atelocollagen, gelatin, proteoglycan, fibronectin, laminin, elastin, entactin and their derivatives. It is done.
  • Artificially synthesized polypeptides and genetically engineered proteins include, but are not limited to, dehydrated condensates of oligopeptides such as Poly (PHG), Poly (PHG / QGIA), Poly (PHG / RGD), Poly (PHG / YIGSR / IKVAV) And the like, such as “Pronectin F” and “Pronectin L” manufactured by Sanyo Chemical Industries, “RetroNectin” manufactured by Takara Shuzo, and the like.
  • the polypeptide having an amino group in the side chain is preferably collagen, atelocollagen, gelatin, fibronectin, Poly (PHG / RGD), derivatives thereof, or a mixture thereof, more preferably collagen, atelocollagen.
  • Gelatin, fibronectin, derivatives thereof and mixtures thereof are particularly preferred.
  • Collagen is one of the proteins that make up the dermis, ligaments, tendons, bones, cartilage, etc., and is the element that makes up the extracellular matrix.
  • the peptide chain of collagen protein has a primary structure “-(glycine)-(amino acid X)-(amino acid Y)-” and glycine repeats every three residues. In many types of collagen, three of these peptide chains gather to form a helical structure and are called tropocollagen.
  • one peptide chain of type I collagen has a sequence that repeats 1014 amino acid residues and has a molecular weight of about 100,000. It is known that there are more than 30 types of human collagen.
  • type I collagen is the main component in dermis, ligaments, tendons, bones, etc.
  • type II collagen is the main component in articular cartilage.
  • type IV collagen is mainly contained in the basement membrane which is the lining structure of all epithelial tissues. The most abundant in the body is type I collagen.
  • collagen Preferably it is soluble collagen. More preferred is collagen that is soluble in water.
  • the collagen may be peptided collagen (collagen fragment).
  • Atelocollagen is collagen obtained by removing telopeptides present at both ends of collagen molecules by enzymatic treatment, and is soluble in water. Atelocollagen may be peptideized.
  • Gelatin means the collagen extracted into water by heating with water for a long time.
  • the gelatin may be peptideized.
  • the peptide of gelatin is the same as that of collagen.
  • Gelatin is a heat-denatured substance of collagen, so its amino acid composition is almost the same as collagen. Although it depends on the origin, there are about 12% acidic groups, and about one third of them are amidated.
  • Gelatin includes acid-treated gelatin, alkali-treated gelatin, and the like depending on the pretreatment step of water extraction. Acid-treated gelatin is treated with inorganic acids such as hydrochloric acid and sulfuric acid for several tens of hours to several days, and the deamidation rate is low, whereas alkali-treated gelatin is almost 100% deamidated in the limestone process for 2 to 3 months. Has been.
  • the polypeptide having an amino group in the side chain is preferably selected from collagen, atelocollagen, gelatin, derivatives thereof or a mixture thereof.
  • the polysaccharide is a sugar in which a monosaccharide molecule is polymerized by glycosidic bonds.
  • the polysaccharide having an amino group in the side chain in the present invention include deacetylated chitin and chitosan (eg, Daichitosan VL manufactured by Dainichi Seika), succinylated chitosan, glycerylated chitosan, carboxymethylated chitosan, hydroxybutyl-hydroxy
  • Examples include chitosan derivatives such as propylated chitosan, derivatives of natural and semi-artificial polysaccharides such as aminated dextran, aminated starch, aminoalkylated pullulan, aminated hyaluronic acid, aminated chondroitin sulfate, and aminated cellulose.
  • the concentration of the polypeptide or polysaccharide or oligosaccharide oxide having an amino group in the side chain depends on the type thereof, taking into account the reactivity or viscosity of the composition solution. It can be set appropriately.
  • the concentration is preferably 0.3% or more, and in the case of gelatin, the concentration is preferably 5% or more.
  • the concentration of the oligosaccharide oxide is usually 0.01% or more, preferably 0.05% or more in the case of raffinose oxide, for example.
  • the crosslinkable composition of the present invention includes other additives such as a crosslinking agent, a reaction accelerator, a retarder, a dispersant (anti-aggregation agent), and a reinforcing material (fiber, filament, whisker, particle, microgel). , Droplets and the like can be contained.
  • Crosslinked body The further form of this invention is a crosslinked body obtained from the said crosslinkable composition.
  • the cross-linked product of the present invention refers to an oligosaccharide oxide-polypeptide complex in which an aldehyde group of an oligosaccharide oxide and an amino group of a polypeptide or polysaccharide are covalently bonded (-polypeptide-oligosaccharide oxide-poly Peptide-) and oligosaccharide oxide-polysaccharide complex (polysaccharide-oligosaccharide oxide-polysaccharide).
  • the covalent bond between the oligosaccharide oxide and the polypeptide is usually a covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the side chain of the polypeptide, and the amino group of the oligosaccharide oxide and the polypeptide. It consists of a mixture of covalent bonds of the terminal amino group, but it may consist only of a covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the side chain of the polypeptide, or it may consist of the aldehyde group and the polysaccharide of the oligosaccharide oxide. It may consist only of a covalent bond of the amino group at the amino terminus of the peptide.
  • the covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide means that the covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide is present in the crosslinked product. It means that there is at least one.
  • the crosslinked product preferably contains an aldehyde group of an oligosaccharide oxide that does not participate in a covalent bond and an amino group of a polypeptide or a polysaccharide.
  • a functional group for interacting with these physiologically active substances may be required.
  • bFGF whose charge is positively biased has an electrostatic interaction with the aldehyde group of the oligosaccharide oxide.
  • the side chain structure of the polypeptide is destroyed by the formation of a covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide. It is desirable to minimize structural changes.
  • a preferred embodiment of the present invention is a state where the crosslinked body is gelled (hereinafter also simply referred to as “gel”).
  • the gel means that the fluidity is lost in the crosslinked body (crosslinkable composition).
  • the cross-linked product in the present invention refers to an oligosaccharide oxide-polypeptide complex, or an aldehyde group of an oligosaccharide oxide and a multi-valent compound by covalently bonding an aldehyde group of an oligosaccharide oxide and an amino group of a polypeptide.
  • the saccharide amino group is covalently bonded to form an oligosaccharide oxide-polysaccharide complex, and the crosslinked product of the present invention includes a case where it is not gelled.
  • the crosslinkable composition By making the crosslinkable composition into a gel, it is possible to obtain a large regenerative tissue scaffolding material having a stable structure, which is particularly preferable.
  • the crosslinked body of the present invention can contain a physiologically active substance.
  • physiologically active substances include growth factors and cytokines, antibodies and the like.
  • growth factors and cytokines include vascular endothelial growth factor (VEGF), acidic fibroblast growth factor (a-FGF), basic fibroblast growth factor.
  • Base fibroblast growth factor b-FGF
  • platelet-derived endothelial cell growth factor PD-ECGF
  • transforming growth factor transforming growth factor, TGF- ⁇ , TGF- ⁇
  • tumor Necrosis factor angiogenin tumor necrosis factor- ⁇ , TNF- ⁇
  • HGF hepatocyte growth factor
  • GM-CSF granulocyte / macrophage-colony stimulating factor
  • insulin Insulin-like growth factor (IGF-I, IGF-II), Ellis Lopoietin (EPO), thrombopoietin (TPO), epidermal growth factor (EGF), heparin binding growth factor (hbgf), nerve growth factor (NGF), muscle formation factor (Muscle morphogenic factor: MMF), bone morphogenetic protein (BMP) and the like.
  • Non-limiting examples of antibodies include antibodies that can stimulate cells such as anti-CD3 antibodies and anti-CD28 antibodies.
  • basic fibroblast growth factor b-FGF
  • VEGF vascular endothelial growth factor
  • PD-ECGF platelet-derived endothelial cell growth factor
  • HGF hepatocyte growth factor
  • GM-CSF granulocyte / macrophage-colony stimulating factor
  • the physiologically active substance may be a cell that secretes a physiologically active substance.
  • the crosslinked body of this invention can contain a cell.
  • the cell type is not limited, but preferably chondrocytes; fibrochondrocytes; bone cells; osteoblasts; osteoclasts; synovial cells; bone marrow cells; mesenchymal cells; Peripheral blood progenitor cells; insulin-producing cells; antibody-producing cells such as B cells or hybridomas; hormone-producing cells; mast cells; chemotransmitter-producing cells (immune cells); genetically modified cells; stem cells (ES cells, EG cells) , IPS cells, and adult (tissue) stem cells); cells that produce bioactive factors of the liver, nerves, thyroid, thymus, adrenal medulla, adrenal cortex, kidney or gastrointestinal tract; and combinations of these cells with other cells Is mentioned.
  • the cells may be in aggregated form (spheroids etc.) or part of tissue.
  • the origin of the cells is not limited, but preferably mammals, more preferably pets such as dogs and cats; humans; livestock animals such as cows, pigs, horses, sheep, goats; monkeys, rabbits, mice, rats And the like, particularly preferably humans.
  • the cells are preferably derived from a transplant subject.
  • the crosslinked body of this invention can contain a culture solution further.
  • the type of the culture solution is variously prepared depending on the type, state, and purpose of the cell. Examples include, but are not limited to, pure water, physiological saline, and body fluid of the host.
  • the crosslinked product of the present invention can be produced by preparing a crosslinkable composition containing an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain, and allowing the crosslinking reaction to proceed. it can. Specifically, a phosphate buffer solution of a polypeptide or polysaccharide having an amino group in the side chain is prepared, pH is adjusted, and an oligosaccharide oxide having an aldehyde group is added thereto to obtain a crosslinkable composition. The reaction is allowed to proceed with stirring for a predetermined time. If the stirring is continued, the solution thickens, and if the stirring is further performed, the fluidity is lost and a gel can be formed.
  • the chemical bonding reaction between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide is considered to be significantly accelerated under alkaline conditions. It is adjusted to 7.0 or more, more preferably 7.6 or more.
  • the reaction temperature can be set according to the type of polypeptide used, but the higher the reaction temperature, the shorter the gelation time.
  • the concentration of the oligosaccharide oxide can be set according to the type of polypeptide used, etc., but the gelation time can be shortened as the concentration of the oligosaccharide oxide increases.
  • Cell culture system The further form of this invention is related with the cell culture system using the bridge
  • an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain are mixed to prepare a crosslinkable composition, its pH is adjusted, and a gel is prepared. Progress. Then, before the gelation progresses, during the progress, and at any time after the completion, the cells and the culture solution are added to culture the cells.
  • the cells when the crosslinkable composition is thickened, the cells are added to and mixed with this, and the cell mixture is injected into a petri dish or the like, and left to stand for a predetermined temperature and time for gelation.
  • the cells can be cultured by coating a disc-shaped gel having a predetermined thickness and diameter with a culture solution.
  • spheroids obtained by seeding cells in a separate step and culturing for a predetermined period are added and mixed, and allowed to stand at a predetermined temperature and time.
  • the cells can be cultured by coating a disc-shaped gel having a predetermined thickness and diameter with a culture solution.
  • Examples of the culture solution include pure water, physiological saline, phosphate buffer, Eagle basal medium (Basal Medium Eagle: BME), Dulbecco's Modified eagle's Medium: DMEM, DMEM: F12 medium, Glasgow Minimum Essential Medium (GMEM), Grace's Insect Medium, Ham's Medium, Iscove's Dulbecco's Medium : IMDM), L-15 (Leibovitz) medium (L-15 (Leibovitz) Medium), McCoy's 5A medium (Mc oy's 5A Medium, Minimum Essential Medium-Eagle (MEM Eagle / E-MEM), 199 Medium (Medium 199), NCTC-109 Medium (NCTC-109 Medium), Richter CM (Richter's CM), RPMI 1640 Medium (RPMI 1640 Medium), insect medium, Weymouth's Medium, William's Medium, body fluid, and the like, but are not limited thereto.
  • Basal Medium Eagle: BME Eagle basal medium
  • the cells, spheroids, and cell culture solution described above can be used.
  • the aforementioned physiologically active substance can be appropriately added to the gel.
  • a reinforcing material can be contained in a gel.
  • a flow system can be incorporated that injects the culture medium into the gel as described in the implantation system below.
  • the cell culture system of the present invention is highly transparent and suitable for cell observation. Therefore, in addition to the components and factors necessary for cell growth assuming transplantation, it is also possible to add other drugs that affect the behavior of cells (good or bad influence) into the gel.
  • the gel of the present invention can exhibit a function as a drug carrier.
  • Transplantation system A further aspect of the present invention is a system and method for transplanting the crosslinked body of the present invention or the cultured cells obtained by the cell culture system of the present invention into the body.
  • cells or tissue are encapsulated in a gel formed from a crosslinkable composition containing an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain. It is a transplant material for constructing a regenerative tissue that is buried and used to be transplanted into the body of a subject.
  • a cross-linked body containing cells a cross-linked body containing cell tissues such as spheroids (for example, a spheroid surrounded by a cross-linked body that is a scaffold material), a plurality of cell tissues
  • a cross-linked body encapsulated for example, a plurality of cellular tissues covered with a cross-linked body in a two-dimensional or three-dimensional manner
  • a cross-linked body not containing cells can be transplanted into a host mother bed.
  • the cross-linked body used in the transplantation system of the present invention has a matrix structure with appropriate gaps, and can maintain a stable structure in vivo because of its good adhesion to vascular wall constituent cells, Since it is decomposed by angiogenic protease, a space for blood vessel growth can be secured, and angiogenesis can be efficiently induced.
  • the crosslinked body preferably contains an angiogenic factor, and particularly preferably bound to a polypeptide in the crosslinked body.
  • the blood vessel can be induced inside the crosslinked body without releasing the angiogenic factor outside the crosslinked body.
  • the transplant system of this invention can also be equipped with the means to supply a culture solution from the exterior to the bridge
  • the culture solution can be injected under pressure into the center (in some cases, a plurality of points) of the gel by a pump or the like.
  • the transplanted cultured cells will express the function for a long time in the human body and become an artificial organ as it is.
  • the expression level of the function can be controlled by the size and shape of the cultured cells, or both.
  • hepatocytes are used as cultured cells, it shows a role as an artificial liver, such as liver enzyme deficiency, hemophilia, coagulopathy, liver failure, fulminant hepatitis, chronic hepatitis, cirrhosis, treatment of patients with hepatectomy, It is used for the essential treatment of each disease mentioned in infectious diseases, etc., or for the purpose of assisting liver function.
  • transplanted tissues include liver, heart, vascular tissue, kidney, lung, pancreas (islet), gastrointestinal tissue, eye (cornea), brain, nerve tissue, bone marrow, bone, tooth, cartilage, and skin. (Hair) and muscles.
  • the transplant system of the present invention becomes a cultured cell transplant animal.
  • the expression level of the function can be controlled by the size and shape of the cultured cells.
  • animals used herein include rats, mice, guinea pigs, marmosets, rabbits, dogs, pigs, chimpanzees, and immunodeficient animals thereof, but are not particularly limited.
  • Such an islet transplanted animal is used for the purpose of, for example, a pancreatic function evaluation system in which a test substance is administered to the pancreatic islet transplanted animal and the influence of the test substance on pancreatic function is determined. Is not to be done.
  • Example 1 A 0.8% atelocollagen (Nitta gelatin, collagen BM, derived from pig, Type-I) phosphate buffer solution (pH 7.6) was prepared on gelled ice (4 ° C.) of the collagen solution. This solution was dispensed into an Eppendorf tube in an amount of 0.3 mL, 0.08 mL of the aqueous solution of sugar oxide (3%) prepared in the conditions (1) and (2) of Synthesis Examples 1 to 5 was added, and stirred. Allowed to stand at ° C. Periodically, the solution was contacted with a glass rod, and the time required from the addition of oxidized sugar to the loss of fluidity was defined as the gel time. For comparison, an experiment was also performed on sugars that had not been subjected to periodate oxidation treatment.
  • the collagen solution prepared at a neutral pH is fibrillated and gelled due to the influence of hydrophobic interaction and the like as the temperature rises.
  • the collagen solution to which raffinose was added turned into a fibrillated and white turbid gel at about 20 minutes at 37 ° C.
  • raffinose oxide was added to the collagen solution, fibrosis was not observed even at 37 ° C., and gelled in about 1 hour while being colorless and transparent. This is probably because the cross-linking of collagen molecules with raffinose oxide limited the association / aggregation of collagens, so that fibrosis was suppressed and a colorless and transparent gel was obtained.
  • the preparation temperature was 4 ° C.
  • the gelation time was as slow as 7 hours, and as the temperature increased, the gelation time tended to be shortened.
  • Example 2 Gelation of gelatin solution (1) Gelation of porcine-derived gelatin with sugar oxide An aqueous solution of gelatin (077-03155 manufactured by Wako Pure Chemical Industries, Ltd., derived from pig) was prepared at 40 ° C., and a phosphate buffer solution was added, and finally 15% A gelatin solution (pH 7.6) was prepared. To 0.3 mL of this solution, 0.06 mL of a 3% sugar oxide aqueous solution was added and stirred, and allowed to stand at 40 ° C.
  • porcine-derived gelatin forms a reversible physical gel at room temperature
  • the gel preparation test was performed at 40 ° C. when the gelatin solution melts. Gelation was observed when raffinose oxide and sucrose oxide were added, but no gelation was observed with glucose oxide, maltose oxide, or flucotose oxide. At 40 ° C., it is expected that the thermal degradation of collagen and the cross-linking reaction by sugar oxide proceed at the same time. Therefore, it is considered that a quick cross-linking reaction is necessary to prepare the gel. Although all of the above sugar oxides are considered to form a crosslinked structure, raffinose oxide and sucrose oxide were recognized to have an ability to gel efficiently.
  • Example 3 Cell culture test At 4 ° C., 0.8% atelocollagen (Nitta gelatin, collagen BM) phosphate buffer solution (pH 7.6, 1 mL) and 3% oxidized raffinose aqueous solution (oxidation conditions of Synthesis Example 1 (1)) (0. 2 mL) was added and stirred. This mixed solution was allowed to stand at 18 ° C. for 4 hours. When the viscosity was increased, 120 ⁇ L was collected, and each cell was added and mixed. A hole ( ⁇ 8 mm) was made in a vinyl sheet having a thickness of 2 mm that was in close contact with the petri dish, and the cell mixture was poured into it, and left to stand at 18 ° C. for 1 hour to cause gelation. Disc-shaped gel (thickness 2 mm, ⁇ 8 mm) was coated with a culture solution and cultured.
  • atelocollagen Nita gelatin, collagen BM
  • HUVEC normal human umbilical vein endothelial cells
  • HUVEC Angio-Proteomie
  • Hepatocytes Hepatocytes were collected from the liver of Wistar rats (300 g) by the collagenase perfusion method. Primer-Surfae 24-well plates (Sumitomo Bakelite) were seeded with hepatocytes (8 ⁇ 10 4 cells / well), and after culturing for 4 days, the spheroids were added to 24 wells in a disc-shaped gel (120 ⁇ L). The culture solution was prepared according to the method of Yoshizato et al. FIG. 2 shows the results of observation with a microscope on the third day after the spheroids were added to the gel.
  • the obtained gel was highly transparent, and it was possible to observe the three-dimensional growth process of cells inside the gel over time. There was no problem with the cytotoxicity of the gel, and in the case of hepatocyte spheroids, a process in which new cells sprout from the spherical cell mass was observed as indicated by the arrows in FIG.
  • hepatocyte spheroids a process in which new cells sprout from the spherical cell mass was observed as indicated by the arrows in FIG.
  • HIUVEC and cardiomyocytes a cell branching structure was observed the day after in-gel culture, and a three-dimensional network structure was constructed after several days.
  • cardiomyocytes the pulsation gradually shifted from the individual pulsation to the pulsation linked to the individual cells, and finally the entire gel pulsated (the part indicated by the arrow in FIG. 3).
  • Example 4 Implantation experiment to rat 0.3 mL of 0.8% atelocollagen (Nitta gelatin, collagen BM) phosphate buffer solution (pH 7.6) was prepared on ice (4 ° C.). To this solution, 0.06 mL of 3% (50 mM) raffinose oxide aqueous solution was added and stirred. 14 ⁇ L each was dispensed into an ePTFE artificial blood vessel (height 1 mm, inner diameter ⁇ 3 mm) cut into a ring and allowed to stand at 18 ° C. for 4 hours 30 minutes to gel. The gel was coated with a HANKS buffer to suppress the progress of gelation and allowed to stand overnight at 37 ° C. under humidity.
  • FIG. 4 shows a schematic flow diagram of the transplantation experiment.
  • Fig. 5 shows the results of observation with an optical microscope. It can be seen that the transplanted gel (portion surrounded by a yellow broken line in the center of the photograph) was replaced with a granule tissue rich in blood vessels in 5 days. Moreover, the case where only the blood vessel has invaded using the gel as a scaffold is also seen, and the effect of inducing the blood vessel is also expected.

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CN112089826A (zh) * 2019-06-18 2020-12-18 杭州生物医药创新研究中心 一种包含活性生物因子的缓释药物及其制备方法
CN112999418A (zh) * 2021-03-02 2021-06-22 华东数字医学工程研究院 医用水凝胶组合物、医用水凝胶及其制备方法
WO2023020256A1 (fr) * 2021-08-14 2023-02-23 中国海洋大学 Hydrogel de polysaccharide biologique, procédé de préparation de celui-ci et application de celui-ci
CN117414462A (zh) * 2023-10-23 2024-01-19 江苏博朗森思医疗器械有限公司 一种用于弥漫性出血的止血颗粒及其制备方法和应用
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KR20240115552A (ko) * 2023-01-19 2024-07-26 주식회사 엘지화학 폴리머 재료
KR20260051259A (ko) * 2024-10-08 2026-04-16 주식회사 엘지화학 폴리머 재료

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CN111902424A (zh) * 2018-04-27 2020-11-06 凸版印刷株式会社 含细胞外基质的组合物及其制造方法、以及三维组织体、三维组织体形成剂
CN112089826A (zh) * 2019-06-18 2020-12-18 杭州生物医药创新研究中心 一种包含活性生物因子的缓释药物及其制备方法
CN112999418A (zh) * 2021-03-02 2021-06-22 华东数字医学工程研究院 医用水凝胶组合物、医用水凝胶及其制备方法
WO2023020256A1 (fr) * 2021-08-14 2023-02-23 中国海洋大学 Hydrogel de polysaccharide biologique, procédé de préparation de celui-ci et application de celui-ci
CN117414462A (zh) * 2023-10-23 2024-01-19 江苏博朗森思医疗器械有限公司 一种用于弥漫性出血的止血颗粒及其制备方法和应用
CN117414462B (zh) * 2023-10-23 2024-12-27 江苏博朗森思医疗器械有限公司 一种用于弥漫性出血的止血颗粒及其制备方法和应用
CN120168700A (zh) * 2025-03-21 2025-06-20 广东云曌医疗科技有限公司 一种多糖基水凝胶及其制备方法和应用

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