WO2005090550A1 - Method of proliferating stem cell - Google Patents

Method of proliferating stem cell Download PDF

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Publication number
WO2005090550A1
WO2005090550A1 PCT/JP2005/004831 JP2005004831W WO2005090550A1 WO 2005090550 A1 WO2005090550 A1 WO 2005090550A1 JP 2005004831 W JP2005004831 W JP 2005004831W WO 2005090550 A1 WO2005090550 A1 WO 2005090550A1
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WIPO (PCT)
Prior art keywords
stem cells
culture
cells
fibrinogen
culture vessel
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PCT/JP2005/004831
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French (fr)
Japanese (ja)
Inventor
Kotaro Yoshimura
Daisuke Matsumoto
Emiko Aiba
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Biomaster, Inc.
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Application filed by Biomaster, Inc. filed Critical Biomaster, Inc.
Priority to JP2006511226A priority Critical patent/JPWO2005090550A1/en
Publication of WO2005090550A1 publication Critical patent/WO2005090550A1/en

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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
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings

Definitions

  • the present invention relates to regenerative medicine. More particularly, the present invention relates to the culture of stem cells.
  • stem cells there are various types of stem cells, and there are stem cells derived from fertilized eggs, embryonic stem cells, and so-called tissue stem cells. Fertilized eggs are divided into three germ layers, endoderm, mesoderm and ectoderm after gastrulation, and ectoderm-derived cells mainly exist in the brain and include neural stem cells.
  • Mesodermal-derived cells are mainly present in bone marrow, and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like.
  • Endoderm-derived cells are mainly present in organs and include hepatic stem cells, spleen stem cells, and the like.
  • mesenchymal cells including fat cells, bones, ligaments, myocardium, and the like have an important function in forming a body skeleton, and therefore, mesenchymal cells such as populations and tissues containing the cells.
  • Expectations for application to regenerative medicine and transplantation medicine for lineage cells are increasing.
  • bone marrow mesenchymal stem cells are divided into various organs of the mesodermal system, and they are receiving attention as a center of regenerative medicine.
  • the conditions for the separation require a rather special medium known to require a special medium containing a differentiation inducer (for example, dexamethasone) (Non-patent Document 1). 1).
  • Mesenchymal stem cells are a type of tissue stem cells, and are extremely small in nature (present in human bone marrow of newborns at a rate of 1 / 10,000 and then rapidly decreasing. It is said to be 1 / 1,000,000 in the elderly). It only exists and is difficult to separate. It has been reported that mesenchymal stem cells separate from mesodermal cells, and thus their application range is expanding more than before. However, the conditions for such sorting are more specific than those described above. Table of mesenchymal stem cells Surface antigens are CD105 (+), CD73 (+), CD29 (+), CD44 (+), CD14 (-), CD3
  • Non-Patent Document 2
  • Stem cells in adipose have attracted much attention because of their abundant sources and higher prevalence than other tissues (eg, bone marrow). However, there are many unknowns regarding the method of treating stem cells.
  • stem cells cannot be obtained in such a large amount, when a large number of stem cells are required, it is necessary to supply a large number of sources.
  • the source of stem cells is limited, and there is a possibility that side effects may occur due to mixing of multiple strains.
  • Patent document 1 International publication WOOOZ53795 pamphlet
  • Patent document 2 International publication WO03Z022988 pamphlet
  • Patent document 3 International publication WO01Z62901 pamphlet
  • Non-patent document 1 Stem cell 'clone research protocol Nakatsuji ed., Yodosha (2001)
  • Non-patent document 2 Zuk, P.A., et al., Tissue Engineering, Vol. 7, 211-228,
  • Non-Patent Document 3 Zuk, P.A., et al., Molecular Biology of the Cell Vol., 13, 4279-4295, 2002
  • An object of the present invention is to provide a method for amplifying a stem cell (for example, a fibroblast stem cell such as a mesenchymal stem cell (particularly derived from fat)).
  • a stem cell for example, a fibroblast stem cell such as a mesenchymal stem cell (particularly derived from fat)
  • fibrinogen represented by fibrin glue or a derivative thereof, extracellular matrix (eg, collagen (eg, type I, type IV, etc.), laminin And fibronectin), and the use of a culture vessel coated with gelatin and the like, and found that stem cells derived from fat and the like can be efficiently amplified.
  • extracellular matrix eg, collagen (eg, type I, type IV, etc.), laminin And fibronectin
  • the present invention provides the following.
  • a culture vessel containing fibrinogen or a derivative thereof for culturing stem cells (1) A culture vessel containing fibrinogen or a derivative thereof for culturing stem cells.
  • the fibrin glue is soluble fibrinogen (I) contained in the fibrin glue. Factor) is converted into insoluble fibrin (fibrin monomer) by thrombin.Fibrin monomer has the adhesive property that the N-terminus and C-terminus of each other polymerize to form a fibrin polymer and form a fibrin clot. , The culture vessel of item 11.
  • the culture container according to item 1 wherein the material of the culture container includes a material selected from the group consisting of glass, silica, silicon, ceramic, silicon dioxide, plastic, metal, natural polymer and synthetic polymer. .
  • a method comprising:
  • the above culture is performed using a medium selected from the group consisting of DMEM, P199, MEM, Hanks balanced salt solution (HBSS), Ham, sF12, BME, RPMI1640, MCDB104 and MCDB153 (KGM). , Item 22.
  • a medium selected from the group consisting of DMEM, P199, MEM, Hanks balanced salt solution (HBSS), Ham, sF12, BME, RPMI1640, MCDB104 and MCDB153 (KGM).
  • the above culture is performed using corticosteroids, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbic acid and its derivatives, Fate, estrogen and its derivatives, progesterone and its derivatives, androgens and its derivatives, growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum , Heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate, linolenic acid, 3-isobutyl-1-methylxanthine, demethylating agent, histone deacetylating agent, activin, cytokinin, hexamethylene bisa Cetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethylsulfoxide (DMSO), ododeoxyperidine (IdU), hydroxyperrea (HU), cytosine arabin
  • a culture container coated with a tissue adhesive for culturing stem cells (32) A culture container coated with a tissue adhesive for culturing stem cells.
  • tissue adhesive is selected from the group consisting of a cyanoacrylate adhesive, a gelatin aldehyde adhesive, and a fibrin glue adhesive.
  • ASC adipose-derived stem cell
  • the extracellular matrix according to item 34 wherein the extracellular matrix is selected from the group consisting of collagen, laminin, fibronectin, tenascin, sitetactin, hexabrachion, entactin, nightzine, vitronectin, gelatin and proteodarican. Culture vessels.
  • a method comprising:
  • a method comprising:
  • the present invention provides a method for efficiently growing and amplifying stem cells. Such a method has not been achieved so far, and can efficiently amplify useful stem cells in cosmetic surgery and the like, and can use it from a small number of raw materials for many uses.
  • FIG. 1 shows the growth rate of stem cells (derived from fat) on day 2 when various coating dishes were used.
  • Fig. 2 shows human fat cell-derived stem cells on a human PRP-coated 6-wellenplate and an uncoated 6-wellenplate using M199 supplemented with 10% FBS when using a PRP-coated dish.
  • the horizontal axis shows, from left, 0% FBS without coating, 10% FBS without coating, 0% FBS with PRP coating, and 10% FBS with PRP coating.
  • the vertical axis is the number of cells.
  • cell is the broadest term used in the art. It is defined in the same way as the meaning, and is a constituent unit of the tissue of a multicellular organism, which is wrapped in a membrane structure that isolates the outside world, has self-renewal ability inside, and has genetic information and its expression mechanism. In the method of the present invention, any cell can be targeted.
  • the number of "cells” used in the present invention can be counted through a light microscope. When counting through an optical microscope, counting is performed by counting the number of nuclei.
  • the tissue is made into a tissue slice, and extracellular matrix (eg, elastin or collagen) and nuclei derived from cells are stained with a dye by performing hematoxylin-eosin (HE) staining.
  • the tissue section can be examined under an optical microscope, and the number of nuclei per specific area (for example, 200 ⁇ 200 ⁇ m) can be counted as the number of cells.
  • the cells used in the present invention may be stem cells (particularly stem cells derived from adipose tissue) or cells derived from any organism as long as there is a counterpart thereof (eg, metal eel, japonicum, chondrichthyes). And teleost fish, amphibians, reptiles, birds, mammals, etc.).
  • stem cells particularly stem cells derived from adipose tissue
  • cells derived from any organism as long as there is a counterpart thereof (eg, metal eel, japonicum, chondrichthyes).
  • such cells are derived from mammals (e.g., monotremes, marsupials, oligodonts, dermis, winged fins, carnivores, insectivores, longnoses, hoofedids, artiodactyla).
  • cells from primates eg, chimpanzees, macaques, humans
  • cells from humans are used, but are not limited thereto.
  • stem cell has a self-renewal ability, and may have a dangling ability (ie, pluripotency).
  • a stem cell can be, but is not limited to, an embryonic stem (ES) cell or a tissue stem cell (also a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell).
  • ES embryonic stem
  • tissue stem cell also a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell
  • artificially produced cells can also be stem cells, as long as they have the above-mentioned ability.
  • Embryonic stem cells are pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981 and have been applied to the production of knockout mice since 1989. Human embryonic stem cells were established in 1998 and are being used for regenerative medicine.
  • Tissue stem cells unlike embryonic stem cells, are cells in which the direction of differentiation is limited, exist at specific positions in tissues, and have an undifferentiated intracellular structure. Thus, tissue stem cells have a low level of pluripotency. Tissue stem cells have a nuclear Z cytoplasmic ratio Poor intracellular organelles are high. Tissue stem cells are generally pluripotent and maintain their proliferative potential for more than one lifetime in individuals whose cell cycle is slow. As used herein, stem cells may preferably be tissue stem cells, such as mesenchymal stem cells. Embryonic stem cells may also be used depending on the context.
  • stem cells when referring to stem cells, a tissue aggregate containing at least a certain amount of stem cells can be used. Obtaining stem cells can be performed using any method known in the art. Therefore, in the present specification, for example, stem cells obtained by adipose tissue harvesting by collagenase treatment (adipocyte-derived stem cells used in Examples) can be used as stem cells, but are not limited thereto.
  • adipocyte-derived stem cells used in Examples
  • tissue stem cells When classified according to the site of origin, tissue stem cells are classified into, for example, skin system, digestive system, bone marrow system, nervous system and the like.
  • Skin tissue tissue stem cells include epidermal stem cells and hair follicle stem cells.
  • tissue stem cells of the conjugation system include knee (common) stem cells, hepatic stem cells, and the like.
  • myeloid stem cells include hematopoietic stem cells and mesenchymal stem cells.
  • Tissue stem cells of the nervous system include neural stem cells and retinal stem cells.
  • mesenchymal stem cell refers to a stem cell found in mesenchyme. In this specification, it may be abbreviated as MSC.
  • MSC mesenchyme
  • the mesenchyme is formed by a population of free cells with stellate or irregular projections filling the gap between epithelial tissues and the accompanying cell stroma, which are observed at each stage of the development of multicellular animals.
  • Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, skeletal muscle cells, smooth muscle cells (including cardiomyocytes), stromal cells, tendon cells, and adipocytes.
  • Mesenchymal stem cells are used to culture or proliferate the collected bone marrow cells, etc., and to differentiate them into chondrocytes or osteoblasts, or to regenerate bone, cartilage, joints, etc., such as alveolar bone and arthrosis.
  • the demand is large.
  • the demand for mesenchymal stem cells is increasing because they can be divided into blood cells and lymphoid cells.
  • adipose stem cell refers to a stem cell derived from adipose tissue. Some of such methods for separating stem cells are known, and can be separated using, for example, the methods described in Non-Patent Document 1, Patent Documents 13 and the like. What is described in these documents The sections are specifically incorporated herein by reference where relevant. As used herein, adipose stem cells refer to all adipose tissue-derived stem cells, including adipose tissue-derived stem cells obtained by this known separation method.
  • adipose stem cells are described in WOOO / 53795, WO03 / 022988, WO01 / 62901, Zuk, PA, et al., Tissue Engineering, Vol. 7, 211-228, 2001, and Zuk, PA, et al. al., Molecular Biology of the Cell Vol., 13, 4279-4295, 2002, or the like or a modification thereof.
  • the lipoaspirate is sufficiently washed with a physiological saline using a 1-liter separating funnel; (2) the lipoaspirate is sufficiently in the upper layer, and the saline is sufficiently in the lower layer. Check that it has separated, and discard the lower layer.
  • ASC adipocyte-derived stem cells
  • PLA processed lipoaspirate
  • somatic cells are cells other than germ cells such as eggs and sperm, and refer to all cells that do not directly transfer their DNA to the next generation. Somatic cells usually have a limited or pluripotent force or disappear. As used herein, somatic cells may be naturally occurring or genetically modified.
  • the term "divided cells” refers to cells whose functions and morphology are specialized (for example, muscle cells, nerve cells, and the like). No performance or I don't know.
  • the differentiated cells include, for example, epidermal cells, splenic parenchymal cells, splenic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, nerve cells, vascular endothelial cells, dyes Cells, smooth muscle cells, fat cells, bone cells, chondrocytes and the like.
  • the sorting cells may be in the form of a population or a tissue.
  • Fibrinogen or a derivative thereof refers to fibrinogen itself or fibrinogen obtained by decomposing fibrinogen by a protease or other degrading enzyme, or a product obtained by modifying the same (for example, a product obtained by chemical modification). ). Fibrinogen or a derivative thereof typically includes, but is not limited to, fibrin glue.
  • fibrin refers to a residual protein (fibrin monomer) in which thrombin acts on fibrinogen to release fibrinopeptides A and B, and a polymer (fibrin monomer) having the protein as a constituent unit. Polymer). It is a colorless or white, fibrous, amorphous, and elastic solid that, upon blood clotting, entraps blood cells and hardens to form a blood clot. Fibrin formed in plasma and in saline solutions that do not inhibit polymerization precipitates and gels, forming a fibrin mass.
  • the polypeptide chain constituting fibrinogen and fibrin consists of three chains a, j8 and ⁇ .
  • Fibrin formed by adding thrombin and Ca2 + to fibrinogen, is soluble in 30% (WZV) urea solution and 1% (W / V) monochloroacetic acid solution.Fibrin is formed by adding Ca2 + to normal plasma. Phosphorus is insoluble in these solutions. The reason is that in the former, fibrinogen (H (A) ⁇ ( ⁇ ) ⁇ ) is released by ⁇ -terminal force fibrinopeptides ⁇ and ⁇ due to the action of calombin.
  • protease refers to an enzyme that hydrolyzes a peptide bond. In the present invention, it can be used to degrade fibrinogen. Hydrolysis of fibrinogen also has the power to produce adhesiveness.
  • a protease that degrades fibrinogen so as to improve the adhesiveness is used as the protease, and examples of such proteases include, for example, thrombin, notroxobin, and the like. Not.
  • protease inhibitor refers to any factor that inhibits the protease's peptide bond degrading activity.
  • protease inhibitors include, but are not limited to, aprotune, 4 (aminomethyl) cyclohexanecarboxylic acid, and the like.
  • tissue adhesive refers to any drug used for bonding living tissues.
  • tissue adhesives include, but are not limited to, cyanoacrylate adhesives, gelatin aldehyde adhesives, and fibrin glue adhesives.
  • cyanacrylate-based adhesive refers to an adhesive utilizing a polymerization reaction of a cyanoacrylate 'monomer. It is excellent in that the bonding strength is high and the bonding speed is fast.
  • gelatin-aldehyde-based adhesive refers to an adhesive utilizing a crosslinking reaction between gelatin (modified biopolymer collagen) and formaldehyde and dartaldehyde. Say. The bonding strength is high enough.
  • fibrin glue or “fibrin glue-based adhesive” refers to an adhesive utilizing a reaction of coagulating blood (a process of fibrin polymerization). This adhesive is not toxic and does not interfere with wound healing, but is not very adhesive due to its flexibility. The present invention has been found to be suitable for the maintenance and growth of stem cells. Without being bound by theory, it is expected that one factor is that the fibrin glue itself becomes a cell culture medium (nutrition). Examples of the fibrin glue include cryoprecipitate, plasma or platelet-containing plasma (PRP) diluted at an appropriate magnification.
  • PRP platelet-containing plasma
  • the magnification of fibrin glue is defined as cryoprecipitate and standard thrombin solution. It is calculated based on what is generated in the liquid. Usually, 1 to 10000 times fibrin glue is used, preferably 5 to 5000 times, more preferably 10 to 1000 times, and still more preferably 50 to 500 times.
  • extracellular matrix is also called “extracellular matrix” and refers to a substance existing between somatic cells regardless of epithelial cells or non-epithelial cells.
  • the extracellular matrix is involved in the composition of the internal environment necessary for the survival of all somatic cells, not just for tissue support.
  • Extracellular matrices are generally produced from connective tissue cells, but some are also secreted from cells that themselves possess basement membranes, such as epithelial and endothelial cells.
  • the fibrous components are classified into fibrous components and fibrous components and elastic fibers.
  • the basic component of the substrate is glycosaminodalican (acid mucopolysaccharide), most of which binds to non-collagenous proteins to form a macromolecule of proteodalican (acid mucopolysaccharide protein complex).
  • glycoproteins such as laminin in basement membrane, microfibril around elastic fibers, fibers, and fibronectin on cell surface are also included in the matrix. Even in specially differentiated tissues, the basic structure is the same. Produced.
  • gelatin is used in the broadest sense used in the art, and refers to an induced protein obtained by treating collagen constituting the skin, tendon or bone of an animal with boiling water. .
  • the term "culture container” refers to any container for culturing cells. Therefore, in the present invention, as long as the culture vessel has a bottom area, any vessel commonly used in the art can be used, for example, a petri dish, a flask, a mold vessel, etc.
  • a container eg, 1 cm 2 or more
  • the container can be made of any material, including glass, silica, silicon, ceramic, silicon dioxide, plastics (eg, polystyrene, polycarbonate, etc.), metals, natural polymers and synthetic polymers. Gain is not limited to them.
  • the term "medium” refers to any substance used for culturing cells. Examples include, but are not limited to, DMEM, P199, MEM, Hanks, equilibrated salt solution (HBSS), Ham, sF12, BME, RPMI1640, MCDB104 and MCDB153 (KGM).
  • the medium includes, for example, corticosteroids, insulin, darcose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbic acid and its derivatives, glycerol phosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives.
  • Derivatives growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, henolin, sodium bicarbonate, HEPES, albumin, transferrin, selenate Salt, linolenic acid, 3-isobutyl-1-methylxanthine, demethylating agent, histone deacetylating agent, activin, cytoforce, hexamethylenebisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethyl sulfoxy (DMSO), iododoxyperidine (IdU), hydroxyperrea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, etc. Components may be added.
  • HMBA hexamethylenebisacetamide
  • DMA dimethylacetamide
  • factor that promotes differentiation into differentiated cells refers to a factor that is known to promote shunting to shunt cells (for example, chemical Substance, temperature, etc.).
  • factors include, for example, various environmental factors, such as temperature, humidity, pH, salt concentration, nutrients, metals, gases, organic solvents, pressure, chemical Examples include, but are not limited to, substances (eg, steroids, antibiotics, etc.) or any combination thereof.
  • DNA demethylating agents such as 5-azacytidine
  • histone deacetylating agents such as trichostatin
  • nuclear receptor ligands such as retinoic acid (ATRA )
  • Vitamin D T3, etc.
  • cell growth factors activin, IGF-
  • an identified cell may be used, but even if the property is unknown, for example, by using a marker, it corresponds to a desired site by using a sorting technique such as FACS.
  • Cells can be prepared. The method of using FACS is described in, eg, Flow Cytometry Freedom Cell Engineering Separate Volume (Shujunsha), Nakauchi, 1999, and the like, and the contents thereof are incorporated herein by reference.
  • transplantation means that the cells, compositions, medicaments, and the like of the present invention are introduced into the body alone or in combination with other therapeutic agents.
  • the stem cells or their derivatives prepared according to the present invention can be transplanted into the body for various purposes (eg, for therapeutic, regenerative, cosmetic, prophylactic, etc.).
  • self refers to an individual or a part thereof (eg, cells, tissues, organs, etc.) derived from the individual.
  • self may broadly include a transplant from another genetically identical individual (eg, an identical twin).
  • the allogeneic refers to an individual who is the same species but is genetically different from another individual to be transplanted or a part thereof (eg, cells, tissues, organs, etc.).
  • genetically Allogeneic individuals can elicit an immune response in transplanted individuals (recipients).
  • examples of such cells include, but are not limited to, cells derived from the parent.
  • heterologous refers to a substance that is transplanted into a heterologous individual.
  • a transplant from a pig is referred to as a xenograft.
  • the coating agent for example, fibrinogen or its derivative, extracellular matrix, etc.
  • the coating agent may be of the same type as the cell to be cultured (especially It may be your own).
  • the donor of stem cells is a human, it is usually necessary to obtain informed consent
  • the providing candidate responds to the head of the providing medical institution whether or not he / she agrees with the providing. Consent requires the signing of informed 'consent' documents, which will be kept securely by the provider. With the consent of a specific candidate Will not communicate the consequences of disagreement to interested parties.
  • kits refers to a unit which is usually divided into two or more sections and provided with a part to be provided (eg, a reagent, a culture vessel, a coating agent, particles, and the like). .
  • This kit form is preferred when it is intended to provide a composition such that it should preferably be mixed or coated shortly before use and should not be provided as a mixture.
  • kits preferably include instructions describing how to provide the provided parts (eg, reagents, culture vessels, coating agents, particles, etc.).
  • Such instructions may be in any medium. Examples of such medium include, but are not limited to, a paper medium, a transmission medium, and a recording medium.
  • Examples of the transmission medium include, but are not limited to, the Internet, an intranet, an extranet, and a LAN.
  • Examples of the recording medium include, but are not limited to, CD-ROM, CD-R, flexible disk, DVD-ROM, MD, mini disk, MO, and memory stick.
  • any culture solution can be used.
  • examples of such culture media include, but are not limited to, DMEM, P199, MEM, HBSS (Hanks balanced salt solution), Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM). Not determined.
  • Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, and ascorbine.
  • BMP ⁇ PDGF and other proliferation Factor pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (selenium suboxide) Sodium), linolenic acid, demethylating agents such as 3-isobutyl-1-methylxanthin, 5-azancytidine; histone deacetylating agents such as trichostatin; activin; sites such as LIF'IL-2'IL-6 Force-in, hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbc AMP), dimethyl sul
  • coating refers to covering the surface of an object with a thin film of a specific substance. Any substance can be used as such a substance.
  • fibrinogen or a derivative thereof, a tissue adhesive, an extracellular matrix, gelatin, and the like can be used for coating, but are not limited thereto.
  • Such coating can be performed by a technique well known in the art, for example, a method of immersing the decellularized tissue in such a biocompatible polymer solution or a method of applying by spraying. But not limited to them.
  • After applying the coating it may be advantageous to perform fixing, preferably.
  • any method such as drying, standing, chemical treatment, and physical treatment can be used.
  • the coating may be fixed by cross-linking, but is not so limited.
  • cross-linking can be accomplished, for example, by chemical treatment, radical reactions (eg, ultraviolet irradiation, exposure to free radical sources, ultrasonic treatment, X-ray irradiation, gamma irradiation, and electron beam irradiation). it can.
  • radical reactions eg, ultraviolet irradiation, exposure to free radical sources, ultrasonic treatment, X-ray irradiation, gamma irradiation, and electron beam irradiation.
  • such substances can include biocompatible molecules.
  • the biocompatible polymer used in the present invention is a biocompatible polymer. Can be degradable.
  • biocompatible refers to the property of being toxic and immunologically rejectable and capable of existing in vivo without any obstacles.
  • biocompatible polymer refers to a polymer that is biocompatible and does not cause toxicity even if it remains.
  • a method for determining whether or not a certain polymer has such biocompatibility uses a test method such as a subcutaneous implantation test in a laboratory animal such as a rat. This test method uses the results of a subcutaneous implantation test.
  • the biocompatibility is low.
  • an artificial blood vessel is transplanted into a specific affected area, such as when transplanted into an animal's blood vessel, the transplant site is observed several days after several months, and the presence or absence of tissue engraftment, inflammation around the transplanted tissue, and adhesion Then, the degree of thrombus formation due to blood coagulation is observed to determine histocompatibility.
  • tissue section of the transplant site is prepared and cells are stained with hematoxylin and 'yesin' staining, and other staining methods' are observed.
  • Granular cells responsible for the immune system are used as an indicator of poor histocompatibility. Determine if there is much invasion or if there is any formation of scar tissue separating the conventional tissue and the transplanted tissue.
  • various cells such as vascular endothelial cells, fibroblasts, and smooth muscle cells have invaded from the conventional tissue and recellularized has occurred. (That is, whether the boundary between the conventional tissue and the transplanted tissue is clear! And whether the transplanted tissue has survived to a degree).
  • the concentration of cytoforce in blood caused by the progress of the inflammatory reaction the concentration of antibody against transplanted tissue recognized as a foreign substance (titer)
  • the concentration of complement components the amount of drug-metabolizing enzymes (P450, etc.) induced by foreign body implantation, etc.
  • P450 drug-metabolizing enzymes
  • test items such as test, blood compatibility test, systemic toxicity test, etc., and they are individual items according to the guidelines of the Ministry of Health, Labor and Welfare, the US National Standards, and the international industrial standard ISO-10993. Another test method is specified!
  • biocompatible polymer examples include polyvinyl alcohol, polylactic acid, polyglycolic acid, polybutylpyrrolidone, polyethylene glycol, gelatin, collagen, ⁇ -polyglutamic acid, silicone, polyvinyl chloride, and polymethyl methacrylate.
  • the polybutyl alcohols in addition to the unmodified ones, there are also those that have been chemically modified to some extent with amino groups, carboxyl groups, acetyl groups, etc., and these are commercially available, and these modified polyvinyl alcohols are commercially available. Can also be used in the present invention.
  • the biocompatible polymer is biodegradable, but not limited thereto.
  • biodegradable refers to the property of being degraded in vivo or by the action of microorganisms when referring to a substance.
  • the biodegradable polymer can be decomposed into water, carbon dioxide, methane, and the like, for example, by hydrolysis.
  • the method of determining whether a substance is biodegradable is based on the bioabsorbability, which is a part of biodegradability, for a few days to several years for experimental animals such as rats, puppies, and dogs.
  • embedding tests and microbial degradation tests use methods such as embedding and disintegration tests of sheet-shaped macromolecules in soil for several days to several years.
  • phosphate buffered saline PBS
  • various buffers such as phosphate buffered saline (PBS) can be used for non-enzymatic degradation of polymers.
  • PBS phosphate buffered saline
  • a dissolution test in an aqueous solution may be carried out using a buffer solution to which the high-molecular hydrolase (protease, glycosidase, lipase, esterase, etc.) is added.
  • Biodegradable polymers include natural and synthetic polymers. Examples of natural polymers include proteins such as collagen and starch, and polysaccharides, and examples of synthetic polymers include aliphatic polyesters such as polyglycolic acid, polylactic acid, and polyethylene succinate. However, they are not limited to them. Does polyvinyl alcohol show weak biodegradability? Thus, in the present specification, it can be recognized as having biodegradability.
  • polymer is used in the same meaning as "molecule” and does not particularly limit the molecular weight.
  • molecule generally refers to a polymer having a molecular weight of 500 or more, but is not limited thereto.
  • the upper limit of the molecular weight of the polymer used in the present invention is a force that is infinite in principle.In the present invention, it is usually handled as a solution, and the molecular weight can be discussed (dynamic light scattering, gel filtration, In the sense that molecular weight measurement such as sedimentation equilibrium using a centrifuge can be applied), a molecular weight of up to about 5,000,000 can be used, but is not limited thereto.
  • the present invention provides a culture vessel for culturing stem cells, comprising fibrinogen or a derivative thereof. It is understood that any fibrinogen or derivative thereof can be used. Therefore, any method known in the art can be used for preparing fibrinogen or a derivative thereof. A typical method for producing fibrin glue is shown below.
  • ACD blood (Terumo quadruple bags) or its equivalent (preferably a koto with less than 4 hours).
  • the blood is processed at 3000 rpm 10 min at 4 ° C (or 4000 rpm for 6 minutes).
  • Buffy coat remove red blood cells and use plasma.
  • This plasma should be frozen at 80 ° C or slowly frozen at 40 ° C with an air cap. (To maximize the yield of fibrin).
  • the yield is increased 1.5 times if frozen and thawed twice in the still state.
  • slower freezing with shaking at 20 ° C and slower thawing in a 4 ° C water bath seems to yield more.
  • Store at 20 ° C. Thaw at 4 ° C for 16-18-24 hours.
  • Storage is performed by quick freezing at -80 ° C or storage at -20 ° C. Thaw at 37 ° C before use.
  • the fibrin concentration at the time of use may be from about lOmgZml to about lOOmgZml. Commercially available products are often 80mgZml.
  • Solution B can be prepared in accordance with Autotransfusion Manual: Dr. Masuhiko Takaori, published by Katseido Shuppan, 1996.
  • Solution B can be prepared with 5000 units of thrombin powder; 0.5M calcium chloride solution 1ml aprotin 3500 units; and distilled water 7.5ml. Aprotune can be obtained from Bayer and other sources.
  • fibrinogen or a derivative thereof is coated on a culture vessel. It is understood that any method known in the art can be used for the coating.
  • such a fibrinogen or a derivative thereof may be coated on the culture vessel at any thickness, but is preferably coated at a thickness of 1 ⁇ m to 100 ⁇ m. , More preferably 5 ⁇ m to 50 ⁇ m. It is understood that if the amount is too small, it may not be able to withstand cell growth, so that it is preferable to have a certain thickness.
  • fibrinogen or a derivative thereof can be used with any molecular weight, but preferably has a molecular weight of 340,000 Da or more, more preferably a fibrin polymer or a fibrin mass. Is used.
  • fibrinogen or a derivative thereof contained in the culture container of the present invention contains fibrin.
  • Fibrin may be present at any concentration but is preferred Or 0.1 mg to 100 mg per 1 cm 2 of the container.
  • the fibrinogen or derivative thereof comprises a protease.
  • protease promotes the degradation of fibrinogen and improves the adhesive ability.
  • the protease may be inactivated.
  • examples of the protease include, but are not limited to, thrombin (for example, ⁇ thrombin and the like) and batroxobin.
  • fibrinogen or a derivative thereof used in the present invention contains a protease inhibitor.
  • a protease inhibitor By including a protease inhibitor, it is also possible to control the adverse effects of the protease. Any protease can be used, but is not limited thereto.
  • examples of the protease inhibitor include, but are not limited to, aprotune, fusane, 4- (aminomethyl) cyclohexanecarboxylic acid, and the like.
  • fibrinogen or a derivative thereof used in the present invention is fibrin glue.
  • the fibrin glue used in the present invention contains components such as fibrin, fibrin monomer, fibrin polymer, fibronectin, and platelets.
  • the fibrin glue used in the present invention is obtained by using thrombin from the N-terminus of the Aa chain of fibrinogen to the fibrinopeptide A (FPA) force.
  • Peptide B (FPB) is cleaved.
  • FPA and FPB are cleaved from one molecule of fibrinogen, and the remaining molecule is fibrin monomer.
  • the fibrin monomer has an adhesive property such that the N-terminal and the C-terminal thereof are polymerized because the polymerization site is exposed, and the fibrin polymer becomes a fibrin mass.
  • fibrin glue can be used at any concentration, but is preferably diluted to 500-fold or less (produced with cryoprecipitate and a standard thrombin solution). U, which is preferred to use). It is more preferable to use fibrin glue diluted 50-fold to 500-fold. But not limited to. More preferably, it may be preferable to use those diluted 100 times or more, for example, about 500 times. This is because the growth efficiency is significantly improved. Dilution of the fibrin glue can be performed using any medium. Preferably, a physiologically compatible medium is used. Examples of such a solution include, but are not limited to, physiological saline.
  • the fibrin glue that can be used in the present invention includes "cryoprecipitate or plasma or platelet-rich plasma (PRP)" and "thrombin in calcium chloride and aprotune solution (trasilol product name). It can be prepared by mixing the “added solution”. Typically, the dilution can be reduced to about 10,000 times for cryoprecipitate and to about 500 times for plasma. With the latter thrombin solution, up to about 1000 times is possible.
  • the composition of the thrombin solution is, for example,
  • 500 times the dilution ratio of the thrombin solution is defined as the thrombin concentration of 0.1.
  • fibrinogen or a derivative thereof used in the present invention may include, for example, fibrin monomer, fibrin polymer, fibrinogen, platelets, and the like.
  • fibrin glue includes (1) "Cryoprecipitate (rich fibrinogen plasma) or plasma platelet-rich plasma (PRP)” and (2) "thrombin solution (thrombin solution). (A solution to which dani calcium and aprotune solution (trasilol product name) are added) (including 5000 units of thrombin in 12 ml) ". Both (1) and (2) can be used after dilution.
  • the stem cells targeted by the present invention may be any stem cells, but are preferably tissue stem cells, more preferably mesenchymal stem cells, for example, stem cells derived from adipose tissue. obtain.
  • tissue stem cells more preferably mesenchymal stem cells, for example, stem cells derived from adipose tissue. obtain.
  • mesenchymal stem cells for example, stem cells derived from adipose tissue. obtain.
  • stem cells derived from lipoaspirates has not been achieved in the prior art (Daniel A. De Ug artea Kouki Morizonoc Amir Elbarbarya Zeni Alfonsob, Patricia A. Zuka Min Zhua Jason L. Dragooa Peter Ashjiana Bert Thomasa,
  • the stem cell to be cultured in the present invention may be an adipocyte-derived stem cell (ASC).
  • ASC adipocyte-derived stem cell
  • the fibrin or a degradation product thereof used in the present invention when dried after coating, is contained at a concentration of 0.1-100 mg / cm 2 by weight per unit area.
  • any material can be used as long as it is suitable for cell culture.
  • the present invention provides a method for culturing a stem cell using the container of the present invention.
  • a method for preparing (or culturing) such a stem cell comprises: A) providing the stem cell; and B) culturing the stem cell in a culture vessel containing a medium and fibrinogen or a derivative thereof. I do.
  • any method known in the art can be used. Such provision includes, but is not limited to, sorting cells by FACS using a marker specific for stem cells as an index.
  • the culture of the stem cells can use any commonly used medium.
  • Such culture conditions include, for example, pH 7.2-7.4, temperature 37 ° C, CO 2
  • the condition of a concentration of 5% can be mentioned, but is not limited thereto.
  • the pH is, for example, around neutral (for example, pH 5-9), the temperature is, for example, normal temperature-37 ° C, and the CO concentration is, for example, 11-10% (for example, 5%). I can list them
  • Examples of the medium that can be used in the method of the present invention include DMEM, P199, and ME. M, Hanks, Equilibrated Salt Solution (HBSS), Ham's F12, BME, RPMI 1640, MCDB 104 and MCDB 153 (KGM) and the like. DMEM and P199 are preferred. This has been shown to be appropriate for stem cell expansion.
  • the medium used in the method of the present invention includes corticosteroids, insulin, dalcose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbic acid and its derivatives, glycerol phosphate, estrogen and its derivatives, progesterone and And its derivatives, androgen and its derivatives, growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heterophosphorus, sodium bicarbonate, HEPES , Albumin, transferrin, selenate, linolenic acid, 3-isobutyl-1-methylxanthine, demethylating agent, histone deacetylating agent, activin, cytokinin, hexamethylene bisacetamide (HMBA), dimethylacetate Amide (DMA), dibutyl cAMP (d bcAMP), dimethyl sulfoxide (DMSO), iodo
  • the cell preparation method of the present invention further includes a step of obtaining a stem cell as a raw material.
  • a method of obtaining include, but are not limited to, separation from a living body or FACS sorting and separation of sample force.
  • the cell preparation method of the present invention can further include a step of differentiating a stem cell. It is understood that the use of the cells thus separated is also included in the scope of the present invention.
  • the present invention provides a method for producing a culture vessel coated with fibrinogen or a derivative thereof for culturing stem cells.
  • the method includes the steps of: A) providing a culture vessel; B) providing fibrinogen or a derivative thereof to the culture vessel; and C) fixing the fibrinogen or derivative thereof to the surface of the culture vessel.
  • the provision of the culture vessel can be realized by a method known in the art. Fibrinogen or a derivative thereof is placed in such a culture vessel. To provide, any known technique can be used, and preferably, coating is advantageous. Such fibrinogen or a derivative thereof is fixed to a culture vessel using any method known in the art.
  • provision of the fibrinogen or a derivative thereof used in the present invention is performed by an aseptic operation. It is also a force that has been found to have a positive effect on culture by being provided by such conditions.
  • the immobilization of the fibrinogen or a derivative thereof of the present invention can be performed by leaving the fibrinogen or the derivative in a dry state for a certain period of time, or by irradiating with ultraviolet rays.
  • the present invention provides a culture vessel coated with a tissue adhesive for culturing stem cells.
  • a tissue adhesive those known in the art can be used, and examples thereof include a cyanoacrylate adhesive, a gelatin aldehyde adhesive, and a fibrin glue adhesive.
  • the culture vessel any form described in the description of the culture vessel containing fibrinogen or a derivative thereof can be adopted.
  • any form described in the description of the culture vessels containing the above-mentioned fibrinogen or a derivative thereof can also be adopted.
  • the cell adhesion agent may be included in the 0.1- lOOmg / cm 2 or more concentrations
  • the present invention provides a culture vessel coated with an extracellular matrix for culturing stem cells.
  • an extracellular matrix those known in the art can be used, and examples thereof include fibronectin, collagen and laminin.
  • any form described in the description of the culture vessel containing the above-mentioned fibrinogen or a derivative thereof can be adopted.
  • any of the forms described in the description of the culture vessels containing fibrinogen or a derivative thereof can be adopted.
  • the extracellular matrix at a concentration of 0. 1- lOOmgZcm 2 or more Can be rare.
  • fibronectin Collagen may be 8 ⁇ g / cm 2 and laminin may be 5-20 ⁇ g / cm 2 .
  • the present invention provides a culture vessel coated with gelatin for culturing stem cells.
  • gelatin those known in the art can be used.
  • the culture vessel any form described in the description of the culture vessel containing the above-mentioned fibrinogen or a derivative thereof can be adopted.
  • the cells to be targeted by these culture vessels any of the forms described in the description of the culture vessels containing fibrinogen or a derivative thereof can be adopted.
  • the present invention provides a method for preparing a stem cell.
  • This method comprises the steps of: A) providing a culture vessel containing a medium and an extracellular matrix and Z or gelatin; B) arranging stem cells in the culture vessel; C) culturing the stem cells for a desired period.
  • the culture vessel may adopt any form described in the description of the aspect including the above fibrinogen or a derivative thereof.
  • any of the forms described in the above description of the aspect including fibrinogen or a derivative thereof can be adopted.
  • the present invention provides an extracellular matrix and
  • a method for producing a culture vessel coated with Z or gelatin comprises: A) providing a culture vessel; B) an extracellular matrix and
  • the culture vessel is described in the above description of the aspect containing fibrinogen or a derivative thereof.
  • any of the forms described above can be employed.
  • any of the forms described in the above description regarding the aspect containing fibrinogen or a derivative thereof can be adopted.
  • the present invention provides the use of fibrinogen or a derivative thereof for preparing a stem cell.
  • Fibrinogen or its derivatives can be used in stem cells (particularly in tissues). It is not known whether it has a proliferative effect on stem cells). Therefore, the present invention provides a novel use of fibrinogen or a derivative thereof.
  • the culture vessel may adopt any form described in the description relating to the aspect containing fibrinogen or a derivative thereof.
  • the cells targeted by these culture vessels can also adopt any of the forms described in the description of the aspect containing the above-mentioned fibrinogen or a derivative thereof.
  • the present invention provides the use of an extracellular matrix and Z or gelatin for preparing a stem cell. It was unknown whether extracellular matrices (especially collagen, laminin, fibronectin, etc.) had a proliferative effect. Therefore, the present invention provides a novel use of fibrinogen or a derivative thereof. Also in this use, the culture container may adopt any form described in the description of the aspect including the above-mentioned fibrinogen or a derivative thereof. For the cells targeted by these culture vessels, any of the forms described in the description relating to the surface containing fibrinogen or a derivative thereof can also be employed.
  • a method for preparing liposuction waste fluid stem cells comprising: A) a step of obtaining a liposuction waste solution; B) a step of obtaining a cell fraction by centrifuging the liposuction waste solution; C) Subjecting the cell fraction to density gradient centrifugation to separate cells by specific gravity; and D) collecting a cell layer having a specific gravity lower than that of red blood cells.
  • the liposuction waste liquid used in the present method may be a liquid sucked together during liposuction (for example, a force including but not limited to tomescent liquid and blood) or a liquid generated by washing a suction fat with a liquid. Waste liquids include, but are not limited to.
  • the liposuction method can be performed using techniques well known to those skilled in the art.
  • the apparatus used for the liposuction method include, but are not limited to, a Keisei degreasing suction device monkey pump SAL76-A (Keisei Medical Industry Co., Ltd., Hongo 3-19-6, Bunkyo-ku, Tokyo).
  • a fluid such as physiological saline containing 0.0001% adrenaline is injected into adipose tissue in an amount equal to twice the expected aspirated fat mass, and then injected into the adipose tissue. This is performed by suctioning with a negative pressure of about 250-900 mmHg using a mm-force Yule (metal suction tube).
  • physiological saline is used as a liquid for washing the sucked fat cells.
  • any liquid other than physiological saline can be used as long as it does not adversely affect the stem cells to be isolated.
  • any isotonic solution such as Ringer's solution and mammalian culture medium (eg, DMEM, MEM, M199, and MCDB153) can be used.
  • the liposuction waste liquid can be treated with an enzyme such as collagenase, if necessary.
  • an enzyme such as collagenase
  • the amount of collagen contained in the liposuction waste liquid is smaller than that of adipose tissue, when liposuction waste liquid is used as a raw material for preparing stem cells, the time required for collagenase treatment, and the amount of Z or enzyme The amount of enzyme required for the treatment is lower than in the case of adipose tissue.
  • the cell fraction and other components for example, plasma, physiological saline injected at the time of surgery, an anesthetic, a hemostatic agent, and Any conditions for separating the leaked lipid component from the adipocytes
  • centrifugation at 400-800 X g for about 5-15 minutes can be used.
  • the cell fraction is separated by its specific gravity, for example, by density gradient centrifugation.
  • a medium usable for density gradient centrifugation is used as the medium.
  • a preferred medium has a specific gravity at 20 ° C of 1.076-1.078 gZml.
  • the preferred pH of the medium is 4.5-7.5.
  • the preferred endotoxin level in the medium is 0.12 E UZml or less.
  • Representative media include, but are not limited to, sucrose, ficoll (a copolymer of sucrose and epichlorohydrin), and percoll (a colloidal silica product having a coating of polybulpyrrolidone).
  • Ficoll is, for example, Ficoll-Paque PLUS (Falmacia Biotech Co., Ltd., Tokyo), Histopaque-1077 (Sigma-Aldrich Japan Co., Ltd., Tokyo), and Lym phoprep (registered trademark) (Nicomed, Oslo) (Norway)).
  • Percoll is called Percoll (Sigma Aldrich Japan Co., Ltd., Tokyo) The product with the name is on the market!
  • the conditions of centrifugation by specific gravity are 400 X g for 30 to 40 minutes when Ficoll-Paque PLUS is used as a medium, and 370 g X g for about 30 minutes when a Histopak is used as a medium.
  • the most abundant cells are usually red blood cells, which can be confirmed as a red cell layer. Since stem cells have a lower specific gravity than red blood cells, when separating stem cells, a cell layer lighter than red blood cells is collected. Preferably, a cell layer with a specific gravity in the range of 1.050-1.075 is collected.
  • the approximate specific gravity of the cells is determined by preparing a density gradient centrifugation medium such as Percoll or Ladygrad with a sodium salt solution or a sucrose solution and collecting the density marker beads together with the collected cells. It is possible to investigate by checking which layer contains the cells among the 5-10 layers separated by beads after centrifuging and centrifuging.
  • a density gradient centrifugation medium such as Percoll or Ladygrad with a sodium salt solution or a sucrose solution
  • the cells separated into layers can be collected using, for example, a pipette.
  • a cell separator for example, a blood component separation device A
  • STEC204 (Amco) can also be used.
  • a medium for culturing the collected cells for example, DMEM, M199, MEM, HBS
  • Powers including, but not limited to, S, Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM).
  • Preferred media include DMEM, and M199.
  • the collected cells are a heterogeneous population of cells, including stem cells that express CD13, 29, 44, 49d, 71, 73, 99, 105, 151, and furthermore, which of CD31, CD34, Alternatively, it includes a stem cell that expresses both.
  • vascular endothelial cells nerve cells, smooth muscle cells, cardiomyocytes, skeletal muscle cells, chondrocytes , Bone cells, adipocytes, ligament cells, and stromal cells.
  • vascular endothelium was collected from the collected cell group using the following method. It is possible to separate and collect precursor cells.
  • the obtained cell group is cultured in a 1% gelatin-coated culture dish for 4 to 5 days using a medium for vascular endothelial cells. 0.25% Trypsin is removed from the culture dish and reacted with anti-PECAM-1 beads. Only the cells that have reacted with the antibody are separated using MACS (magnetic cell separation system) or FACS (flow cytometry). . The separated cells are cultured again in a medium for vascular endothelial cells ⁇ 1% gelatin-coated culture dish.
  • MACS magnetic cell separation system
  • FACS flow cytometry
  • vascular endothelial progenitor cells By culturing the cells in another culture dish, cells differentiated into vascular endothelial progenitor cells can be isolated.
  • Hutley LJ, et al Human adipose tissue endothelial cells promote preadipocyte proliferation. Am J Physiol Endocrinol Metab. 2001 Nov; 281 (5): E1037-44.).
  • a system for preparing stem cells comprising: A) means for obtaining a liposuction waste liquid; B) means for centrifuging the liposuction waste liquid to obtain a cell fraction. And C) means for subjecting the cell fraction to density gradient centrifugation to separate cells by specific gravity.
  • Means for obtaining a liposuction waste liquid include a means for sucking fat from the inside of a living body by decompression.
  • Such means include liposuction surgery (liposuction surgery with a manual negative pressure syringe and liposuction surgery with a motorized liposuction device), and delipidation by incising the skin
  • liposuction surgery liposuction surgery with a manual negative pressure syringe and liposuction surgery with a motorized liposuction device
  • delipidation by incising the skin it is not limited to these.
  • Examples of a means for obtaining a cell fraction by centrifuging a liposuction waste liquid and a means for subjecting a cell fraction to density gradient centrifugation to separate cells by specific gravity include a centrifugal separator.
  • the conditions include a centrifugation force of 200 to 1200 X g for 3 to 60 minutes, preferably 260 to 900 X g for 3 to 30 minutes.
  • Perform centrifugation more preferably 400-800 ⁇ g, for 3-15 minutes, most preferably 400 ⁇ g, 5-10 minutes.
  • the conditions are as follows: 00—1200 X g, 10-60 min, separation power S, preferably 260-900 X g, 10-60 min centrifugation, preferably 370-1100 X g, 10-60 min Perform centrifugation, more preferably at 400-800 X g for 10-40 min, separation, and most preferably centrifugation at 400 X g for 30-40 min.
  • the system also includes means for collecting a cell layer having a specific gravity as required.
  • the cell layer collected is a cell layer having a lower specific gravity than red blood cells.
  • Such means include, but are not limited to, manual pitchers, manual aspirators, automatic pitchers, and automatic aspirators.
  • stem cell of the present invention various screening cells and Z or progenitor cells can be obtained. Specifically, the following method is used.
  • the present invention provides a method for preparing a differentiated cell from a stem cell.
  • Stem cell force The obtained divided cells include not only cells that have been finally divided but also precursor cells.
  • Stem cell force derived from liposuction waste fluid can be prepared by (1) a method of adding a differentiation inducer (eg, dexamethasone) to a medium, and (2) a method of preparing a desired site. And a method of culturing with the differentiated cells.
  • a differentiation inducer eg, dexamethasone
  • differentiation inducers that induce stem cell differentiation include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, Proliferation including ascorbic acid and its derivatives, glycerophosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, aFGF-bFGF-EGF-IGF-TGF ⁇ ECGF.BMPPDGF Factor, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenite (selenium suboxide) Sodium), linolenic acid, 3-iso Kisame Chill -1 over methylxanthine, 5-demethylating agent such Azanshichijin histone de Asechiru agents such as tric,
  • any culture solution can be used for the cell mixture of the present invention as long as the maintenance of the cells to be mixed and the differentiation into differentiated cells corresponding to the desired site are maintained.
  • a culture medium include, but are not limited to, DMEM, M199, MEM, HBSS (Hanks' Balanced salt solution), Ham, sF12, BME, RPMI1640, MCDB104, MCDB153 (KGM) and the like.
  • Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, and ascorbate.
  • glycerophosphate glycerophosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, aFGF'bFGF'EGF, IGF-TGF jS 'ECGF'BMP'PDGF and other growth factors , Pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (sodium selenite) Linolenic acid, 3-isobutyl-1-methylxanthi) , Demethylating agents such as 5-azanticidin, histone deacetylating agents such as trichostatin, activin, cytokins such as LIF'IL-2'IL-6, hexamethylene bisacetoamide (HMBA), Dimethylacetoamide (DMA), dibutyl cAMP (dbcAMP), dimethylsulfoxide (DMSO),
  • Induction of differentiation of stem cells derived from liposuction waste fluid into adipocytes is performed, for example, by culturing in a DMEM medium supplemented with isobutyl-methylxanthine, dexamethasone, insulin, and indomethacin. Not limited.
  • Induction of stem cells from liposuction waste fluid into chondrocytes can be performed, for example, by adding 1% FBS. Force performed by culturing in a DMEM medium containing insulin, ascorbate 2-phosphate, and TGF-jS 1 in a medium that is not limited thereto.
  • Induction of stem cells derived from liposuction waste fluid into bone cells is performed, for example, by adding dexamethasone, ascorbate 2-phosphate and j8-glycerophosphate to a DMEM medium containing 10% FBS.
  • the power produced by culturing in tanned medium but not limited to
  • Induction of stem cells derived from liposuction waste fluid into muscle cells was performed, for example, by adding dexamethasone and Hyde-mouth cortisone to a DMEM medium containing 10% FBS and 5% HS (horse serum).
  • the force exerted by culturing in the medium is not limited to this.
  • adipose stem cells were prepared from lipoaspirates from humans who had given consent to this experiment.
  • the lipoaspirate was thoroughly washed with saline using a 1-liter separatory funnel. It was confirmed that the lipoaspirate was sufficiently separated in the upper layer and the physiological saline was sufficiently separated in the lower layer. The lower layer was discarded, and this was repeated until the physiological saline was almost transparent to the naked eye. In this example, the test was performed five times.
  • the lipoaspirate was weighed with 10 ml of 0.075% collagenase ZPBS (Gibco) in the same volume as 10 ml and incubated at 37 ° C. for 30 minutes with good stirring. To this sample, the same amount of DMEM supplemented with 10% serum was added, and centrifuged at 1200 ⁇ g for 10 minutes.
  • ASCs adipose-derived stem cells
  • a tomescent solution (a physiological saline solution containing 0.0001% adrenaline), which is equal to or larger than the expected amount of aspirated fat, is injected into the subcutaneous fat layer (tumescent method).
  • Liposuction surgery was performed using a 2-3 mm force-Yure (metal suction tube). Liposuction surgery is well known in the art, and its method is described, for example, in Cosmetic Surgery Practice 2, edited by Masanari Takada, Ryusaburo Tanino, Yoshiaki Hosaka, Bunkodo, P429-469.
  • suction fat was washed with physiological saline. Of the total 5-10 liters of effluent generated by washing, only the first 1-2 liters, rich in cellular components, were used for further processing.
  • the waste solution was treated using one of the following two methods to prepare a stem cell suspension.
  • Neither of the following two methods requires a treatment using an enzyme such as collagenase, and therefore differs from adipose tissue-derived stem cells prepared by a conventional method in that there is no contamination with an enzyme such as collagenase.
  • Liposuction waste liquid (usually about 2 to 4 liters) was centrifuged at 400 X g for 10 minutes.
  • the precipitated cells (mostly erythrocytes) were transferred to several 50 ml polypropylene tubes and centrifuged again (400 ⁇ g, 5 minutes).
  • Processing was performed three times by a centrifugal separation method using a cell separator (blood component separator ASTEC204, Amco Co., Ltd.) to remove light-weight platelet components, heavy-weight red blood cells, and granulocyte components as much as possible.
  • a cell separator blood component separator ASTEC204, Amco Co., Ltd.
  • the approximate specific gravity of the cells is determined by preparing a density gradient centrifugation medium such as Percoll or Ladygrad with a sodium chloride solution or a sucrose solution, and collecting the density marker beads together with the collected cells. It is possible to investigate by checking which layer contains the cells among the 5-10 layers separated by beads after centrifuging and centrifuging.
  • a density gradient centrifugation medium such as Percoll or Ladygrad with a sodium chloride solution or a sucrose solution
  • Example 3 The stem cells collected in Example 3 were characterized using FACS by the following procedure. [0153] About 5 ml of the cell suspension was washed twice with a staining medium (SM; PBS containing 0.5% serum albumin and 0.05% NaN). Cells were counted as needed.
  • SM staining medium
  • gZml was added to a labeled antibody (labeled with phycoerythrin (PE), araphycocynin (APC) and Z or fluorescein isothiocynate (FITC)).
  • PE phycoerythrin
  • APC araphycocynin
  • FITC fluorescein isothiocynate
  • the cells were washed, and the concentration of the cell suspension was adjusted to about 5 ⁇ 10 5 cells / ml using SM.
  • FACS Vantage (Becton Dickinson) was used. Using the label of the antibody as an index, the expression of various CD proteins in the isolated stem cells was analyzed. As a result, as shown in Table 1, it was found that stem cells derived from the fat sucking waste liquid expressed CD90 and CD49d.
  • the isolated stem cells were subcultured twice in DMEM medium. Passaging was performed at 80% confluence. The cells after the two subcultures were analyzed by FACS in the same procedure as described above. The results are shown in Table 1 below.
  • the stem cells prepared from the liposuction waste liquid contained CD31 and 34 positive cells, although they were mesenchymal stem cells, unlike the adipose-derived stem cell group prepared by the conventional method. Therefore, it can be understood that the stem cells prepared by the method of the present invention are a group of cells that can be easily and highly efficiently transferred to the vascular endothelium (angiogenesis). Furthermore, since the expression of CD, which was used as an index in this study, was confirmed after two subcultures, the stem cells of the present invention did not change most of their phenotypes even after about two subcultures. Is understood.
  • stem cells were collected from waste fluids from a plurality of subjects and characterized. The results are shown below.
  • the adipose stem cell of the present invention comprises at least one selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151, and SH3.
  • the adipose stem cell of the present invention comprises at least one selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151, and SH3.
  • the stem cell population was negative for CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144.
  • the adipose stem cell of the present invention is a cell that does not express at least one of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144. It is.
  • CD31, CD34, CD36, CD45, CD106, and CD117 tended to lose their expression as the culture period became longer. Therefore, when subculture was continued, CD106 expression observed before subculture was sometimes not observed.
  • the stem cells prepared as in the above example were cultured on a coated dish as shown in Table 3 below.
  • the culture conditions were as follows.
  • Fibrin glue was prepared as follows.
  • ACD bleeding (Terumo quadruple bags) or its equivalent (a koto using less than 4 hours seems to be preferred) was used.
  • the blood was processed at 3000 rpm 10 min at 4 ° C (or 4000 rpm for 6 minutes). Buffy coat, red blood cells were removed and plasma was used.
  • the plasma was snap frozen at 80 ° C or slowly frozen at 40 ° C with an air cap or the like. (To maximize the yield of fibrin). Here, if freeze-thaw is repeated twice while still standing, the yield will be 1.5 times. Alternatively, slower freezing with shaking at -20 ° C and slow thawing in a 4 ° C water bath yielded more yield. Store at 20 ° C. Thawing was performed at 4 ° C for 16-18-24 hours.
  • Thrombin powder 5000 units; 0.5 M calcium chloride solution 1 ml; aprotune 3500 units; distilled water 7.5 ml of solution B was diluted 50 times and 500 times with distilled water.
  • This fibrin glue can also be obtained from commercially available Japanese organs (product name: Tissile) or Fujisawa Pharmaceutical (product name: Bolhir) or Hoechst (product name: beriplast).
  • the above-mentioned fibrin glue was coated on a culture vessel as follows.
  • a 10 cm dish FALCON 35-3003 Tissue Culture Dish, Becton Dickinson
  • the coating method is as follows.
  • stem cells were cultured under the following conditions.
  • Example 6 it is confirmed whether or not other stem cells (such as mesenchymal stem cells derived from bone marrow) also have a proliferation promoting activity.
  • Bone marrow-derived mesenchymal stem cells are Prepared by sorting in S. Using a cell sorter, using CD34 +, CD117 (c kit) +, Sea-1 + as an indicator of undivided, SH-2 (CD73), SH
  • Example 6 The dish used in Example 6 is used. As a result, it becomes clear that proliferation of bone marrow-derived stem cells also increases.
  • the pluripotency of the cells prepared in Examples 6 and 7 is confirmed.
  • the stem cell of the present invention is differentiated by a known method in this field, it becomes clear that the differentiation ability is maintained.
  • Example 6 it is confirmed whether other stem cells (such as ES cells) also have a growth promoting activity.
  • ES cells are established by techniques known in the art.
  • Example 6 The dish used in Example 6 is used. As a result, it becomes clear that proliferation of bone marrow-derived stem cells also increases.
  • Cells Human fatty fibroblast-derived stromal cells (fat cell-derived stem cells)
  • PRP coating dish Solution A (Thrombin 5000U, 0.5M CaCl 1ml, apro
  • human adipocyte-derived stem cells (A) were seeded on a human PRP-coated 6-well plate and a non-coated 6-well plate at 2 ⁇ 10 4 cells. After 4 days, the medium was replaced, and after 7 days, the number of cells was measured.
  • the present invention makes it possible to easily increase the proliferation rate of stem cells (particularly, stem cells derived from fat). Therefore, the present invention has utility in medical, pharmaceutical, agricultural, etc. fields utilizing stem cells.

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Abstract

A method of amplifying stem cells (for example, tissue stem cells such as mesenchymal stem cells (in particular derived from fat)). There is provided a culture vessel coated with fibrinogen whose typical example is fibrin glue, or its derivative, or an extracellular matrix (for example, collagen (e.g., type I, type IV, etc.), laminin, fibronectin, etc.), or gelatin, or the like. Further, there is provided a method of preparing stem cells, comprising the steps of (A) providing stem cells, and (B) growing the stem cells in a culture vessel containing a culture medium and fibrinogen or its derivative.

Description

明 細 書  Specification
幹細胞の増殖法  Stem cell expansion method
技術分野  Technical field
[0001] 本発明は、再生医療に関する。より詳細には、本発明は、幹細胞の培養に関する。  The present invention relates to regenerative medicine. More particularly, the present invention relates to the culture of stem cells.
背景技術  Background art
[0002] 幹細胞の利用を中心とした、再生医療は、ここ数年で、力なりの発展を遂げてきた。  [0002] Regenerative medicine, centered on the use of stem cells, has developed in the last few years.
従来であれば、存在しないと考えられていた、組織幹細胞が種々の組織力も発見さ れ、同定されてきた。このように、再生医療による疾患治療が最近注目を浴びている  Conventionally, tissue stem cells, which were thought to be nonexistent, have also been discovered and identified with various tissue forces. Thus, regenerative medicine disease treatment has recently attracted attention
[0003] 幹細胞は、種々存在し、受精卵に由来する幹細胞、胚性幹細胞の他、組織幹細胞 と呼ばれるものがある。受精卵は、原腸陥入の後、内胚葉、中胚葉および外胚葉の 3 つの胚葉に分かれ、外胚葉由来の細胞は、主に脳に存在し、神経幹細胞などが含ま れる。中胚葉由来の細胞は、主に骨髄に存在し、血管幹細胞、造血幹細胞および間 葉系幹細胞などが含まれる。内胚葉由来の細胞は主に臓器に存在し、肝幹細胞、脾 幹細胞などが含まれる。 [0003] There are various types of stem cells, and there are stem cells derived from fertilized eggs, embryonic stem cells, and so-called tissue stem cells. Fertilized eggs are divided into three germ layers, endoderm, mesoderm and ectoderm after gastrulation, and ectoderm-derived cells mainly exist in the brain and include neural stem cells. Mesodermal-derived cells are mainly present in bone marrow, and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like. Endoderm-derived cells are mainly present in organs and include hepatic stem cells, spleen stem cells, and the like.
[0004] 脂肪細胞、骨、靭帯、心筋などを含む間葉系細胞は、身体の骨格を形成するに重 要な働きを有していることから、その細胞を含む集団、組織など、間葉系細胞の再生 医療および移植医療への応用の期待が高まっている。特に、骨髄間葉系幹細胞は、 中胚葉系の種々の臓器に分ィ匕することが報告されるようになっており、再生医療の中 心として注目を浴びている。しかし、その分ィ匕の条件は、分化誘導剤（例えば、デキ サメサゾンなど）を含む特殊な培地を必要とすることが知られるかなり特殊なものを必 要とするとされて ヽる (非特許文献 1)。  [0004] Mesenchymal cells including fat cells, bones, ligaments, myocardium, and the like have an important function in forming a body skeleton, and therefore, mesenchymal cells such as populations and tissues containing the cells. Expectations for application to regenerative medicine and transplantation medicine for lineage cells are increasing. In particular, it has been reported that bone marrow mesenchymal stem cells are divided into various organs of the mesodermal system, and they are receiving attention as a center of regenerative medicine. However, it is said that the conditions for the separation require a rather special medium known to require a special medium containing a differentiation inducer (for example, dexamethasone) (Non-patent Document 1). 1).
[0005] 間葉系幹細胞は、組織幹細胞の一種であり、天然にはごく少量 (ヒト新生児の骨髄 に 1万分の 1存在し、その後急速に減少する。高齢者では 200万分の 1といわれる） 存在するだけであり、分離することが困難である。間葉系幹細胞は、中胚葉以外にも 分ィ匕することが報告されていることから、その応用範囲は従来以上に広がっている。 しかし、そのような分ィ匕の条件は、上述のもの以上に特殊である。間葉系幹細胞の表 面抗原は、 CD105 ( + )、 CD73 ( + )、 CD29 ( + )、 CD44 ( + )、 CD14 (— )、 CD3[0005] Mesenchymal stem cells are a type of tissue stem cells, and are extremely small in nature (present in human bone marrow of newborns at a rate of 1 / 10,000 and then rapidly decreasing. It is said to be 1 / 1,000,000 in the elderly). It only exists and is difficult to separate. It has been reported that mesenchymal stem cells separate from mesodermal cells, and thus their application range is expanding more than before. However, the conditions for such sorting are more specific than those described above. Table of mesenchymal stem cells Surface antigens are CD105 (+), CD73 (+), CD29 (+), CD44 (+), CD14 (-), CD3
4 (一）、 CD45 (一)であるとされて!/、る。 4 (one), CD45 (one) !!
[0006] 一方、脂肪にも幹細胞があることが分力つてきた (特許文献 1一 3、非特許文献 2—[0006] On the other hand, it has been a powerful factor that fat also has stem cells (Patent Documents 1 to 3, Non-Patent Document 2—
3)。脂肪にある幹細胞は、他の組織 (例えば、骨髄）に比べて、その供給源が豊富で あり、存在率も多いようであることから、その利用が注目されている。しかし、幹細胞の 処置方法については、未知な部分が多い。 3). Stem cells in adipose have attracted much attention because of their abundant sources and higher prevalence than other tissues (eg, bone marrow). However, there are many unknowns regarding the method of treating stem cells.
[0007] 特に、このような幹細胞は、それほど多量に取れな 、ことから、大量に幹細胞を必 要とする場合は、多数の供給源力 供給する必要がある。しかし、幹細胞の供給源は 限られており、さらに、複数の系統が混じることによる副作用が生じる恐れもある。 [0007] In particular, since such stem cells cannot be obtained in such a large amount, when a large number of stem cells are required, it is necessary to supply a large number of sources. However, the source of stem cells is limited, and there is a possibility that side effects may occur due to mixing of multiple strains.
[0008] 他方、幹細胞を効率よく増殖する方法に対することが対応策として考えられる。しか し、幹細胞を、その多能性を維持しながら増殖する (増幅する）方法は、ほとんど知ら れておらず、特に、脂肪などに由来する間葉系幹細胞については、増殖方法は皆無 であることが現状である。 [0008] On the other hand, a method for efficiently proliferating stem cells can be considered as a countermeasure. However, there is almost no known method of proliferating (amplifying) stem cells while maintaining their pluripotency, and there is no proliferating method for mesenchymal stem cells derived from fats and the like. That is the current situation.
特許文献 1：国際公開 WOOOZ53795パンフレット  Patent document 1: International publication WOOOZ53795 pamphlet
特許文献 2：国際公開 WO03Z022988パンフレット  Patent document 2: International publication WO03Z022988 pamphlet
特許文献 3：国際公開 WO01Z62901パンフレット  Patent document 3: International publication WO01Z62901 pamphlet
非特許文献 1 :幹細胞'クローン 研究プロトコール 中辻編、羊土社 (2001) 非特許文献 2 :Zuk, P. A. , et al.、 Tissue Engineering, Vol. 7, 211—228、 Non-patent document 1: Stem cell 'clone research protocol Nakatsuji ed., Yodosha (2001) Non-patent document 2: Zuk, P.A., et al., Tissue Engineering, Vol. 7, 211-228,
2001 2001
非特許文献 3 :Zuk, P. A. , et al.、 Molecular Biologyof the Cell Vol. , 1 3, 4279-4295, 2002  Non-Patent Document 3: Zuk, P.A., et al., Molecular Biology of the Cell Vol., 13, 4279-4295, 2002
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] 本発明は、幹細胞 (例えば、間葉系幹細胞 (特に脂肪由来)のような糸且織幹細胞)を 増幅する方法を提供することを課題とする。 [0009] An object of the present invention is to provide a method for amplifying a stem cell (for example, a fibroblast stem cell such as a mesenchymal stem cell (particularly derived from fat)).
課題を解決するための手段  Means for solving the problem
[0010] 上記課題は、本発明者らがフイブリンのりに代表されるフイブリノゲンまたはその誘 導体、あるいは細胞外マトリクス (例えば、コラーゲン (例えば、 I型、 IV型など）、ラミニ ン、フイブロネクチンなど）、ゼラチンなどをコーティングした培養容器を用いることによ つて、脂肪などに由来する幹細胞が効率よく増幅することを見出したことによって完 成された。 [0010] The above-mentioned problems have been solved by the present inventors by considering fibrinogen represented by fibrin glue or a derivative thereof, extracellular matrix (eg, collagen (eg, type I, type IV, etc.), laminin And fibronectin), and the use of a culture vessel coated with gelatin and the like, and found that stem cells derived from fat and the like can be efficiently amplified.
従って、本発明は、以下を提供する。  Accordingly, the present invention provides the following.
( 1)フイブリノゲンまたはその誘導体を含む、幹細胞を培養するための培養容器。 (1) A culture vessel containing fibrinogen or a derivative thereof for culturing stem cells.
(2)上記フイブリノゲンまたはその誘導体は、上記培養容器にコーティングされる、項 目 1に記載の培養容器。 (2) The culture container according to item 1, wherein the fibrinogen or a derivative thereof is coated on the culture container.
(3)上記フイブリノゲンまたはその誘導体は、上記培養容器に 50 μ m— 300 μ mの 厚さでコーティングされる、項目 1に記載の培養容器。  (3) The culture vessel according to item 1, wherein the fibrinogen or a derivative thereof is coated on the culture vessel with a thickness of 50 μm to 300 μm.
(4)上記フイブリノゲンまたはその誘導体は、フイブリンを含む、項目 1に記載の培養 谷器。  (4) The culture well according to item 1, wherein the fibrinogen or a derivative thereof contains fibrin.
(5)上記フイブリンは、フイブリンモノマー、フイブリンポリマーおよびフィブリン塊から なる群より選択される形態を含む、項目 1に記載の培養容器。  (5) The culture container according to item 1, wherein the fibrin has a form selected from the group consisting of a fibrin monomer, a fibrin polymer, and a fibrin clot.
(6)上記フイブリノゲンまたはその誘導体は、プロテアーゼを含む、項目 1に記載の培 養容器。  (6) The culture container according to item 1, wherein the fibrinogen or a derivative thereof contains a protease.
(7)上記フイブリノゲンまたはその誘導体は、トロンビンまたはバトロキソビンを含む、 項目 1に記載の培養容器。  (7) The culture container according to item 1, wherein the fibrinogen or a derivative thereof includes thrombin or batroxobin.
(8)上記フイブリノゲンまたはその誘導体は、 exトロンビンを含む、項目 1に記載の培 養容器。  (8) The culture container according to item 1, wherein the fibrinogen or a derivative thereof contains ex-thrombin.
(9)上記フイブリノゲンまたはその誘導体は、プロテアーゼ阻害剤を含む、項目 1に記 載の培養容器。  (9) The culture vessel according to item 1, wherein the fibrinogen or a derivative thereof contains a protease inhibitor.
( 10)上記プロテアーゼ阻害剤は、ァプロチュンおよび Zまたは 4 (アミノメチル)シク 口へキサンカルボン酸を含む、項目 9に記載の培養容器。  (10) The culture container according to item 9, wherein the protease inhibitor comprises aprotune and Z or 4 (aminomethyl) cyclohexacarboxylic acid.
( 11)上記フイブリノゲンまたはその誘導体は、フイブリンのりである、項目 1に記載の 培養容器。  (11) The culture vessel according to item 1, wherein the fibrinogen or a derivative thereof is fibrin glue.
( 12)上記フイブリンのりは、フイブリンモノマー、フイブリンポリマー、フイブリノゲンまた は血小板の全部もしくは一部を成分として含む、項目 11に記載の培養容器。  (12) The culture container according to item 11, wherein the fibrin glue contains, as a component, all or a part of fibrin monomer, fibrin polymer, fibrinogen or platelets.
( 13)上記フイブリンのりは、上記フイブリンのり中に含まれる可溶性のフイブリノゲン (I 因子)が、トロンビンによって不溶性のフイブリン (フイブリンモノマー）に転換される力 フイブリンモノマーは互いの N末端と C末端が重合して、フイブリンポリマーとなり、フィ ブリン塊を形成していくという接着性を有する、項目 11に記載の培養容器。 (13) The fibrin glue is soluble fibrinogen (I) contained in the fibrin glue. Factor) is converted into insoluble fibrin (fibrin monomer) by thrombin.Fibrin monomer has the adhesive property that the N-terminus and C-terminus of each other polymerize to form a fibrin polymer and form a fibrin clot. , The culture vessel of item 11.
(14)上記フイブリンのりは、クリオプレシピテート、血漿または血小板含有血漿 (PRP )を 500倍以下に希釈したものである、項目 11に記載の培養容器。  (14) The culture container according to item 11, wherein the fibrin glue is prepared by diluting cryoprecipitate, plasma or platelet-containing plasma (PRP) to 500 times or less.
(15)上記フイブリンのりは、クリオプレシピテート、血漿または血小板含有血漿 (PRP )を 50倍一 500倍に希釈したものである、項目 11に記載の培養容器。  (15) The culture container according to item 11, wherein the fibrin glue is prepared by diluting cryoprecipitate, plasma or platelet-containing plasma (PRP) 50-fold to 500-fold.
(16)上記幹細胞は、脂肪組織由来である、項目 1に記載の培養容器。  (16) The culture vessel according to item 1, wherein the stem cells are derived from adipose tissue.
(17)上記幹細胞は、間葉系幹細胞である、項目 1に記載の培養容器。  (17) The culture vessel according to item 1, wherein the stem cells are mesenchymal stem cells.
(18)上記幹細胞は、脂肪吸引物に由来する、項目 1に記載の培養容器。  (18) The culture container according to item 1, wherein the stem cells are derived from a lipoaspirate.
(19)上記幹細胞は、脂肪細胞由来幹細胞である、項目 1に記載の培養容器。 (19) The culture vessel according to item 1, wherein the stem cells are fat cell-derived stem cells.
(20)上記フイブリンまたはその分解物は、 0. 1— lOOmgZcm2の濃度で含まれるこ とを特徴とする、項目 1に記載の培養容器。 (20) The culture container according to item 1, wherein the fibrin or a decomposition product thereof is contained at a concentration of 0.1 to 100 mgZcm 2 .
(21)上記培養容器の材質は、ガラス、シリカ、シリコン、セラミック、二酸化珪素、ブラ スチック、金属、天然ポリマーおよび合成ポリマーからなる群より選択される材料を含 む、項目 1に記載の培養容器。  (21) The culture container according to item 1, wherein the material of the culture container includes a material selected from the group consisting of glass, silica, silicon, ceramic, silicon dioxide, plastic, metal, natural polymer and synthetic polymer. .
(22)幹細胞を調製するための方法であって、  (22) a method for preparing a stem cell,
A)幹細胞を提供する工程；および  A) providing stem cells; and
B)上記幹細胞を、培地ならびにフイブリノゲンまたはその誘導体を含む培養容器中 で培養する工程、  B) culturing the stem cells in a culture vessel containing a medium and fibrinogen or a derivative thereof,
を包含する、方法。 A method comprising:
(23)上記培養は、 pH7. 2-7. 4、温度 37°C、および CO濃度 5%という条件下で  (23) The above culture was performed under the conditions of pH 7.2-7.4, temperature of 37 ° C, and CO concentration of 5%.
2  2
培養される、項目 22に記載の方法。 23. The method according to item 22, which is cultured.
(24)上記培養は、 DMEM、 P199、 MEM、 Hanks平衡化塩溶液（HBSS)、 Ham ，s F12、 BME、 RPMI1640、 MCDB104および MCDB153 (KGM)からなる群 より選択される培地を用いて行われる、項目 22に記載の方法。  (24) The above culture is performed using a medium selected from the group consisting of DMEM, P199, MEM, Hanks balanced salt solution (HBSS), Ham, sF12, BME, RPMI1640, MCDB104 and MCDB153 (KGM). , Item 22.
(25)上記培養は、副腎皮質ステロイド、インスリン、グルコース、インドメタシン、イソ ブチルーメチルキサンチン（IBMX)、ァスコルビン酸およびその誘導体、グリセ口ホス フェート、エストロゲンおよびその誘導体、プロゲステロンおよびその誘導体、アンド口 ゲンおよびその誘導体、増殖因子、下垂体エキス、松果体エキス、レチノイン酸、ビタ ミン D、甲状腺ホルモン、子牛血清、馬血清、ヒト血清、へパリン、炭酸水素ナトリウム 、 HEPES、アルブミン、トランスフェリン、セレン酸塩、リノレン酸、 3 イソブチルー 1— メチルキサンチン、脱メチル化剤、ヒストン脱ァセチル化剤、ァクチビン、サイト力イン、 へキサメチレンビスァセトアミド（HMBA)、ジメチルァセトアミド（DMA)、ジブチル c AMP (dbcAMP)、ジメチルスルホキシド（DMSO)、ョードデォキシゥリジン（IdU)、 ヒドロキシゥレア（HU)、シトシンァラビノシド (AraC)、マイトマイシン C (MMC)、酪 酸ナトリウム (NaBu)、ポリプレンおよびセレニウム力もなる群より選択される成分のう ち少なくとも 1つを含む培養液中で行われる、項目 22に記載の方法。 (25) The above culture is performed using corticosteroids, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbic acid and its derivatives, Fate, estrogen and its derivatives, progesterone and its derivatives, androgens and its derivatives, growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum , Heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate, linolenic acid, 3-isobutyl-1-methylxanthine, demethylating agent, histone deacetylating agent, activin, cytokinin, hexamethylene bisa Cetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethylsulfoxide (DMSO), ododeoxyperidine (IdU), hydroxyperrea (HU), cytosine arabino Cid (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polypropylene and selenium Carried out in a culture medium containing at least one is the component caries Chi selected from the group The method of claim 22.
(26)さらに、上記幹細胞を入手する工程を包含する、項目 22に記載の方法。  (26) The method according to item 22, further comprising a step of obtaining the stem cell.
(27)さらに、上記幹細胞を分化させる工程を包含する、項目 22に記載の方法。 (27) The method according to item 22, further comprising a step of differentiating the stem cell.
(28)上記フイブリノゲンまたはその誘導体は、上記培養容器にコーティングされる、 項目 22に記載の方法。 (28) The method according to item 22, wherein the fibrinogen or a derivative thereof is coated on the culture container.
(29)幹細胞を培養するための、フイブリノゲンまたはその誘導体がコーティングされ た培養容器を生産するための方法であって、  (29) A method for producing a culture vessel coated with fibrinogen or a derivative thereof for culturing stem cells,
A)培養容器を提供する工程；  A) providing a culture vessel;
B)上記培養容器にフイブリノゲンまたはその誘導体を提供する工程;および B) providing fibrinogen or a derivative thereof to the culture vessel; and
C)上記フイブリノゲンまたはその誘導体を上記培養容器の表面に固定する工程、 を包含する、方法。 C) fixing the fibrinogen or a derivative thereof to the surface of the culture vessel.
(30)上記フイブリノゲンまたはその誘導体の提供は、無菌操作にて行われる、項目 2 9に記載の方法。  (30) The method according to item 29, wherein the provision of the fibrinogen or a derivative thereof is performed by an aseptic operation.
(31)上記固定は、乾燥状態で一定時間放置すること、または紫外線照射という特徴 を有する、項目 29に記載の方法。  (31) The method according to item 29, wherein the fixing is characterized by being left in a dry state for a predetermined time or by irradiating with ultraviolet rays.
(32)幹細胞を培養するための、組織接着剤がコーティングされた培養容器。  (32) A culture container coated with a tissue adhesive for culturing stem cells.
(33)上記組織接着剤は、シァノアクリレート系接着剤、ゼラチン アルデヒド系接着 剤およびフイブリングルー系接着剤からなる群より選択される、項目 32に記載の培養 (34)幹細胞を培養するための、細胞外マトリクスがコーティングされた培養容器。(33) The culture according to item 32, wherein the tissue adhesive is selected from the group consisting of a cyanoacrylate adhesive, a gelatin aldehyde adhesive, and a fibrin glue adhesive. (34) A culture vessel coated with an extracellular matrix for culturing stem cells.
(35)上記幹細胞は、脂肪組織由来である、項目 34に記載の培養容器。 (35) The culture container according to item 34, wherein the stem cells are derived from adipose tissue.
(36)上記幹細胞は、間葉系幹細胞である、項目 34に記載の培養容器。  (36) The culture vessel according to item 34, wherein the stem cells are mesenchymal stem cells.
(37)上記幹細胞は、脂肪細胞由来幹 (ASC ; adipose— derived stem cell)細胞 である、項目 34に記載の培養容器。  (37) The culture container according to item 34, wherein the stem cell is an adipose-derived stem cell (ASC) cell.
(38)上記幹細胞は、脂肪吸引物に由来する、項目 34に記載の培養容器。  (38) The culture container according to item 34, wherein the stem cells are derived from a lipoaspirate.
(39)上記細胞外マトリクスは、コラーゲン、ラミニン、フイブロネクチン、テネイシン、サ イトタクチン、へキサブラキオン、ェンタクチン、ナイトジ工ン、ビトロネクチン、ゼラチン およびプロテオダリカン力もなる群より選択される、項目 34に記載の培養容器。 (39) The extracellular matrix according to item 34, wherein the extracellular matrix is selected from the group consisting of collagen, laminin, fibronectin, tenascin, sitetactin, hexabrachion, entactin, nightzine, vitronectin, gelatin and proteodarican. Culture vessels.
(40)上記細胞外マトリクスは、 3 μ g/cm2— 20 μ g/cm2の濃度で含まれることを特 徴とする、項目 34に記載の培養容器。 (40) The culture container according to item 34, wherein the extracellular matrix is contained at a concentration of 3 μg / cm 2 to 20 μg / cm 2 .
(41)幹細胞を培養するための、ゼラチンがコーティングされた培養容器。  (41) A culture vessel coated with gelatin for culturing stem cells.
(42)幹細胞を調製するための方法であって、  (42) A method for preparing a stem cell,
A)培地、ならびに細胞外マトリクスおよび Zまたはゼラチンを含む培養容器を提供 する工程；  A) providing a medium and a culture vessel containing the extracellular matrix and Z or gelatin;
B)幹細胞を上記培養容器に配置する工程；  B) placing the stem cells in the culture vessel;
C)上記幹細胞を所望の期間培養する工程、  C) a step of culturing the stem cells for a desired period,
を包含する、方法。 A method comprising:
(43)幹細胞を培養するための、細胞外マトリクスおよび Zまたはゼラチンがコーティ ングされた培養容器を生産するための方法であって、  (43) A method for producing a culture vessel coated with an extracellular matrix and Z or gelatin for culturing stem cells,
A)培養容器を提供する工程；  A) providing a culture vessel;
B)上記培養容器に細胞外マトリクスおよび Zまたはゼラチンを提供する工程;およ び  B) providing extracellular matrix and Z or gelatin to the culture vessel; and
C)上記細胞外マトリクスおよび Zまたはゼラチンを上記培養容器の表面に固定す る工程、  C) fixing the extracellular matrix and Z or gelatin on the surface of the culture vessel,
を包含する、方法。 A method comprising:
(44)フイブリノゲンまたはその誘導体の、幹細胞を調製するための使用。  (44) Use of fibrinogen or a derivative thereof for preparing a stem cell.
(45)細胞外マトリクスおよび Zまたはゼラチンの、幹細胞を調製するための使用。 [0012] 従って、本発明のこれらおよび他の利点は、添付の図面を参照して、以下の詳細な 説明を読みかつ理解すれば、当業者には明白〖こなることが理解される。 (45) Use of extracellular matrix and Z or gelatin for preparing stem cells. [0012] Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description, with reference to the accompanying drawings.
発明の効果  The invention's effect
[0013] 本発明は、幹細胞を効率よく増殖 Z増幅するための方法を提供する。このような方 法は、従来達成されておらず、美容整形などにおいて有用な幹細胞を効率よく増幅 し、少ない原料から多くの用途に利用することができる。  [0013] The present invention provides a method for efficiently growing and amplifying stem cells. Such a method has not been achieved so far, and can efficiently amplify useful stem cells in cosmetic surgery and the like, and can use it from a small number of raw materials for many uses.
図面の簡単な説明  Brief Description of Drawings
[0014] [図 1]図 1は、各種コーティングディッシュを用いた場合の、 2日目の幹細胞 (脂肪由 来)の増殖速度を示す。  FIG. 1 shows the growth rate of stem cells (derived from fat) on day 2 when various coating dishes were used.
[図 2]図 2は、 PRPコーティングディッシュを用いた場合の、 FBS10%添加した M199 を用いて、ヒト PRPコート 6ウエノレープレート，非コーティング 6ウエノレープレートにヒト脂 肪細胞由来幹細胞 (A)を 2 X 104細胞/ゥエルにて播種した。 4日後に培地交換、 7 日後に細胞数を測定した結果を示す。横軸は、左から、コーティングなし 0%FBS, コーティングなし 10%FBS、 PRPコーティング 0%FBS、 PRPコーティング 10%FBS を示す。縦軸は、細胞数である。 [Fig. 2] Fig. 2 shows human fat cell-derived stem cells on a human PRP-coated 6-wellenplate and an uncoated 6-wellenplate using M199 supplemented with 10% FBS when using a PRP-coated dish. Was seeded at 2 × 10 4 cells / well. The results obtained by replacing the medium after 4 days and measuring the number of cells after 7 days are shown. The horizontal axis shows, from left, 0% FBS without coating, 10% FBS without coating, 0% FBS with PRP coating, and 10% FBS with PRP coating. The vertical axis is the number of cells.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0015] 以下、本発明を説明する。本明細書の全体にわたり、単数形の表現は、特に言及 しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形 の冠詞 (例えば、英語の場合は「a」、「an」、「the」など）は、特に言及しない限り、そ の複数形の概念をも含むことが理解されるべきである。また、本明細書において使用 される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられるこ とが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用さ れる全ての専門用語および科学技術用語は、本発明の属する分野の当業者によつ て一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書 (定義を含め て）が優先する。  Hereinafter, the present invention will be described. It should be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. Therefore, it is to be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of the plural unless specifically stated otherwise. . It is to be understood that the terms used in the present specification are used in a meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. . In case of conflict, the present specification, including definitions, will control.
[0016] (用語の定義）  [0016] (Definition of terms)
以下に本明細書において特に使用される用語の定義を列挙する。  The definitions of terms used particularly in the present specification are listed below.
[0017] 本明細書において使用される「細胞」は、当該分野において用いられる最も広義の 意味と同様に定義され、多細胞生物の組織の構成単位であって、外界を隔離する膜 構造に包まれ、内部に自己再生能を備え、遺伝情報およびその発現機構を有する 生命体をいう。本発明の方法においては、どのような細胞でも対象とされ得る。本発 明で使用される「細胞」の数は、光学顕微鏡を通じて計数することができる。光学顕 微鏡を通じて計数する場合は、核の数を数えることにより計数を行う。当該組織を組 織切片スライスとし、へマトキシリンーェォシン (HE)染色を行うことにより細胞外マトリ タス (例えば、エラスチンまたはコラーゲン)および細胞に由来する核を色素によって 染め分ける。この組織切片を光学顕微鏡にて検鏡し、特定の面積 (例えば、 200 X 200 μ m)あたりの核の数を細胞数と見積って計数することができる。 [0017] As used herein, "cell" is the broadest term used in the art. It is defined in the same way as the meaning, and is a constituent unit of the tissue of a multicellular organism, which is wrapped in a membrane structure that isolates the outside world, has self-renewal ability inside, and has genetic information and its expression mechanism. In the method of the present invention, any cell can be targeted. The number of "cells" used in the present invention can be counted through a light microscope. When counting through an optical microscope, counting is performed by counting the number of nuclei. The tissue is made into a tissue slice, and extracellular matrix (eg, elastin or collagen) and nuclei derived from cells are stained with a dye by performing hematoxylin-eosin (HE) staining. The tissue section can be examined under an optical microscope, and the number of nuclei per specific area (for example, 200 × 200 μm) can be counted as the number of cells.
[0018] 本発明において使用される細胞は、幹細胞 (特に、脂肪組織に由来する幹細胞)ま たはその対応物がある限り、どの生物由来の細胞 (例えば、メタラウナギ類、ャッメゥ ナギ類、軟骨魚類、硬骨魚類、両生類、爬虫類、鳥類、哺乳動物など)でも用いるこ とができる。好ましくは、そのような細胞は、哺乳動物 (例えば、単孔類、有袋類、貧歯 類、皮翼類、翼手類、食肉類、食虫類、長鼻類、奇蹄類、偶蹄類、管歯類、有鱗類、 海牛類、クジラ目、霊長類、齧歯類、ゥサギ目など）由来の細胞が用いられる。 1つの 実施形態では、霊長類 (たとえば、チンパンジー、二ホンザル、ヒト）由来の細胞、特 にヒト由来の細胞が用いられるがそれに限定されない。 [0018] The cells used in the present invention may be stem cells (particularly stem cells derived from adipose tissue) or cells derived from any organism as long as there is a counterpart thereof (eg, metal eel, japonicum, chondrichthyes). And teleost fish, amphibians, reptiles, birds, mammals, etc.). Preferably, such cells are derived from mammals (e.g., monotremes, marsupials, oligodonts, dermis, winged fins, carnivores, insectivores, longnoses, hoofedids, artiodactyla). , Rodents, squamata, squids, cetaceans, cetaceans, primates, rodents, and cyperidae). In one embodiment, cells from primates (eg, chimpanzees, macaques, humans), especially cells from humans, are used, but are not limited thereto.
[0019] 本明細書において「幹細胞」とは、自己複製能を有し、多分ィ匕能 (すなわち多能性） [0019] As used herein, the term "stem cell" has a self-renewal ability, and may have a dangling ability (ie, pluripotency).
(「pluripotency」）を有する細胞をいう。幹細胞は通常、組織が傷害を受けたときに その組織を再生することができる。本明細書では幹細胞は、胚性幹 (ES)細胞または 組織幹細胞 (組織性幹細胞、組織特異的幹細胞または体性幹細胞とも ヽぅ）であり得 るがそれらに限定されない。また、上述の能力を有している限り、人工的に作製した 細胞もまた、幹細胞であり得る。胚性幹細胞とは初期胚に由来する多能性幹細胞を いう。胚性幹細胞は、 1981年に初めて榭立され、 1989年以降ノックアウトマウス作 製にも応用されている。 1998年にはヒト胚性幹細胞が榭立されており、再生医学に も利用されつつある。組織幹細胞は、胚性幹細胞とは異なり、分化の方向が限定さ れている細胞であり、組織中の特定の位置に存在し、未分化な細胞内構造をしてい る。従って、組織幹細胞は多能性のレベルが低い。組織幹細胞は、核 Z細胞質比が 高ぐ細胞内小器官が乏しい。組織幹細胞は、概して、多分化能を有し、細胞周期が 遅ぐ個体の一生以上に増殖能を維持する。本明細書において使用される場合は、 幹細胞は好ましくは間葉系幹細胞のような組織幹細胞であり得る力 状況に応じて胚 性幹細胞も使用され得る。 ("Pluripot e ncy"). Stem cells can usually regenerate tissue when it is damaged. As used herein, a stem cell can be, but is not limited to, an embryonic stem (ES) cell or a tissue stem cell (also a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell). In addition, artificially produced cells can also be stem cells, as long as they have the above-mentioned ability. Embryonic stem cells are pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981 and have been applied to the production of knockout mice since 1989. Human embryonic stem cells were established in 1998 and are being used for regenerative medicine. Tissue stem cells, unlike embryonic stem cells, are cells in which the direction of differentiation is limited, exist at specific positions in tissues, and have an undifferentiated intracellular structure. Thus, tissue stem cells have a low level of pluripotency. Tissue stem cells have a nuclear Z cytoplasmic ratio Poor intracellular organelles are high. Tissue stem cells are generally pluripotent and maintain their proliferative potential for more than one lifetime in individuals whose cell cycle is slow. As used herein, stem cells may preferably be tissue stem cells, such as mesenchymal stem cells. Embryonic stem cells may also be used depending on the context.
[0020] 本明細書にぉ 、て幹細胞と!/、うときは、幹細胞を少なくとも一定量含む組織集合物 をさし得ることが理解される。幹細胞の入手は、当該分野において周知の任意の方 法を用いて行うことができる。したがって、本明細書では、幹細胞は、例えば、コラゲ ナーゼ処理して脂肪組織力 採取した幹細胞 (実施例において使用される脂肪細胞 由来幹細胞など)を用いることができるがそれらに限定されない。  [0020] In this specification, it is understood that when referring to stem cells, a tissue aggregate containing at least a certain amount of stem cells can be used. Obtaining stem cells can be performed using any method known in the art. Therefore, in the present specification, for example, stem cells obtained by adipose tissue harvesting by collagenase treatment (adipocyte-derived stem cells used in Examples) can be used as stem cells, but are not limited thereto.
[0021] 由来する部位により分類すると、組織幹細胞は、例えば、皮膚系、消化器系、骨髄 系、神経系などに分けられる。皮膚系の組織幹細胞としては、表皮幹細胞、毛嚢幹 細胞などが挙げられる。消ィ匕器系の組織幹細胞としては、膝 (共通)幹細胞、肝幹細 胞などが挙げられる。骨髄系の糸且織幹細胞としては、造血幹細胞、間葉系幹細胞な どが挙げられる。神経系の組織幹細胞としては、神経幹細胞、網膜幹細胞などが挙 げられる。  When classified according to the site of origin, tissue stem cells are classified into, for example, skin system, digestive system, bone marrow system, nervous system and the like. Skin tissue tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of tissue stem cells of the conjugation system include knee (common) stem cells, hepatic stem cells, and the like. Examples of myeloid stem cells include hematopoietic stem cells and mesenchymal stem cells. Tissue stem cells of the nervous system include neural stem cells and retinal stem cells.
[0022] 本明細書において「間葉系幹細胞」とは、間葉に見出される幹細胞をいう。本明細 書では MSCと略されることがある。ここで、間葉とは、多細胞動物の発生各期に認め られる、上皮組織間の間隙をうめる星状または不規則な突起をもつ遊離細胞の集団 と，それに伴う細胞間質によって形成される組織をいう。間葉系幹細胞は、増殖能と、 骨細胞、軟骨細胞、骨格筋細胞、平滑筋細胞（心筋細胞を含む）、ストローマ細胞、 腱細胞、脂肪細胞への分化能を有する。間葉系幹細胞は、患者力 採取した骨髄細 胞等を培養または増殖、軟骨細胞あるいは骨芽細胞に分化させるために使用され、 または歯槽骨、関節症等の骨、軟骨、関節などの再建材料として使用されており、そ の需要は大きい。また、間葉系幹細胞は、血液細胞、リンパ系細胞へも分ィ匕し得るこ とから、その需要がますます高まっている。  [0022] As used herein, "mesenchymal stem cell" refers to a stem cell found in mesenchyme. In this specification, it may be abbreviated as MSC. Here, the mesenchyme is formed by a population of free cells with stellate or irregular projections filling the gap between epithelial tissues and the accompanying cell stroma, which are observed at each stage of the development of multicellular animals. Refers to an organization. Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, skeletal muscle cells, smooth muscle cells (including cardiomyocytes), stromal cells, tendon cells, and adipocytes. Mesenchymal stem cells are used to culture or proliferate the collected bone marrow cells, etc., and to differentiate them into chondrocytes or osteoblasts, or to regenerate bone, cartilage, joints, etc., such as alveolar bone and arthrosis. The demand is large. In addition, the demand for mesenchymal stem cells is increasing because they can be divided into blood cells and lymphoid cells.
[0023] 本明細書において「脂肪幹細胞」とは、脂肪組織に由来する幹細胞をいう。このよう な幹細胞の分離方法の一部は公知であり、例えば、非特許文献 1、特許文献 1一 3な どに記載される方法を利用して分離することができる。これらの文献に記載された事 項は、本明細書において特に関連する場所が参考として援用される。本明細書にお ける脂肪幹細胞は、この公知の分離方法によって得られる脂肪組織由来幹細胞を含 む、すべての脂肪組織由来幹細胞のことを指す。 [0023] In the present specification, "adipose stem cell" refers to a stem cell derived from adipose tissue. Some of such methods for separating stem cells are known, and can be separated using, for example, the methods described in Non-Patent Document 1, Patent Documents 13 and the like. What is described in these documents The sections are specifically incorporated herein by reference where relevant. As used herein, adipose stem cells refer to all adipose tissue-derived stem cells, including adipose tissue-derived stem cells obtained by this known separation method.
[0024] ここで、脂肪幹細胞は、 WOOO/53795, WO03/022988, WO01/62901, Zuk, P. A. , et al.、 Tissue Engineering, Vol. 7, 211—228、 2001、ならび に Zuk, P. A. , et al.、 Molecular Biologyof the Cell Vol. , 13, 4279—4 295、 2002などに記載される方法またはその改変を利用して調製することができる。 具体的には、例えば、（1)脂肪吸引物を 1リットル大の分液漏斗を用いて生理食塩水 で十分に洗浄し；（2)上層に脂肪吸引物、下層に生理食塩水が十分に分離したのを 確認し、下層を捨てる。肉眼で見て生理食塩水がほぼ透明になるまでこれを繰り返し ; (3)脂肪吸引物と同量の 0. 075%コラゲナーゼ /PBSをカ卩え、 37°Cでよく攪拌し ながら 30分間インキュベートし；（4)上記の試料に等量の 10%血清加 DMEMをカロ え；（5)上記の試料を 1200gで 10分間遠心分離し；（6)ペレットに 0. 16M NH C1  [0024] Here, adipose stem cells are described in WOOO / 53795, WO03 / 022988, WO01 / 62901, Zuk, PA, et al., Tissue Engineering, Vol. 7, 211-228, 2001, and Zuk, PA, et al. al., Molecular Biology of the Cell Vol., 13, 4279-4295, 2002, or the like or a modification thereof. Specifically, for example, (1) the lipoaspirate is sufficiently washed with a physiological saline using a 1-liter separating funnel; (2) the lipoaspirate is sufficiently in the upper layer, and the saline is sufficiently in the lower layer. Check that it has separated, and discard the lower layer. Repeat until the physiological saline is almost transparent to the naked eye; (3) Add 0.075% collagenase / PBS in the same amount as the lipoaspirate and incubate for 30 minutes at 37 ° C with good mixing (4) Add 10% serum-containing DMEM to the above sample with the same amount of DMEM; (5) Centrifuge the above sample at 1200g for 10 minutes; (6) Add 0.16M NH C1 to the pellet.
4 Four
ZPBSを加えて懸濁し、室温で 10分間インキュベートし；（7)上記の試料を口径 100 μ mのメッシュを用いて吸引ろ過し；および（8)ろ過物を 1200gで 5分間遠心分離す ることによって調製することができる。ここで、調製量に応じて、上記プロトコールをス ケールアップまたはスケールダウンすることは、当業者の技術範囲内である。このよう な方法によって調製された細胞は、脂肪細胞由来幹細胞 (ASC； adipose— derived stem cell)または PLA (processed lipoaspirate)細胞とも呼ばれる。本明細書 において、 ASCと PLA細胞とは、交換可能に用いられ、その調製法に限定されるこ となく脂肪細胞に由来する幹細胞一般を指すことが理解される。従って、 ASCおよび PLA細胞は、上記以外の調製法で調製された幹細胞であり得る。 Suspend with ZPBS and incubate at room temperature for 10 minutes; (7) Suction-filter the above sample using a 100 μm mesh; and (8) Centrifuge the filtrate at 1200 g for 5 minutes. Can be prepared by Here, it is within the skill of a person skilled in the art to scale up or down the above protocol depending on the amount of preparation. The cells prepared by such a method are also called adipocyte-derived stem cells (ASCs) or PLA (processed lipoaspirate) cells. As used herein, ASC and PLA cells are used interchangeably and are understood to refer to adipocyte-derived stem cells in general, without being limited to their preparation. Therefore, ASC and PLA cells may be stem cells prepared by a preparation method other than those described above.
[0025] 本明細書において「体細胞」とは、卵子、***などの生殖細胞以外の細胞であり、 その DNAを次世代に直接引き渡さない全ての細胞をいう。体細胞は通常、多能性 が限定されている力または消失している。本明細書において使用される体細胞は、 天然に存在するものであってもよぐ遺伝子改変されたものであってもよい。  [0025] In the present specification, "somatic cells" are cells other than germ cells such as eggs and sperm, and refer to all cells that do not directly transfer their DNA to the next generation. Somatic cells usually have a limited or pluripotent force or disappear. As used herein, somatic cells may be naturally occurring or genetically modified.
[0026] 本明細書にぉ 、て「分ィ匕 (した)細胞」とは、機能および形態が特殊化した細胞 (例 えば、筋細胞、神経細胞など）をいい、幹細胞とは異なり、多能性はないか、またはほ とんどない。分化した細胞としては、例えば、表皮細胞、脾実質細胞、脾管細胞、肝 細胞、血液細胞、心筋細胞、骨格筋細胞、骨芽細胞、骨格筋芽細胞、神経細胞、血 管内皮細胞、色素細胞、平滑筋細胞、脂肪細胞、骨細胞、軟骨細胞などが挙げられ る。本発明において用いられる場合、分ィ匕細胞は、集団または組織の形態であって ちょい。 [0026] As used herein, the term "divided cells" refers to cells whose functions and morphology are specialized (for example, muscle cells, nerve cells, and the like). No performance or I don't know. The differentiated cells include, for example, epidermal cells, splenic parenchymal cells, splenic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, nerve cells, vascular endothelial cells, dyes Cells, smooth muscle cells, fat cells, bone cells, chondrocytes and the like. When used in the present invention, the sorting cells may be in the form of a population or a tissue.
[0027] 本明細書にぉ 、て「フイブリノゲンまたはその誘導体」とは、フイブリノゲンそのもの およびフイブリノゲンをプロテアーゼなどの分解酵素によって分解したもの、ならびに それを改変したもの（例えば、化学的修飾を行ったもの）をいう。フイブリノゲンまたは その誘導体としては、代表的に、フイブリンのりが挙げられるがそれに限定されない。  [0027] As used herein, the term "fibrinogen or a derivative thereof" refers to fibrinogen itself or fibrinogen obtained by decomposing fibrinogen by a protease or other degrading enzyme, or a product obtained by modifying the same (for example, a product obtained by chemical modification). ). Fibrinogen or a derivative thereof typically includes, but is not limited to, fibrin glue.
[0028] 本明細書において「フイブリン」とは、フイブリノゲンにトロンビンが作用してフイブリノ ペプチド Aおよび Bを遊離した残余のタンパク質 (フイブリンモノマー)およびこのタン ノ ク質を構成単位とする高分子 (フイブリンポリマー)の総称である。無色あるいは白色 、繊維状無定形で弾性のある固体であり、血液凝固の際に血球をからめて硬化し血 餅となる。血漿中および重合阻止作用をもたない塩類溶液中で生成したフイブリンは 析出してゲル状となり、フイブリン塊を形成する。フイブリノゲンおよびフイブリンを構成 するポリペプチド鎖は、 a、 j8および γの 3本鎖からなる。フイブリノゲンにトロンビン および Ca2+を加えて生成したフイブリンは、 30% (WZV)尿素溶液および 1% (W/ V)モノクロル酢酸溶液に可溶性である力 正常血漿に Ca2+を加えて生成したフイブ リンはこれらの溶液に不溶性である。その理由は，前者ではフイブリノゲン（ひ（A) β ( Β) γ ) カ讣ロンビンの作用で Ν末端力 フイブリノペプチド Αおよび Βを遊離してフィ[0028] In the present specification, the term "fibrin" refers to a residual protein (fibrin monomer) in which thrombin acts on fibrinogen to release fibrinopeptides A and B, and a polymer (fibrin monomer) having the protein as a constituent unit. Polymer). It is a colorless or white, fibrous, amorphous, and elastic solid that, upon blood clotting, entraps blood cells and hardens to form a blood clot. Fibrin formed in plasma and in saline solutions that do not inhibit polymerization precipitates and gels, forming a fibrin mass. The polypeptide chain constituting fibrinogen and fibrin consists of three chains a, j8 and γ. Fibrin, formed by adding thrombin and Ca2 + to fibrinogen, is soluble in 30% (WZV) urea solution and 1% (W / V) monochloroacetic acid solution.Fibrin is formed by adding Ca2 + to normal plasma. Phosphorus is insoluble in these solutions. The reason is that in the former, fibrinogen (H (A) β (Β) γ) is released by 作用 -terminal force fibrinopeptides Α and で due to the action of calombin.
2 2
ブリンモノマー（α β γ )となり，さらに Ca2+の存在下で水素結合により重合してフィ ブリンポリマーとなりゲルィ匕した状態となるのに対し，後者ではさらに血漿中に含有さ れて 、て y -グルタミル基転移酵素作用を示す第 VIII因子の作用を受けて，フイブリ ンモノマー分子間に架橋が生じ、イソペプチド結合が形成され、フイブリンモノマーを 構成単位とする高分子（α β γ ) が生成される。フイブリンはプラスミンによって分解 される。他方，フイブリノゲンはある種のへビ毒にも加水分解され，フイブリノペプチド Αだけを遊離して脱 Αフィブリンとなりゲルィ匕する。 Brin monomer (alpha beta gamma), and the relative further become a state of being Gerui spoon becomes fibrin polymer polymerized by hydrogen bonding in the presence of Ca 2+, the latter is further contained in the plasma, Te y Under the action of factor VIII, which exhibits the action of -glutamyltransferase, cross-linking occurs between fibrin monomer molecules, isopeptide bonds are formed, and a macromolecule (αβγ) having fibrin monomer as a constituent unit is generated. You. Fibrin is degraded by plasmin. On the other hand, fibrinogen is also hydrolyzed into certain snake venoms, releasing only fibrinopeptides and defibrinating to form geri.
[0029] 本明細書にお!、て「プロテアーゼ」とは、ペプチド結合を加水分解する酵素を!、う。 本発明では、フイブリノゲンを分解するために用いられ得る。フイブリノゲンを加水分 解することによって、接着性が出てくる力もである。好ましくは、プロテアーゼとしては 、フイブリノゲンの接着性を改善するような分解を行うものが使用され、そのような例と しては、例えば、トロンビン、ノ トロキソビンなどを挙げることができるがそれらに限定さ れない。 [0029] In this specification, "protease" refers to an enzyme that hydrolyzes a peptide bond. In the present invention, it can be used to degrade fibrinogen. Hydrolysis of fibrinogen also has the power to produce adhesiveness. Preferably, a protease that degrades fibrinogen so as to improve the adhesiveness is used as the protease, and examples of such proteases include, for example, thrombin, notroxobin, and the like. Not.
[0030] 本明細書において、プロテアーゼ阻害剤とは、プロテアーゼのペプチド結合分解 活性を阻害する任意の因子をいう。そのようなプロテアーゼ阻害剤としては、例えば、 ァプロチュン、 4 (アミノメチル）シクロへキサンカルボン酸などを挙げることができる がそれらに限定されない。  [0030] As used herein, the term "protease inhibitor" refers to any factor that inhibits the protease's peptide bond degrading activity. Examples of such protease inhibitors include, but are not limited to, aprotune, 4 (aminomethyl) cyclohexanecarboxylic acid, and the like.
[0031] 本明細書にぉ ヽて「組織接着剤」とは、生体組織を接着するために用いられる任意 の薬剤をいう。そのような組織接着剤としては、例えば、シァノアクリレート系接着剤、 ゼラチン アルデヒド系接着剤、フイブリングルー系接着剤などを挙げることができる 力 Sそれらに限定されない。本発明では、組織接着能を有する物質を培養容器にコー ティングすることによって、幹細胞の増殖が顕著に改善したこと 、う効果を有する。  [0031] As used herein, the term "tissue adhesive" refers to any drug used for bonding living tissues. Examples of such tissue adhesives include, but are not limited to, cyanoacrylate adhesives, gelatin aldehyde adhesives, and fibrin glue adhesives. In the present invention, by coating a substance having a tissue adhesive ability on a culture vessel, the proliferation of stem cells is remarkably improved.
[0032] 本明細書において、「シァノアクリレート系接着剤」とは、シァノアクリレート'モノマー の重合反応を利用した接着剤をいう。接着強度が高ぐしかも接着速度が速い点で 優れている。  [0032] In the present specification, "cyanacrylate-based adhesive" refers to an adhesive utilizing a polymerization reaction of a cyanoacrylate 'monomer. It is excellent in that the bonding strength is high and the bonding speed is fast.
[0033] 本明細書にぉ 、て、「ゼラチン-アルデヒド系接着剤」とは、ゼラチン (生体高分子の コラーゲンが変性したもの）とホルムアルデヒド、ダルタルアルデヒドの架橋反応を利 用した接着剤をいう。接着強度は十分高い。  [0033] In the present specification, "gelatin-aldehyde-based adhesive" refers to an adhesive utilizing a crosslinking reaction between gelatin (modified biopolymer collagen) and formaldehyde and dartaldehyde. Say. The bonding strength is high enough.
[0034] 本明細書において、「フイブリンのり」または「フイブリングルー系接着剤」とは、血液 が凝固する反応 (フイブリンの重合過程)を利用した接着剤をいう。この接着剤は、毒 性がなく創傷治癒を阻害しないが、柔軟であるため接着力がそれほど強くない。幹細 胞の維持および増殖に適して ヽることが本発明にお ヽて見出された。理論に束縛さ れないが、フイブリンのり自体が細胞の培地（栄養）となることが一つの要因であること が予想される。フイブリンのりとしては、例えば、クリオプレシピテート、血漿または血小 板含有血漿 (PRP)を適宜の倍率に希釈したものを挙げることができる。  [0034] In the present specification, "fibrin glue" or "fibrin glue-based adhesive" refers to an adhesive utilizing a reaction of coagulating blood (a process of fibrin polymerization). This adhesive is not toxic and does not interfere with wound healing, but is not very adhesive due to its flexibility. The present invention has been found to be suitable for the maintenance and growth of stem cells. Without being bound by theory, it is expected that one factor is that the fibrin glue itself becomes a cell culture medium (nutrition). Examples of the fibrin glue include cryoprecipitate, plasma or platelet-containing plasma (PRP) diluted at an appropriate magnification.
[0035] 本明細書においてフイブリンのりの倍率は、クリオプレシピテートと標準トロンビン溶 液で生成されたものを基準として算出される。通常 1倍一 10000倍のフイブリンのりが 用いられ、好ましくは 5— 5000倍、より好ましくは 10— 1000倍、さらに好ましくは 50 一 500倍のものが使用される。 [0035] In the present specification, the magnification of fibrin glue is defined as cryoprecipitate and standard thrombin solution. It is calculated based on what is generated in the liquid. Usually, 1 to 10000 times fibrin glue is used, preferably 5 to 5000 times, more preferably 10 to 1000 times, and still more preferably 50 to 500 times.
[0036] 本明細書において「細胞外マトリクス」 (ECM)とは「細胞外基質」とも呼ばれ、上皮 細胞、非上皮細胞を問わず体細胞（somatic cell)の間に存在する物質をいう。細 胞外マトリクスは、組織の支持だけでなぐすべての体細胞の生存に必要な内部環境 の構成に関与する。細胞外マトリクスは一般に、結合組織細胞から産生されるが、一 部は上皮細胞や内皮細胞のような基底膜を保有する細胞自身からも分泌される。線 維成分とその間を満たす基質とに大別され、線維成分としては膠原線維および弾性 線維がある。基質の基本構成成分はグリコサミノダリカン (酸性ムコ多糖)であり、その 大部分は非コラーゲン性タンパクと結合してプロテオダリカン (酸性ムコ多糖 タンパ ク複合体)の高分子を形成する。このほかに、基底膜のラミニン、弾性線維周囲のミク ロフイブリル（microfibril)、線維、細胞表面のフイブロネクチンなどの糖タンパクも基 質に含まれる。特殊に分化した組織でも基本構造は同一で、例えば硝子軟骨では軟 骨芽細胞によって特徴的に大量のプロテオダリカンを含む軟骨基質が産生され、骨 では骨芽細胞によって石灰沈着が起こる骨基質が産生される。  [0036] In the present specification, "extracellular matrix" (ECM) is also called "extracellular matrix" and refers to a substance existing between somatic cells regardless of epithelial cells or non-epithelial cells. The extracellular matrix is involved in the composition of the internal environment necessary for the survival of all somatic cells, not just for tissue support. Extracellular matrices are generally produced from connective tissue cells, but some are also secreted from cells that themselves possess basement membranes, such as epithelial and endothelial cells. The fibrous components are classified into fibrous components and fibrous components and elastic fibers. The basic component of the substrate is glycosaminodalican (acid mucopolysaccharide), most of which binds to non-collagenous proteins to form a macromolecule of proteodalican (acid mucopolysaccharide protein complex). In addition, glycoproteins such as laminin in basement membrane, microfibril around elastic fibers, fibers, and fibronectin on cell surface are also included in the matrix. Even in specially differentiated tissues, the basic structure is the same. Produced.
[0037] 本明細書において、「ゼラチン」とは、当該分野において用いられる最広義で用い られ、動物の皮、腱または骨などを構成するコラーゲンを熱湯で処理して得られる誘 導タンパク質をいう。  [0037] In the present specification, "gelatin" is used in the broadest sense used in the art, and refers to an induced protein obtained by treating collagen constituting the skin, tendon or bone of an animal with boiling water. .
[0038] 本明細書にぉ 、て、「培養容器」とは、細胞を培養するための任意の容器を 、う。従 つて、本発明では、培養容器は底面積を有する限り、当該分野において通常使用さ れるような任意の容器を用いることができ、例えば、シャーレ、フラスコ、型容器など、 好ましくは底面積が広い (例えば、 1cm2以上)容器が使用され得るが特定の面積に 限定されるわけではない。その容器の材質もまた、どのような材料を利用してもよぐ ガラス、シリカ、シリコン、セラミック、二酸化珪素、プラスチック (例えば、ポリスチレン、 ポリカーボネートなど）、金属、天然ポリマーおよび合成ポリマーなどが用いられ得る がそれらに限定されない。 [0038] As used herein, the term "culture container" refers to any container for culturing cells. Therefore, in the present invention, as long as the culture vessel has a bottom area, any vessel commonly used in the art can be used, for example, a petri dish, a flask, a mold vessel, etc. A container (eg, 1 cm 2 or more) can be used, but is not limited to a particular area. The container can be made of any material, including glass, silica, silicon, ceramic, silicon dioxide, plastics (eg, polystyrene, polycarbonate, etc.), metals, natural polymers and synthetic polymers. Gain is not limited to them.
[0039] 本明細書において「培地」とは、細胞を培養するために用いられる任意の物質をい い、 DMEM、 P199、 MEM、 Hanks，平衡化塩溶液（HBSS)、 Ham， s F12、 BM E、 RPMI1640、 MCDB104および MCDB153 (KGM)などを挙げることができる がそれに限定されない。培地には、例えば、副腎皮質ステロイド、インスリン、ダルコ ース、インドメタシン、イソブチルーメチルキサンチン（IBMX)、ァスコルビン酸および その誘導体、グリセ口ホスフェート、エストロゲンおよびその誘導体、プロゲステロンお よびその誘導体、アンドロゲンおよびその誘導体、増殖因子、下垂体エキス、松果体 エキス、レチノイン酸、ビタミン D、甲状腺ホルモン、子牛血清、馬血清、ヒト血清、へ ノ《リン、炭酸水素ナトリウム、 HEPES、アルブミン、トランスフェリン、セレン酸塩、リノ レン酸、 3—イソブチルー 1ーメチルキサンチン、脱メチル化剤、ヒストン脱ァセチル化剤 、ァクチビン、サイト力イン、へキサメチレンビスァセトアミド（HMBA)、ジメチルァセト アミド（DMA)、ジブチル cAMP (dbcAMP)、ジメチルスルホキシド（DMSO)、ョー ドデォキシゥリジン（IdU)、ヒドロキシゥレア（HU)、シトシンァラビノシド (AraC)、マイ トマイシン C (MMC)、酪酸ナトリウム（NaBu)、ポリブレン、セレニウムなどの補助成 分を加えても良い。 [0039] As used herein, the term "medium" refers to any substance used for culturing cells. Examples include, but are not limited to, DMEM, P199, MEM, Hanks, equilibrated salt solution (HBSS), Ham, sF12, BME, RPMI1640, MCDB104 and MCDB153 (KGM). The medium includes, for example, corticosteroids, insulin, darcose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbic acid and its derivatives, glycerol phosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives. Derivatives, growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, henolin, sodium bicarbonate, HEPES, albumin, transferrin, selenate Salt, linolenic acid, 3-isobutyl-1-methylxanthine, demethylating agent, histone deacetylating agent, activin, cytoforce, hexamethylenebisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethyl sulfoxy (DMSO), iododoxyperidine (IdU), hydroxyperrea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, etc. Components may be added.
本明細書にぉ 、て「分化細胞への分化を促進する因子」または「分化促進因子」と は、分ィ匕細胞への分ィ匕を促進することが知られている因子 (例えば、化学物質、温度 など）であれば、どのような因子であってもよい。そのような因子としては、例えば、種 々の環境要因を挙げることができ、そのような因子としては、例えば、温度、湿度、 pH 、塩濃度、栄養、金属、ガス、有機溶媒、圧力、化学物質 (例えば、ステロイド、抗生 物質など)などまたはそれらの任意の組み合わせが挙げられるがそれらに限定されな い。そのような因子のうち代表的なものとしては、 DN A脱メチル化剤（5—ァザシチジ ンなど）、ヒストン脱ァセチル化剤（トリコスタチンなど）、核内レセプターリガンド (例え ば、レチノイン酸 (ATRA)、ビタミン D、 T3など）、細胞増殖因子（ァクチビン、 IGF—  As used herein, the term “factor that promotes differentiation into differentiated cells” or “differentiation promoting factor” refers to a factor that is known to promote shunting to shunt cells (for example, chemical Substance, temperature, etc.). Such factors include, for example, various environmental factors, such as temperature, humidity, pH, salt concentration, nutrients, metals, gases, organic solvents, pressure, chemical Examples include, but are not limited to, substances (eg, steroids, antibiotics, etc.) or any combination thereof. Representative of such factors are DNA demethylating agents (such as 5-azacytidine), histone deacetylating agents (such as trichostatin), nuclear receptor ligands (such as retinoic acid (ATRA ), Vitamin D, T3, etc.), cell growth factors (activin, IGF-
3  Three
1、 FGF, PDGFゝ TGF— j8、 BMP2/4など）、サイト力イン（LIFゝ IL— 2、 IL— 6など )、へキサメチレンビスァセトアミド、ジメチルァセトアミド、ジブチル cAMP、ジメチォ ルスルホキシド、ョードデォキシゥリジン、ヒドロキシル尿素、シトシンァラビノシド、マイ トマイシン C、酪酸ナトリウム、ァフイディコリン、フルォロデオキシゥリジン、ポリプレン、 セレンなどが挙げられるがそれらに限定されない。しかし、従来は、分化促進因子とし て、分ィ匕した細胞は想定されていな力 た。分化した細胞は、むしろ、分化を抑制す るような因子を放出すると考えられていた力もである。 1, FGF, PDGF ゝ TGF-j8, BMP2 / 4, etc., cytotoxicity (LIF ゝ IL-2, IL-6, etc.), hexamethylene bisacetamide, dimethylacetamide, dibutyl cAMP, dimethyl Examples include, but are not limited to, sulfoxide, rhododoxy peridine, hydroxyl urea, cytosine arabinoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxy peridine, polypropylene, selenium, and the like. However, conventionally, as a differentiation promoting factor, Therefore, the divided cells had unexpected power. Rather, differentiated cells are also thought to release factors that inhibit differentiation.
[0041] 本発明において使用される代表的な細胞を判別するために有用なマーカーとして は、例えば、（1)脂肪:細胞質内におけるトリグリセリドの存在、 OilRed— O染色、ダリ セロホスフエートデヒドロケナーゼ (Glycerophosphate dehydrogenase = GPDH )活性、細胞質内の GLUT4 、 Ap2 (脂肪酸結合タンパク質）、 LPL (リポタンパク質 リパーゼ）、 PPAR y 1, 2 (ペルォキシソーム増殖活性化レセプター γ 1, 2)、ゃレプ チン (Leptin)の発現；（2)骨細胞、骨組織:アルカリフォスファターゼの存在、骨石灰 ィ匕（カルシウムの沈着）の程度を確認する；ォステオカルシン（Osteocalcin)、ォステ ォポンチン（Osteopontin)、ォステオネクチン（Osteonectin)の発現；（3)軟骨細 胞、軟骨組織:ムコ多糖の存在、タイプ 2コラーゲン、コンドロイチン 4ーサルフエ一ト（ chondroitin— 4 sulfate)の発現 ·存在；（4)骨格筋細胞：細胞質内のミオシンの豊 富な存在などが挙げられるがそれらに限定されない。  [0041] Markers useful for discriminating representative cells used in the present invention include, for example, (1) fat: presence of triglyceride in cytoplasm, OilRed-O staining, dali cellophosphate dehydrokenase (Glycerophosphate dehydrogenase = GPDH) activity, cytoplasmic GLUT4, Ap2 (fatty acid binding protein), LPL (lipoprotein lipase), PPAR y 1, 2 (peroxisome proliferator-activated receptor γ 1, 2), pereptin (Leptin) (2) Osteocytes and bone tissue: Confirm the presence of alkaline phosphatase and the degree of bone calcification (calcification of calcium); Expression of osteocalcin, osteopontin, and osteonectin; (3) Cartilage cells and cartilage tissue: presence of mucopolysaccharide, type 2 collagen, chondroitin 4-sulfate (chondroitin—4 sulfa (4) Skeletal muscle cells: abundant presence of myosin in the cytoplasm, but is not limited thereto.
[0042] 細胞は、同定された細胞を用いてもよいが、性質が未知の細胞であったとしても、 例えば、マーカーを用いることによって、 FACSなどの分別技術を用いて所望の部位 に対応する細胞を調製することができる。 FACSの使用方法は、フローサイトメトリー 自由自在 細胞工学別冊 (秀潤社)、中内編、 1999などに記載されており、本明細 書においてその内容を参考として援用する。  [0042] As the cell, an identified cell may be used, but even if the property is unknown, for example, by using a marker, it corresponds to a desired site by using a sorting technique such as FACS. Cells can be prepared. The method of using FACS is described in, eg, Flow Cytometry Freedom Cell Engineering Separate Volume (Shujunsha), Nakauchi, 1999, and the like, and the contents thereof are incorporated herein by reference.
[0043] 本明細書において「移植」とは、本発明の細胞、組成物、医薬などを、単独で、また は他の治療剤と組み合わせて体内に移入することを意味する。本発明により調製さ れた幹細胞またはその分ィ匕産物は、種々の目的 (例えば、治療、再生、美容、予防 目的など)で体内に移植することができる。  [0043] As used herein, "transplantation" means that the cells, compositions, medicaments, and the like of the present invention are introduced into the body alone or in combination with other therapeutic agents. The stem cells or their derivatives prepared according to the present invention can be transplanted into the body for various purposes (eg, for therapeutic, regenerative, cosmetic, prophylactic, etc.).
[0044] 本明細書において自己または自家とは、ある個体についていうとき、その個体に由 来する個体またはその一部（例えば、細胞、組織、臓器など）をいう。本明細書にお いて自己というときは、広義には遺伝的に同じ他個体 (例えば一卵性双生児）からの 移植片をも含み得る。  [0044] In the present specification, the term "self" or "self" refers to an individual or a part thereof (eg, cells, tissues, organs, etc.) derived from the individual. As used herein, the term self may broadly include a transplant from another genetically identical individual (eg, an identical twin).
[0045] 本明細書において同種（同種異系）とは、同種であっても遺伝的には異なる他個体 カゝら移植される個体またはその一部 (例えば、細胞、組織、臓器など)をいう。遺伝的 に異なることから、同種異系のものは、移植された個体 (レシピエント）において免疫 反応を惹起し得る。そのような細胞などの例としては、親由来の細胞などが挙げられ るがそれらに限定されない。 [0045] In the present specification, the allogeneic (allogeneic) refers to an individual who is the same species but is genetically different from another individual to be transplanted or a part thereof (eg, cells, tissues, organs, etc.). Say. genetically Allogeneic individuals can elicit an immune response in transplanted individuals (recipients). Examples of such cells include, but are not limited to, cells derived from the parent.
[0046] 本明細書において異種とは、異種個体力 移植されるものをいう。従って、例えば、 ヒトがレシピエントである場合、ブタからの移植物は異種移植物という。  [0046] In the present specification, the term "heterologous" refers to a substance that is transplanted into a heterologous individual. Thus, for example, where a human is the recipient, a transplant from a pig is referred to as a xenograft.
[0047] 本発明にお 、て使用されるコーティング剤（例えば、フイブリノゲンまたはその誘導 体、細胞外マトリクスなど）としては、培養が企図される細胞と、異種であってもよぐ同 種 (特に自家)であってもよ 、。  [0047] In the present invention, the coating agent (for example, fibrinogen or its derivative, extracellular matrix, etc.) used in the present invention may be of the same type as the cell to be cultured (especially It may be your own).
[0048] 幹細胞の提供者がヒトである場合、通常インフォームド 'コンセントを得る必要がある  [0048] If the donor of stem cells is a human, it is usually necessary to obtain informed consent
[0049] ヒト受精胚の提供候補者については、幹細胞研究に凍結胚を提供依頼にあたって のインフォームド 'コンセントによる同意が与えられるかどうかを、次のような手順に従 つて確認する。不妊治療の開始から凍結胚の廃棄決定に至る手続き、および関連文 書、その後に始まる幹細胞研究への提供に関するプロセスの例示的概要を示す。 [0049] For human fertilized embryo donor candidates, it is confirmed whether or not informed consent is given when requesting the supply of frozen embryos for stem cell research, according to the following procedure. Here is an exemplary outline of the process from the start of fertility treatment to the decision to discard frozen embryos, as well as related documentation, followed by a process to provide for stem cell research.
[0050] (1) 不妊治療の結果作られて凍結保存されているヒト受精胚が、子宮へ移植され ることなく廃棄させることが決定するまでは、幹細胞研究とは全く無関係な不妊治療 プロセスとして患者と不妊治療担当医師による臨床的問題である。  [0050] (1) Until it is decided that human fertilized embryos produced and cryopreserved as a result of infertility treatment should be discarded without being transplanted to the uterus, this is a fertility treatment process completely unrelated to stem cell research. It is a clinical problem with the patient and the fertility physician.
[0051] (2) 廃棄させることが決定した凍結胚について、不妊治療担当医師が中立的立 場を保ちながら、幹細胞の研究について説明を受けるかどうか提供候補者に質問す る。  [0051] (2) For the frozen embryo that is decided to be discarded, the physician in charge of infertility treatment asks the donor candidate whether or not to receive an explanation on the study of stem cells while maintaining a neutral position.
[0052] (3) 説明を受けたいとの意思を示した提供候補者に対して、榭立機関の説明者（ 榭立責任者以外)が幹細胞とはなにか、将来の医療への応用の可能性、研究内容 の概要、幹細胞株榭立によって提供者は利益も不利益も受けないこと、提供者のプ ライパシーは保護されること、などについて十分に説明する。これらの説明は、全く中 立の立場で行われる。  [0052] (3) For a candidate who has indicated that he / she wants to receive an explanation, the explainer of the institution (other than the person in charge of establishment) will be able to apply stem cell to what is future medical treatment. Give a thorough explanation of the nature of the research, the outline of the research, the fact that the donor will not benefit or disadvantage from the establishment of the stem cell line, and the privacy of the donor will be protected. These explanations are given in a completely neutral way.
[0053] (4) 提供候補者は説明を受けた後に、提供医療機関の長に対して提供に同意す るかどうかを回答する。同意はインフォームド 'コンセントの書類への署名を必要とし、 その書類は提供医療機関が厳重に保管する。特定の提供候補者による同意あるい は不同意に関する結果は榭立関係者には伝えない。 [0053] (4) After receiving the explanation, the providing candidate responds to the head of the providing medical institution whether or not he / she agrees with the providing. Consent requires the signing of informed 'consent' documents, which will be kept securely by the provider. With the consent of a specific candidate Will not communicate the consequences of disagreement to interested parties.
[0054] (5) 提供候補者による同意の署名力 一ヶ月間以上の猶予期間をおいて、提供 候補者の意思に変更がない場合は、凍結胚を榭立機関に移送する。その際、複数 の提供者力もの凍結胚を同時に引渡すとともに、凍結胚容器力 は提供者を同定で きるラベルなどを完全に除去しておく。従って、榭立機関の説明担当者が複数の提 供候補者と面会はするが、その氏名などの個人情報は知ることがなぐまたそれらの 候補のうち誰が同意して提供者となった力も知ることがないので、提供者の匿名性は 保証され得る。さらに、複数の提供者からの凍結胚を使ってその一部のみ力 細胞 株が樹立されるので、どの提供者の胚カも実際に幹細胞株が樹立されたかは榭立機 関と提供医療機関の両者ともに知ることができな ヽようにする。  (5) Signing power of consent by donor candidate After a grace period of at least one month, if there is no change in the intention of the donor candidate, the frozen embryo is transferred to the institution. At that time, multiple frozen embryos from the donor are delivered at the same time, and the label for identifying the donor is completely removed from the frozen embryo container. Therefore, the instructor of the institution will meet with multiple donors, but will not know the personal information such as their names, nor will they know who has agreed to become a donor. Since there is no such thing, the anonymity of the provider can be guaranteed. In addition, since only a part of the cell lines are established using frozen embryos from multiple donors, it is important to note that the embryonic cell lines of all donors actually established the stem cell lines. So that they cannot both know.
[0055] 本明細書において「キット」とは、通常 2つ以上の区画に分けて、提供されるべき部 分 (例えば、試薬、培養容器、コーティング剤、粒子など)が提供されるユニットをいう 。混合されて提供されるべきでなぐ使用直前に混合してまたはコーティングして使用 することが好ましいような組成物の提供を目的とするときに、このキットの形態は好まし い。そのようなキットは、好ましくは、提供される部分 (例えば、試薬、培養容器、コー ティング剤、粒子など)をどのように処理すべきかを記載する説明書を備えていること が有利である。このような説明書は、どのような媒体であってもよぐ例えば、そのよう な媒体としては、紙媒体、伝送媒体、記録媒体などが挙げられるがそれらに限定され ない。伝送媒体としては、例えば、インターネット、イントラネット、ェクストラネット、 LA Nなどが挙げられるがそれらに限定されない。記録媒体としては、 CD— ROM、 CD— R、フレキシブルディスク、 DVD— ROM、 MD、ミニディスク、 MO、メモリースティック などが挙げられるがそれらに限定されない。  [0055] As used herein, the term "kit" refers to a unit which is usually divided into two or more sections and provided with a part to be provided (eg, a reagent, a culture vessel, a coating agent, particles, and the like). . This kit form is preferred when it is intended to provide a composition such that it should preferably be mixed or coated shortly before use and should not be provided as a mixture. Advantageously, such kits preferably include instructions describing how to provide the provided parts (eg, reagents, culture vessels, coating agents, particles, etc.). Such instructions may be in any medium. Examples of such medium include, but are not limited to, a paper medium, a transmission medium, and a recording medium. Examples of the transmission medium include, but are not limited to, the Internet, an intranet, an extranet, and a LAN. Examples of the recording medium include, but are not limited to, CD-ROM, CD-R, flexible disk, DVD-ROM, MD, mini disk, MO, and memory stick.
[0056] 本発明では、任意の培養液を用いることができる。そのような培養液としては、例え ば、 DMEM、 P199、 MEM、 HBSS (Hanks平衡化塩溶液）、 Ham' s F12、 BM E、 RPMI1640、 MCDB104、 MCDB153 (KGM)などが挙げられるがそれらに限 定されない。このような培養液には、デキサメタゾン（dexamethasone)などの副腎皮 質ステロイド、インスリン、グルコース、インドメタシン、イソブチルーメチルキサンチン（I BMX)、ァスコルべ一トー 2—ホスフェート（ascorbate— 2— phosphate)、ァスコルビン 酸およびその誘導体、グリセ口ホスフェート（glycerophosphate)、エストロゲンおよ びその誘導体、プロゲステロンおよびその誘導体、アンドロゲンおよびその誘導体、 a FGF - bFGF - EGF - IGF - TGF β · ECGF.BMP· PDGFをはじめとする増殖因子、 下垂体エキス、松果体エキス、レチノイン酸、ビタミン D、甲状腺ホルモン、子牛血清 、馬血清、ヒト血清、へパリン、炭酸水素ナトリウム、 HEPES、アルブミン、トランスフエ リン、セレン酸（亜セレン酸ナトリウムなど）、リノレン酸、 3—イソブチルー 1ーメチルキサ ンチン、 5—ァザンシチジンなどの脱メチル化剤、トリコスタチンなどのヒストン脱ァセチ ル化剤、ァクチビン、 LIF'IL— 2'IL— 6などのサイト力イン、へキサメチレンビスァセト アミド（HMBA)、ジメチルァセトアミド（DMA)、ジブチル cAMP (dbcAMP)、ジメチ ルスルホキシド（DMSO)、ョードデォキシゥリジン（IdU)、ヒドロキシゥレア（HU)、シ トシンァラビノシド (AraC)、マイトマイシン C (MMC)、酪酸ナトリウム（NaBu)、ポリブ レン、セレニウムなどを 1つまたはその組み合わせとして含ませておいてもよい。 [0056] In the present invention, any culture solution can be used. Examples of such culture media include, but are not limited to, DMEM, P199, MEM, HBSS (Hanks balanced salt solution), Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM). Not determined. Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, and ascorbine. Acid and its derivatives, glycerophosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, a FGF-bFGF-EGF-IGF-TGF β · ECGF. BMP · PDGF and other proliferation Factor, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (selenium suboxide) Sodium), linolenic acid, demethylating agents such as 3-isobutyl-1-methylxanthin, 5-azancytidine; histone deacetylating agents such as trichostatin; activin; sites such as LIF'IL-2'IL-6 Force-in, hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbc AMP), dimethyl sulfoxide (DMSO), eododexperidine (IdU), hydroxyperrea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu) , Polybrene, selenium, or the like may be included as one or a combination thereof.
[0057] 本明細書において「コーティング」とは、物体表面を特定の物質の薄膜でおおうこと をいう。そのような物質としては、任意の物質を用いることができる。本発明では、特に 、フイブリノゲンまたはその誘導体、組織接着剤、細胞外マトリクス、ゼラチンなどをコ 一ティングに用いることができるがそれらに限定されない。このようなコーティングは、 当該分野において周知の技術によって実施することができ、例えば、そのような生体 適合性高分子溶液中に脱細胞化組織を浸す方法、あるいは、スプレーなどで塗布 する方法などが挙げられるがそれらに限定されない。コーティングを行った後は、好 ましくは固定を行うことが有利であり得る。固定には、例えば、乾燥、放置、化学処理 、物理的処理など任意の方法を用いることができる。例えば、コーティングは、架橋を することによって固定され得るがそれに限定されない。そのような架橋は、例えば、化 学処理、ラジカル反応 (例えば、紫外線照射、フリーラジカル供給源への暴露、超音 波処理、 X線照射、 γ線照射および電子線照射など）によって行うことができる。  [0057] As used herein, "coating" refers to covering the surface of an object with a thin film of a specific substance. Any substance can be used as such a substance. In the present invention, in particular, fibrinogen or a derivative thereof, a tissue adhesive, an extracellular matrix, gelatin, and the like can be used for coating, but are not limited thereto. Such coating can be performed by a technique well known in the art, for example, a method of immersing the decellularized tissue in such a biocompatible polymer solution or a method of applying by spraying. But not limited to them. After applying the coating, it may be advantageous to perform fixing, preferably. For fixing, any method such as drying, standing, chemical treatment, and physical treatment can be used. For example, the coating may be fixed by cross-linking, but is not so limited. Such cross-linking can be accomplished, for example, by chemical treatment, radical reactions (eg, ultraviolet irradiation, exposure to free radical sources, ultrasonic treatment, X-ray irradiation, gamma irradiation, and electron beam irradiation). it can.
[0058] コーティングには、本発明の特定の物質に加えて、他の適切なさらなる物質を用い ることができることが理解される。例えば、そのような物質には、生体適合性分子を挙 げることができる。  [0058] It will be appreciated that, in addition to the specific materials of the present invention, other suitable additional materials may be used for the coating. For example, such substances can include biocompatible molecules.
[0059] 好ましい実施形態において、本発明において用いられる生体適合性高分子は、生 分解性であり得る。 [0059] In a preferred embodiment, the biocompatible polymer used in the present invention is a biocompatible polymer. Can be degradable.
[0060] 本明細書にぉ 、て「生体適合性」とは、毒性および免疫学的拒絶能がな!、ために 生体内で障害なく存在することができる性質を 、う。  [0060] As used herein, the term "biocompatible" refers to the property of being toxic and immunologically rejectable and capable of existing in vivo without any obstacles.
[0061] 本明細書にぉ 、て「生体適合性高分子」とは、生体適合性のょ 、高分子を 、い、具 体的には、残存しても毒性を生じないことをいう。ある高分子がそのような生体適合性 を有しているかどうかを判定する方法は、本明細書においては、ラット等の実験動物 皮下への埋植試験等の試験法を使用する。この試験法では、皮下埋植試験の結果 [0061] As used herein, the term "biocompatible polymer" refers to a polymer that is biocompatible and does not cause toxicity even if it remains. In the present specification, a method for determining whether or not a certain polymer has such biocompatibility uses a test method such as a subcutaneous implantation test in a laboratory animal such as a rat. This test method uses the results of a subcutaneous implantation test.
、比較的急性の免疫反応やアレルギー反応等が起き、腫れたり、発赤もしくは発熱し たりする場合には肉眼的に生体適合性が低いことが解る。さらに人工血管を動物の 血管に移植した場合など、特定の患部に移植した場合には、数日力も数ケ月後に、 移植箇所を観察し、組織の生着の有無、移植組織周辺の炎症、癒着、血液凝固によ る血栓形成などの程度を観察して、組織適合性の判定を行う。この他、移植部位の 組織切片を作成してへマトキシリン'ェォシン染色その他の染色法にて細胞を染色' 観察し、組織適合性の低さの指標としては免疫系を担当する顆粒性の細胞が多く侵 入しているかどうか、もしくは従来組織と移植組織の間に両者を隔てる瘢痕組織の形 成が認められるか否かを判定する。また、特に脱細胞化組織の組織適合性の高さの 指標としては、血管内皮細胞、繊維芽細胞、平滑筋細胞など各種の細胞が従来組 織より侵入し、再細胞化が起きているかどうか (すなわち従来組織と移植組織の境界 が明確でな!、程度に移植組織が生着して ヽるかどうか）を判定する。 However, if a relatively acute immune reaction or allergic reaction occurs, and swelling, redness, or fever occurs, the biocompatibility is low. In addition, when an artificial blood vessel is transplanted into a specific affected area, such as when transplanted into an animal's blood vessel, the transplant site is observed several days after several months, and the presence or absence of tissue engraftment, inflammation around the transplanted tissue, and adhesion Then, the degree of thrombus formation due to blood coagulation is observed to determine histocompatibility. In addition, a tissue section of the transplant site is prepared and cells are stained with hematoxylin and 'yesin' staining, and other staining methods' are observed.Granular cells responsible for the immune system are used as an indicator of poor histocompatibility. Determine if there is much invasion or if there is any formation of scar tissue separating the conventional tissue and the transplanted tissue. In particular, as an indicator of the degree of histocompatibility of the decellularized tissue, whether or not various cells such as vascular endothelial cells, fibroblasts, and smooth muscle cells have invaded from the conventional tissue and recellularized has occurred. (That is, whether the boundary between the conventional tissue and the transplanted tissue is clear! And whether the transplanted tissue has survived to a degree).
[0062] また、上記の組織や細胞の形状観察による判定以外にも、炎症反応の進行に起因 する血液中のサイト力インの濃度、異物として認識された移植組織に対する抗体の濃 度 (力価)、補体成分の濃度、異物埋入により誘導された薬物代謝酵素 (P450等)の 酵素量、等の生物化学的な定量値とその移植前後の変動の追跡をもって、組織適 合性の指標とする場合もある。  [0062] In addition to the above-described determination by observing the shapes of tissues and cells, the concentration of cytoforce in blood caused by the progress of the inflammatory reaction, the concentration of antibody against transplanted tissue recognized as a foreign substance (titer) ), The concentration of complement components, the amount of drug-metabolizing enzymes (P450, etc.) induced by foreign body implantation, etc. In some cases,
[0063] 医療器具類の材料については、その毒性を評価して医療器具への使用を合理的 に規制する為に、細胞毒性試験、感作性試験、刺激性試験、埋植試験、遺伝毒性 試験、血液適合性試験、全身毒性試験、などの試験項目があり、厚生労働省のガイ ドライン、米国の National Standards,国際的産業基準の ISO— 10993等により個 別の試験法が規定されて!、る。 [0063] In order to evaluate the toxicity of the materials for medical devices and to reasonably regulate their use in medical devices, cytotoxicity tests, sensitization tests, irritation tests, implant tests, genotoxicity tests, etc. There are test items such as test, blood compatibility test, systemic toxicity test, etc., and they are individual items according to the guidelines of the Ministry of Health, Labor and Welfare, the US National Standards, and the international industrial standard ISO-10993. Another test method is specified!
[0064] 生体適合性高分子としては、例えば、ポリビニルアルコール、ポリ乳酸、ポリグリコー ル酸、ポリビュルピロリドン、ポリエチレングリコール、ゼラチン、コラーゲン、 γ—ポリグ ルタミン酸、シリコーン、ポリ塩化ビニル、ポリメタクリル酸メチル、ポリテトラフルォロェ チレン、ポリエステル、ポリプロピレン、ポリウレタン、セルロース、ポリスチレン、ナイ口 ン、ポリカーボネート、ピリサルホン、ポリアクリロニトリル、コラーゲン、ゼラチン、フイブ リン、キチン、キトサン、アルギン酸、デンプン、 γ -ポリグルタミン酸、ポリ力プロラクト ン、ポリヒドロキシ酪酸などが挙げられるがそれらに限定されない。ここで、ポリビュル アルコールの中には、未修飾のものの他に、アミノ基、カルボキシル基、ァセチル基 などにより若干の程度化学修飾されたものもあり、それらは市販されており、これらの 改変ポリビニルアルコールもまた本発明にお 、て使用することができる。好ましくは、 生体適合性高分子は、生分解性であるがそれに限定されな ヽ。  Examples of the biocompatible polymer include polyvinyl alcohol, polylactic acid, polyglycolic acid, polybutylpyrrolidone, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid, silicone, polyvinyl chloride, and polymethyl methacrylate. , Polytetrafluoroethylene, polyester, polypropylene, polyurethane, cellulose, polystyrene, nylon, polycarbonate, pyrisulfone, polyacrylonitrile, collagen, gelatin, fibrin, chitin, chitosan, alginic acid, starch, γ-polyglutamic acid, poly Examples include, but are not limited to, force prolactone, polyhydroxybutyric acid, and the like. Here, among the polybutyl alcohols, in addition to the unmodified ones, there are also those that have been chemically modified to some extent with amino groups, carboxyl groups, acetyl groups, etc., and these are commercially available, and these modified polyvinyl alcohols are commercially available. Can also be used in the present invention. Preferably, the biocompatible polymer is biodegradable, but not limited thereto.
[0065] 本明細書において「生分解性」とは、物質について言及するとき、生体内で，あるい は微生物の作用により分解される性質をいう。生分解性の高分子は、例えば、加水 分解により，水，二酸化炭素、メタンなどに分解され得る。本明細書では、生分解性 であるかどうかを判定する方法は、生分解性の一部である生体吸収性に関しては、ラ ット、ゥサギ、ィヌなど実験動物への数日間から数年間にわたる埋植試験、微生物に よる分解の試験に関しては、シート状の高分子の土壌中での数日間から数年間にわ たる埋入 '崩壊試験などの方法を使用する。移植に関する場合、動物における試験 を使用することが好ましい。また、上記の試験方法に準ずるより簡便な代替試験法と しては、高分子の非酵素的分解についてはリン酸緩衝液生理食塩水（PBS)などの 各種緩衝液を、高分子の酵素分解については当該高分子の加水分解酵素 (プロテ ァーゼ、グリコシダーゼ、リパーゼ、エステラーゼ等）を添加した緩衝液を、それぞれ 用いて水溶液中での溶解試験を行うこともある。生分解性の高分子には、天然およ び合成高分子がある。天然高分子の例としては，コラーゲン，デンプンなどのタンパ ク質、多糖類が挙げられ、そして合成高分子の例としてはポリグリコール酸、ポリ乳酸 、ポリエチレンスクシナートなどの脂肪族ポリエステルが挙げられるがそれらに限定さ れない。ポリビニルアルコールは、生分解性については、弱い生分解性を示すことか ら、本明細書では、生分解性を有するものとして認識され得る。 [0065] As used herein, the term "biodegradable" refers to the property of being degraded in vivo or by the action of microorganisms when referring to a substance. The biodegradable polymer can be decomposed into water, carbon dioxide, methane, and the like, for example, by hydrolysis. In this specification, the method of determining whether a substance is biodegradable is based on the bioabsorbability, which is a part of biodegradability, for a few days to several years for experimental animals such as rats, puppies, and dogs. For a wide range of embedding tests and microbial degradation tests, use methods such as embedding and disintegration tests of sheet-shaped macromolecules in soil for several days to several years. For transplantation, it is preferable to use tests in animals. As a simpler alternative test method similar to the above test method, various buffers such as phosphate buffered saline (PBS) can be used for non-enzymatic degradation of polymers. For, a dissolution test in an aqueous solution may be carried out using a buffer solution to which the high-molecular hydrolase (protease, glycosidase, lipase, esterase, etc.) is added. Biodegradable polymers include natural and synthetic polymers. Examples of natural polymers include proteins such as collagen and starch, and polysaccharides, and examples of synthetic polymers include aliphatic polyesters such as polyglycolic acid, polylactic acid, and polyethylene succinate. However, they are not limited to them. Does polyvinyl alcohol show weak biodegradability? Thus, in the present specification, it can be recognized as having biodegradability.
[0066] 本明細書において「高分子」は、「分子」と同様の意味で用いられ、特に分子量を限 定しない。高分子について分子量を限定して議論する場合、通常、分子量が 500以 上のものを指すがそれに限定されな 、。本発明にお 、て用いられる高分子の分子量 の上限は、原理的には無限大である力 本発明では、通常、溶液として取り扱い、分 子量を議論できる (動的光散乱、ゲル濾過、遠心分離器による沈降平衡などの分子 量測定が適用できるという意味で)のは分子量 500万程度までが使用され得るがそ れに限定されない。  [0066] In the present specification, "polymer" is used in the same meaning as "molecule" and does not particularly limit the molecular weight. When discussing a polymer with a limited molecular weight, it generally refers to a polymer having a molecular weight of 500 or more, but is not limited thereto. In the present invention, the upper limit of the molecular weight of the polymer used in the present invention is a force that is infinite in principle.In the present invention, it is usually handled as a solution, and the molecular weight can be discussed (dynamic light scattering, gel filtration, In the sense that molecular weight measurement such as sedimentation equilibrium using a centrifuge can be applied), a molecular weight of up to about 5,000,000 can be used, but is not limited thereto.
[0067] (発明を実施するための好ましい実施形態）  (Preferred embodiment for carrying out the invention)
以下に本発明の好ましい実施形態を説明する。以下に提供される実施形態は、本 発明のよりよい理解のために提供されるものであり、本発明の範囲は以下の記載に 限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参 酌して、本発明の範囲内で適宜改変を行うことができることは明らかである。  Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is apparent that those skilled in the art can appropriately make modifications within the scope of the present invention in view of the description in the present specification.
[0068] 1つの局面において、本発明は、フイブリノゲンまたはその誘導体を含む、幹細胞を 培養するための培養容器を提供する。フイブリノゲンまたはその誘導体としては、任 意のものを使用することができることが理解される。従って、フイブリノゲンまたはその 誘導体の調製には、当該分野において公知の任意の方法を用いることができる。フィ ブリンのりの代表的な作製法を以下に示す。  [0068] In one aspect, the present invention provides a culture vessel for culturing stem cells, comprising fibrinogen or a derivative thereof. It is understood that any fibrinogen or derivative thereof can be used. Therefore, any method known in the art can be used for preparing fibrinogen or a derivative thereof. A typical method for producing fibrin glue is shown below.
[0069] (A液）  [0069] (Solution A)
ACD加血 (テルモ 4連バッグ）またはその等価物（4時間以内のものを使用する琴 が好ましいようである）を使用する。この血液を、 4°C 3000rpm lOmin (あるいは 4000rpm 6分）で処理する。バフィ一コート、赤血球を取り除き血漿を使用する。こ の血漿は、 80°Cで急速冷凍するか、あるいは、 40°Cでエアキャップなどを巻いて ゆっくり凍結させる。（フイブリンの収量を出来るだけ多くするため）。ここで、静置した 状態で凍結'解凍を 2度繰り返すと収量は 1. 5倍になるとも言われる。あるいは、 20 °Cで振盪しながら緩速凍結し、 4°C水槽で緩速解凍したほうが、収量が多いようであ る。 20°Cで保存する。解凍は、 4°Cで 16— 18— 24時間程度行う。  Use ACD blood (Terumo quadruple bags) or its equivalent (preferably a koto with less than 4 hours). The blood is processed at 3000 rpm 10 min at 4 ° C (or 4000 rpm for 6 minutes). Buffy coat, remove red blood cells and use plasma. This plasma should be frozen at 80 ° C or slowly frozen at 40 ° C with an air cap. (To maximize the yield of fibrin). Here, it is said that the yield is increased 1.5 times if frozen and thawed twice in the still state. Alternatively, slower freezing with shaking at 20 ° C and slower thawing in a 4 ° C water bath seems to yield more. Store at 20 ° C. Thaw at 4 ° C for 16-18-24 hours.
[0070] 使用時には、 4°C 2300rpm 40分で遠心分離して上清 (FFP)を除き、沈澱を使 用する。この際、最後の遠沈の後、 4°Cの部屋で逆さにしておくとさらに収量が上がる ようである。また、振盪したほうが、収量が多くなるようである。再凍結後 4°C水槽中で 解凍したほうが室温解凍のものよりも収量が多いようである。 [0070] When used, remove the supernatant (FFP) by centrifugation at 4 ° C at 2300 rpm for 40 minutes, and use the precipitate. Use. At this time, it seems that the yield will be further increased if it is turned upside down in a 4 ° C room after the last centrifugation. Also, it appears that the shaking results in a higher yield. After refreezing, thawing in a 4 ° C water bath seems to have higher yields than thawing at room temperature.
[0071] 保存は、—80°Cで急速冷凍するか、あるいは、—20°Cで保存する。使用時には、 37 °Cで解凍し使用する。 [0071] Storage is performed by quick freezing at -80 ° C or storage at -20 ° C. Thaw at 37 ° C before use.
[0072] 使用時のフイブリン濃度は、 lOmgZml程度から lOOmgZml程度のものが使用さ れ得る。市販のものは 80mgZmlが多い。  [0072] The fibrin concentration at the time of use may be from about lOmgZml to about lOOmgZml. Commercially available products are often 80mgZml.
[0073] < BW > [0073] <BW>
B液は、自己血輸血マ-ユアル:高折益彦 先生 著 克誠堂出版、 1996に準じて 調製することができる。 B液は、トロンビン末 5000単位； 0. 5M塩化カルシウム液 lml ァプロチュン 3500単位;蒸留水 7. 5mlで調製することができる。ァプロチュンは、 B ayerなど力 入手することができる。  Solution B can be prepared in accordance with Autotransfusion Manual: Dr. Masuhiko Takaori, published by Katseido Shuppan, 1996. Solution B can be prepared with 5000 units of thrombin powder; 0.5M calcium chloride solution 1ml aprotin 3500 units; and distilled water 7.5ml. Aprotune can be obtained from Bayer and other sources.
[0074] あるいは、ダルコン酸 Ca (カルチコール） 5ml ;トロンビン 10000単位を用いても良 V、。ァプロチュンの代わりに「フサン」を用いても良!、。  [0074] Alternatively, 5 ml of calcium dalconate (calcicol); You can use "Fusan" instead of Aprotune!
[0075] 使用時には、 A液および B液を混ぜて用いることが望ましい。  [0075] At the time of use, it is desirable to use the solution A and the solution B in combination.
[0076] 1つの実施形態において、フイブリノゲンまたはその誘導体は、培養容器にコーティ ングされる。コーティングの方法は、当該分野において公知の任意の方法を用いるこ とができることが理解される。  [0076] In one embodiment, fibrinogen or a derivative thereof is coated on a culture vessel. It is understood that any method known in the art can be used for the coating.
[0077] 1つの実施形態において、このようなフイブリノゲンまたはその誘導体は、培養容器 にどのような厚さでコーティングされても良いが、好ましくは、 1 μ m— 100 μ mの厚さ でコーティングされ、より好ましくは 5 μ m— 50 μ mの厚さでコーティングされる。少な すぎると、細胞の増殖に耐えられない可能性があることから、ある程度の厚みがあるこ とが好ま ヽことが理解される。  [0077] In one embodiment, such a fibrinogen or a derivative thereof may be coated on the culture vessel at any thickness, but is preferably coated at a thickness of 1 μm to 100 μm. , More preferably 5 μm to 50 μm. It is understood that if the amount is too small, it may not be able to withstand cell growth, so that it is preferable to have a certain thickness.
[0078] 1つの実施形態において、フイブリノゲンまたはその誘導体は、どのような分子量の ものでも用いることができるが、好ましくは、分子量が 34万 Da以上であり、より好ましく はフイブリンポリマーまたはフイブリン塊となったものが使用される。  [0078] In one embodiment, fibrinogen or a derivative thereof can be used with any molecular weight, but preferably has a molecular weight of 340,000 Da or more, more preferably a fibrin polymer or a fibrin mass. Is used.
[0079] 1つの実施形態において、本発明の培養容器に含まれるフイブリノゲンまたはその 誘導体は、フイブリンを含む。フイブリンは、どのような濃度で含まれても良いが好まし くは容器 lcm2あたり 0. lmg— lOOmg含まれることが好ましくあり得る。 [0079] In one embodiment, fibrinogen or a derivative thereof contained in the culture container of the present invention contains fibrin. Fibrin may be present at any concentration but is preferred Or 0.1 mg to 100 mg per 1 cm 2 of the container.
[0080] 別の実施形態において、フイブリノゲンまたはその誘導体は、プロテアーゼを含む。 [0080] In another embodiment, the fibrinogen or derivative thereof comprises a protease.
プロテアーゼを含むことにより、フイブリノゲンの分解が進み、接着能力が改善するか らである。なお、使用時にはプロテアーゼは失活していてもよい。プロテアーゼとして は、例えば、トロンビン (例えば、 αトロンビンなど)またはバトロキソビンなどを挙げるこ とができるがそれに限定されない。  This is because the inclusion of the protease promotes the degradation of fibrinogen and improves the adhesive ability. At the time of use, the protease may be inactivated. Examples of the protease include, but are not limited to, thrombin (for example, α thrombin and the like) and batroxobin.
[0081] 好ま 、実施形態では、本発明にお 、て使用されるフイブリノゲンまたはその誘導 体は、プロテアーゼ阻害剤を含む。プロテアーゼ阻害剤を含むことによって、プロテ ァーゼの悪影響をコントロールすることができる力もである。プロテアーゼとしては任 意のものを使用することができるがそれに限定されない。 [0081] Preferably, in an embodiment, fibrinogen or a derivative thereof used in the present invention contains a protease inhibitor. By including a protease inhibitor, it is also possible to control the adverse effects of the protease. Any protease can be used, but is not limited thereto.
[0082] 別の実施形態において、プロテアーゼ阻害剤としては、ァプロチュン、フサン、 4- ( アミノメチル)シクロへキサンカルボン酸などを挙げることができるがそれに限定されな い。 [0082] In another embodiment, examples of the protease inhibitor include, but are not limited to, aprotune, fusane, 4- (aminomethyl) cyclohexanecarboxylic acid, and the like.
[0083] 好ましくは、本発明において使用されるフイブリノゲンまたはその誘導体は、フイブリ ンのりである。  [0083] Preferably, fibrinogen or a derivative thereof used in the present invention is fibrin glue.
[0084] 別の実施形態において、本発明において使用されるフイブリンのりは、フイブリン、 フイブリンモノマー、フイブリンポリマー、フイブロネクチン、血小板などの成分を含む。  [0084] In another embodiment, the fibrin glue used in the present invention contains components such as fibrin, fibrin monomer, fibrin polymer, fibronectin, and platelets.
[0085] 好ましい実施形態において、本発明において使用されるフイブリンのりは、トロンビ ンによってフイブリノゲンの A a鎖の N末端からフイブリノペプチド A (FPA)力 B j8鎖 の N末端カもフイブリノペプチド B (FPB)が切断される。フイブリノゲン 1分子から 2分 子ずつの FPAと FPBが切断されて残った分子がフイブリンモノマーである。本発明に おけるフイブリンのりは、フイブリンモノマーは重合部位が露出しているため互いの N 末端と C末端が重合する程度の接着性を有し、フイブリンポリマ一一フイブリン塊とな る。  [0085] In a preferred embodiment, the fibrin glue used in the present invention is obtained by using thrombin from the N-terminus of the Aa chain of fibrinogen to the fibrinopeptide A (FPA) force. Peptide B (FPB) is cleaved. Two molecules of FPA and FPB are cleaved from one molecule of fibrinogen, and the remaining molecule is fibrin monomer. In the fibrin glue of the present invention, the fibrin monomer has an adhesive property such that the N-terminal and the C-terminal thereof are polymerized because the polymerization site is exposed, and the fibrin polymer becomes a fibrin mass.
[0086] 本発明において使用される場合、フイブリンのりは、どのような濃度でも用いることが できるが、好ましくは、 500倍以下に希釈された (クリオプレシピテートと標準トロンビン 溶液で生成されたものを基準として定義される)ものを用いることが好ま U、。ある ヽ は、フイブリンのりは、 50倍一 500倍に希釈されたものを用いることがより好ましくあり 得るが、それに限定されない。より好ましくは、 100倍以上、例えば、約 500倍に希釈 されたものを用いることが好ましくあり得る。増殖効率が格段に向上するからである。 フイブリンのりの希釈は、任意の媒体を用いることができる力 好ましくは、生理的に 適合された媒体を用いることが好ましい。そのような溶液としては、例えば、生理食塩 水などを挙げることができるがそれに限定されない。 [0086] When used in the present invention, fibrin glue can be used at any concentration, but is preferably diluted to 500-fold or less (produced with cryoprecipitate and a standard thrombin solution). U, which is preferred to use). It is more preferable to use fibrin glue diluted 50-fold to 500-fold. But not limited to. More preferably, it may be preferable to use those diluted 100 times or more, for example, about 500 times. This is because the growth efficiency is significantly improved. Dilution of the fibrin glue can be performed using any medium. Preferably, a physiologically compatible medium is used. Examples of such a solution include, but are not limited to, physiological saline.
[0087] 本発明において使用され得るフイブリンのりは、「クリオプレシピテートあるいは血漿 あるいは血小板含有血漿（platelet— rich plasma： PRP)」と「トロンビンに塩化カル シゥム、ァプロチュン液 (製品名トラジロール)を添加した溶液」を混和して作製され得 る。希釈は、代表的にクリオプレシピテートは、 10000倍程度まで、血漿は、 500倍 程度まで希釈可能。後者のトロンビン溶液の方で、 1000倍程度までが可能である。ト ロンビン溶液の組成は、例えば、 [0087] The fibrin glue that can be used in the present invention includes "cryoprecipitate or plasma or platelet-rich plasma (PRP)" and "thrombin in calcium chloride and aprotune solution (trasilol product name). It can be prepared by mixing the “added solution”. Typically, the dilution can be reduced to about 10,000 times for cryoprecipitate and to about 500 times for plasma. With the latter thrombin solution, up to about 1000 times is possible. The composition of the thrombin solution is, for example,
'トロンビン 5000単位を蒸留水 7. 5mlで溶解  '' Dissolve 5000 units of thrombin in 7.5 ml of distilled water
•0. 5M 塩化カルシウム 1ml  • 0.5M calcium chloride 1ml
.ァプロチュン 35000単位（トラジローノレ 3. 5ml)  .A Protune 35,000 units (3.5 g)
で、総量 12mlとすることによって調製することができる。  To make the total amount 12 ml.
[0088] 従って、トロンビン溶液の希釈倍率の 500倍とは、トロンビンの濃度で定義すると 0. Therefore, 500 times the dilution ratio of the thrombin solution is defined as the thrombin concentration of 0.1.
83単位,!!11でぁる。  83 credits, !! 11.
[0089] 従って、本発明において用いられるフイブリノゲンまたはその誘導体は、例えば、フ イブリンモノマー、フイブリンポリマー、フイブリノゲン、血小板などを含み得る。  [0089] Therefore, fibrinogen or a derivative thereof used in the present invention may include, for example, fibrin monomer, fibrin polymer, fibrinogen, platelets, and the like.
[0090] 例えば、フイブリンのりは、（1)「クリオプレシピテート (濃厚フイブリノゲン血漿)ある いは血漿血小板含有血漿（platelet— rich plasma : PRP)」と（2)「トロンビン溶液 (トロンビンに塩ィ匕カルシウム、ァプロチュン液 (製品名トラジロール)を添加した溶液） (トロンビン 5000単位を 12mlに含む）」を混和して作製することができる。 (1)および (2)ともに希釈して使用することが可能である。  [0090] For example, fibrin glue includes (1) "Cryoprecipitate (rich fibrinogen plasma) or plasma platelet-rich plasma (PRP)" and (2) "thrombin solution (thrombin solution). (A solution to which dani calcium and aprotune solution (trasilol product name) are added) (including 5000 units of thrombin in 12 ml) ". Both (1) and (2) can be used after dilution.
[0091] 本発明が対象とする幹細胞は、どのような幹細胞でも良いが、好ましくは組織幹細 胞であり、より好ましくは、間葉系幹細胞であり、例えば、脂肪組織由来の幹細胞であ り得る。特に、そのような細胞として、脂肪吸引物に由来する幹細胞を用いることがで きたことは、従来技術において達成できていなかったことであり（Daniel A. De Ug artea Kouki Morizonoc Amir Elbarbarya Zeni Alfonsob, Patricia A . Zuka Min Zhua Jason L. Dragooa Peter Ashjiana Bert Thomasa,[0091] The stem cells targeted by the present invention may be any stem cells, but are preferably tissue stem cells, more preferably mesenchymal stem cells, for example, stem cells derived from adipose tissue. obtain. In particular, the ability to use stem cells derived from lipoaspirates as such cells has not been achieved in the prior art (Daniel A. De Ug artea Kouki Morizonoc Amir Elbarbarya Zeni Alfonsob, Patricia A. Zuka Min Zhua Jason L. Dragooa Peter Ashjiana Bert Thomasa,
Prosper Benhaima Irvin Chenc John Fraserb Marc H. Hedricka, Cells Tissues Organs 2003 ; 174 : 101- 109)、従来では達成できなかった効 果を有するといえる。なお、この文献では、ダブリング時間が 78 ( ± 26)時間とされて おり、本発明より顕著に遅い。好ましくは、本発明で培養されるべき幹細胞は、脂肪 細胞由来幹細胞（ASC ; adipose— derived stem cell)であり得る。 Prosper Benhaima Irvin Chenc John Fraserb Marc H. Hedricka, Cells Tissues Organs 2003; 174: 101-109), which can be said to have effects that could not be achieved conventionally. In this document, the doubling time is set to 78 (± 26) hours, which is significantly slower than the present invention. Preferably, the stem cell to be cultured in the present invention may be an adipocyte-derived stem cell (ASC).
[0092] 1つの実施形態において、本発明において用いられる、フイブリンまたはその分解 物は、コーティング後に乾燥した場合、単位面積あたりの重量で、 0. 1-lOOmg/c m2の濃度で含まれる。 [0092] In one embodiment, the fibrin or a degradation product thereof used in the present invention, when dried after coating, is contained at a concentration of 0.1-100 mg / cm 2 by weight per unit area.
[0093] 本発明の培養容器の材質としては、細胞培養に適切なものであれば、任意の材料 を使用することができ、例えば、ガラス、シリカ、シリコン、セラミック、二酸化珪素、ブラ スチック、金属、天然ポリマーおよび合成ポリマーからなる群より選択される材料を用 いることがでさる。  [0093] As the material of the culture container of the present invention, any material can be used as long as it is suitable for cell culture. For example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal And a material selected from the group consisting of natural polymers and synthetic polymers.
[0094] 別の局面において、本発明は、本発明の容器を用いて幹細胞を培養する方法を提 供する。そのような幹細胞を調製 (または培養)するための方法は、 A)幹細胞を提供 する工程；および B)該幹細胞を、培地ならびにフイブリノゲンまたはその誘導体を含 む培養容器中で培養する工程、を包含する。  [0094] In another aspect, the present invention provides a method for culturing a stem cell using the container of the present invention. A method for preparing (or culturing) such a stem cell comprises: A) providing the stem cell; and B) culturing the stem cell in a culture vessel containing a medium and fibrinogen or a derivative thereof. I do.
[0095] 幹細胞の提供は、当該分野において公知の任意の手法を用いることができる。そ のような提供には、例えば、幹細胞に特異的なマーカーを指標に FACSにより細胞 をソートすることなどが挙げられるがそれらに限定されない。  [0095] For providing the stem cells, any method known in the art can be used. Such provision includes, but is not limited to, sorting cells by FACS using a marker specific for stem cells as an index.
[0096] 本発明の方法において、幹細胞の培養は、通常使用される任意の培地を用いるこ とができる。そのような培養条件としては、例えば、 pH7. 2-7. 4、温度 37°C、 CO  [0096] In the method of the present invention, the culture of the stem cells can use any commonly used medium. Such culture conditions include, for example, pH 7.2-7.4, temperature 37 ° C, CO 2
2 濃度 5%という条件を挙げることができるがそれらに限定されない。 pHとしては、例え ば、中性付近 (例えば、 pH5— 9など）、温度としては、例えば、常温一 37°Cなど、 C O濃度としては、例えば、 1一 10% (例えば、 5%)などを挙げることができるがそれら 2 The condition of a concentration of 5% can be mentioned, but is not limited thereto. The pH is, for example, around neutral (for example, pH 5-9), the temperature is, for example, normal temperature-37 ° C, and the CO concentration is, for example, 11-10% (for example, 5%). I can list them
2 2
に限定されない。  It is not limited to.
[0097] 本発明の方法において使用され得る培地としては、例えば、 DMEM、 P199、 ME M、 Hanks，平衡化塩溶液（HBSS)、 Ham' s F12、 BME、 RPMI1640、 MCDB 104および MCDB153 (KGM)などを挙げることができるがそれらに限定されない。 DMEMおよび P199が好ましい。幹細胞の増殖に適切であることが示されているか らである。 [0097] Examples of the medium that can be used in the method of the present invention include DMEM, P199, and ME. M, Hanks, Equilibrated Salt Solution (HBSS), Ham's F12, BME, RPMI 1640, MCDB 104 and MCDB 153 (KGM) and the like. DMEM and P199 are preferred. This has been shown to be appropriate for stem cell expansion.
[0098] 本発明の方法において用いられる培地は、副腎皮質ステロイド、インスリン、ダルコ ース、インドメタシン、イソブチルーメチルキサンチン（IBMX)、ァスコルビン酸および その誘導体、グリセ口ホスフェート、エストロゲンおよびその誘導体、プロゲステロンお よびその誘導体、アンドロゲンおよびその誘導体、増殖因子、下垂体エキス、松果体 エキス、レチノイン酸、ビタミン D、甲状腺ホルモン、子牛血清、馬血清、ヒト血清、へ ノ《リン、炭酸水素ナトリウム、 HEPES、アルブミン、トランスフェリン、セレン酸塩、リノ レン酸、 3—イソブチルー 1ーメチルキサンチン、脱メチル化剤、ヒストン脱ァセチル化剤 、ァクチビン、サイト力イン、へキサメチレンビスァセトアミド（HMBA)、ジメチルァセト アミド（DMA)、ジブチル cAMP (dbcAMP)、ジメチルスルホキシド（DMSO)、ョー ドデォキシゥリジン（IdU)、ヒドロキシゥレア（HU)、シトシンァラビノシド (AmC)、マイ トマイシン C (MMC)、酪酸ナトリウム（NaBu)、ポリプレンおよびセレニウムなどをさら に加えることができる。  [0098] The medium used in the method of the present invention includes corticosteroids, insulin, dalcose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbic acid and its derivatives, glycerol phosphate, estrogen and its derivatives, progesterone and And its derivatives, androgen and its derivatives, growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heterophosphorus, sodium bicarbonate, HEPES , Albumin, transferrin, selenate, linolenic acid, 3-isobutyl-1-methylxanthine, demethylating agent, histone deacetylating agent, activin, cytokinin, hexamethylene bisacetamide (HMBA), dimethylacetate Amide (DMA), dibutyl cAMP (d bcAMP), dimethyl sulfoxide (DMSO), iododeoxyperidine (IdU), hydroxyperrea (HU), cytosine arabinoside (AmC), mitomycin C (MMC), sodium butyrate (NaBu), polypropylene And selenium and the like can be further added.
[0099] 1つの実施形態にお!、て、本発明の細胞調製法では、原料となる幹細胞を入手す る工程をさらに包含する。そのような入手の方法は、生体からの分離、あるいは、サン プル力 の FACSソート、分離などを挙げることができるがそれらに限定されない。  [0099] In one embodiment, the cell preparation method of the present invention further includes a step of obtaining a stem cell as a raw material. Examples of such a method of obtaining include, but are not limited to, separation from a living body or FACS sorting and separation of sample force.
[0100] 別の実施形態において、本発明の細胞調製法は、幹細胞を分化させる工程をさら に包含することができる。そのようにして分ィ匕させた細胞を用いることも本発明の範囲 内に入ることが理解される。  [0100] In another embodiment, the cell preparation method of the present invention can further include a step of differentiating a stem cell. It is understood that the use of the cells thus separated is also included in the scope of the present invention.
[0101] 別の局面において、本発明は、幹細胞を培養するための、フイブリノゲンまたはその 誘導体がコーティングされた培養容器を生産するための方法を提供する。この方法 は、 A)培養容器を提供する工程； B)該培養容器にフイブリノゲンまたはその誘導体 を提供する工程；および C)該フイブリノゲンまたはその誘導体を該培養容器の表面 に固定する工程、を包含する。培養容器の提供は、当該分野において周知の方法 により実現することができる。このような培養容器にフイブリノゲンまたはその誘導体を 提供することは、任意公知技術を用いることができ、好ましくは、コーティングすること が有利である。そのようなフイブリノゲンまたはその誘導体は、培養容器に当該分野 において公知の任意の方法を用いて固定される。 [0101] In another aspect, the present invention provides a method for producing a culture vessel coated with fibrinogen or a derivative thereof for culturing stem cells. The method includes the steps of: A) providing a culture vessel; B) providing fibrinogen or a derivative thereof to the culture vessel; and C) fixing the fibrinogen or derivative thereof to the surface of the culture vessel. . The provision of the culture vessel can be realized by a method known in the art. Fibrinogen or a derivative thereof is placed in such a culture vessel. To provide, any known technique can be used, and preferably, coating is advantageous. Such fibrinogen or a derivative thereof is fixed to a culture vessel using any method known in the art.
[0102] 好ましい実施形態において、本発明において使用されるフイブリノゲンまたはその 誘導体の提供は、無菌操作にて行われる。そのような条件によって提供されること〖こ よって、培養に対してプラスの効果があることが判明した力もである。  [0102] In a preferred embodiment, provision of the fibrinogen or a derivative thereof used in the present invention is performed by an aseptic operation. It is also a force that has been found to have a positive effect on culture by being provided by such conditions.
[0103] 別の好ましい実施形態において、本発明のフイブリノゲンまたはその誘導体の固定 は、乾燥状態で一定時間放置すること、または紫外線照射下などにより行うことがで きる。  [0103] In another preferred embodiment, the immobilization of the fibrinogen or a derivative thereof of the present invention can be performed by leaving the fibrinogen or the derivative in a dry state for a certain period of time, or by irradiating with ultraviolet rays.
[0104] 1つの局面において、本発明は、幹細胞を培養するための、組織接着剤がコーティ ングされた培養容器を提供する。このような組織接着剤は、当該分野において公知 のものを使用することができ、例えば、シァノアクリレート系接着剤、ゼラチン アルデ ヒド系接着剤およびフイブリングルー系接着剤などを挙げることができる。培養容器に つ!、ては、上記フイブリノゲンまたはその誘導体を含む培養容器に関する説明にお V、て記載される任意の形態を採用することができる。これらの培養容器が対象とする 細胞にっ 、てもまた、上記フイブリノゲンまたはその誘導体を含む培養容器に関する 説明にお 、て記載される任意の形態を採用することができる。  [0104] In one aspect, the present invention provides a culture vessel coated with a tissue adhesive for culturing stem cells. As such a tissue adhesive, those known in the art can be used, and examples thereof include a cyanoacrylate adhesive, a gelatin aldehyde adhesive, and a fibrin glue adhesive. Regarding the culture vessel, any form described in the description of the culture vessel containing fibrinogen or a derivative thereof can be adopted. For the cells targeted by these culture vessels, any form described in the description of the culture vessels containing the above-mentioned fibrinogen or a derivative thereof can also be adopted.
[0105] ここで、好ましくは、上記細胞接着剤は、 0.1— lOOmg/cm2以上の濃度で含まれ得る [0105] Here, preferably, the cell adhesion agent may be included in the 0.1- lOOmg / cm 2 or more concentrations
[0106] 別の局面において、本発明は、幹細胞を培養するための、細胞外マトリクスがコー ティングされた培養容器を提供する。このような細胞外マトリクスは、当該分野におい て公知のものを使用することができ、例えば、フイブロネクチン、コラーゲンおよびラミ ニンなどを挙げることができる。培養容器については、上記フイブリノゲンまたはその 誘導体を含む培養容器に関する説明にお ヽて記載される任意の形態を採用するこ とができる。これらの培養容器が対象とする細胞についてもまた、上記フイブリノゲン またはその誘導体を含む培養容器に関する説明にお 、て記載される任意の形態を 採用することができる。 [0106] In another aspect, the present invention provides a culture vessel coated with an extracellular matrix for culturing stem cells. As such an extracellular matrix, those known in the art can be used, and examples thereof include fibronectin, collagen and laminin. Regarding the culture vessel, any form described in the description of the culture vessel containing the above-mentioned fibrinogen or a derivative thereof can be adopted. For the cells targeted by these culture vessels, any of the forms described in the description of the culture vessels containing fibrinogen or a derivative thereof can be adopted.
[0107] ここで、好ましくは、上記細胞外マトリクスは、 0. 1— lOOmgZcm2以上の濃度で含 まれ得る。例えば、より好ましくは、フイブロネクチンが 1一 2. 5 μ
Figure imgf000029_0001
コラーゲン カ^ー 8 μ g/cm2、ラミニンが 5— 20 μ g/cm2であってもよい。 [0107] Here, containing preferably, the extracellular matrix, at a concentration of 0. 1- lOOmgZcm 2 or more Can be rare. For example, it is more preferable that fibronectin
Figure imgf000029_0001
Collagen may be 8 μg / cm 2 and laminin may be 5-20 μg / cm 2 .
[0108] 別の局面において、本発明は、幹細胞を培養するための、ゼラチンがコーティング された培養容器を提供する。このようなゼラチンは、当該分野において公知のものを 使用することができる。培養容器については、上記フイブリノゲンまたはその誘導体を 含む培養容器に関する説明において記載される任意の形態を採用することができる 。これらの培養容器が対象とする細胞についてもまた、上記フイブリノゲンまたはその 誘導体を含む培養容器に関する説明にお ヽて記載される任意の形態を採用するこ とがでさる。 [0108] In another aspect, the present invention provides a culture vessel coated with gelatin for culturing stem cells. As such gelatin, those known in the art can be used. As the culture vessel, any form described in the description of the culture vessel containing the above-mentioned fibrinogen or a derivative thereof can be adopted. For the cells to be targeted by these culture vessels, any of the forms described in the description of the culture vessels containing fibrinogen or a derivative thereof can be adopted.
[0109] 別の局面において、本発明は、幹細胞を調製するための方法を提供する。この方 法は、 A)培地、ならびに細胞外マトリクスおよび Zまたはゼラチンを含む培養容器を 提供する工程； B)幹細胞を該培養容器に配置する工程； C)該幹細胞を所望の期間 培養する工程、を包含する。この方法でもまた、培養容器については、上記フイブリノ ゲンまたはその誘導体を含む局面に関する説明において記載される任意の形態を 採用することができる。これらの培養容器が対象とする細胞についてもまた、上記フィ ブリノゲンまたはその誘導体を含む局面に関する説明において記載される任意の形 態を採用することができる。  [0109] In another aspect, the present invention provides a method for preparing a stem cell. This method comprises the steps of: A) providing a culture vessel containing a medium and an extracellular matrix and Z or gelatin; B) arranging stem cells in the culture vessel; C) culturing the stem cells for a desired period. Include. Also in this method, the culture vessel may adopt any form described in the description of the aspect including the above fibrinogen or a derivative thereof. For the cells targeted by these culture vessels, any of the forms described in the above description of the aspect including fibrinogen or a derivative thereof can be adopted.
[0110] 他の局面において、本発明は、幹細胞を培養するための、細胞外マトリクスおよび [0110] In another aspect, the present invention provides an extracellular matrix and
Zまたはゼラチンがコーティングされた培養容器を生産するための方法を提供する。 この方法は、 A)培養容器を提供する工程； B)該培養容器に細胞外マトリクスおよびA method for producing a culture vessel coated with Z or gelatin is provided. The method comprises: A) providing a culture vessel; B) an extracellular matrix and
Zまたはゼラチンを提供する工程；および c)該細胞外マトリクスおよび Zまたはゼラ チンを該培養容器の表面に固定する工程、を包含する。この方法でもまた、培養容 器については、上記フイブリノゲンまたはその誘導体を含む局面に関する説明におProviding Z or gelatin; and c) fixing the extracellular matrix and Z or gelatin to the surface of the culture vessel. Also in this method, the culture vessel is described in the above description of the aspect containing fibrinogen or a derivative thereof.
V、て記載される任意の形態を採用することができる。これらの培養容器が対象とする 細胞についてもまた、上記フイブリノゲンまたはその誘導体を含む局面に関する説明 において記載される任意の形態を採用することができる。 V, any of the forms described above can be employed. Regarding the cells targeted by these culture vessels, any of the forms described in the above description regarding the aspect containing fibrinogen or a derivative thereof can be adopted.
[0111] 他の局面において、本発明は、フイブリノゲンまたはその誘導体の、幹細胞を調製 するための使用を提供する。フイブリノゲンまたはその誘導体が、幹細胞 (特に、組織 幹細胞）に対して増殖効果を有するかどうか知られていな力つた。従って、本発明は 、フイブリノゲンまたはその誘導体の新規用途を提供することになる。この使用でもま た、培養容器については、上記フイブリノゲンまたはその誘導体を含む局面に関する 説明において記載される任意の形態を採用することができる。これらの培養容器が対 象とする細胞についてもまた、上記フイブリノゲンまたはその誘導体を含む局面に関 する説明において記載される任意の形態を採用することができる。 [0111] In another aspect, the present invention provides the use of fibrinogen or a derivative thereof for preparing a stem cell. Fibrinogen or its derivatives can be used in stem cells (particularly in tissues). It is not known whether it has a proliferative effect on stem cells). Therefore, the present invention provides a novel use of fibrinogen or a derivative thereof. Also in this use, the culture vessel may adopt any form described in the description relating to the aspect containing fibrinogen or a derivative thereof. The cells targeted by these culture vessels can also adopt any of the forms described in the description of the aspect containing the above-mentioned fibrinogen or a derivative thereof.
[0112] 別の局面において、本発明は、細胞外マトリクスおよび Zまたはゼラチンの、幹細 胞を調製するための使用を提供する。細胞外マトリクス (特に、コラーゲン、ラミニン、 フイブロネクチンなど）が増殖効果を有するかどうか知られていな力つた。従って、本 発明は、フイブリノゲンまたはその誘導体の新規用途を提供することになる。この使用 でもまた、培養容器については、上記フイブリノゲンまたはその誘導体を含む局面に 関する説明において記載される任意の形態を採用することができる。これらの培養容 器が対象とする細胞につ!、てもまた、上記フイブリノゲンまたはその誘導体を含む局 面に関する説明において記載される任意の形態を採用することができる。  [0112] In another aspect, the present invention provides the use of an extracellular matrix and Z or gelatin for preparing a stem cell. It was unknown whether extracellular matrices (especially collagen, laminin, fibronectin, etc.) had a proliferative effect. Therefore, the present invention provides a novel use of fibrinogen or a derivative thereof. Also in this use, the culture container may adopt any form described in the description of the aspect including the above-mentioned fibrinogen or a derivative thereof. For the cells targeted by these culture vessels, any of the forms described in the description relating to the surface containing fibrinogen or a derivative thereof can also be employed.
[0113] (脂肪吸引廃液力 の幹細胞の調製方法） [0113] (Method for Preparing Stem Cells for Liposuction Wastewater)
本発明の 1つの局面において、脂肪吸引廃液力 幹細胞を調製する方法であって 、A)脂肪吸引廃液を得る工程; B)該脂肪吸引廃液を遠心分離して細胞画分を得る 工程； C)該細胞画分を密度勾配遠心分離にかけて比重による細胞分離を行う工程； および、 D)赤血球よりも比重が軽い細胞層を収集する工程、を含む、方法が提供さ れる。本方法において用いる脂肪吸引廃液としては、脂肪吸引時に一緒に吸引され た液体 (例えば、トウメセント液および血液が挙げられる力 これらに限定されない）ま たは、吸引脂肪を液体で洗浄する際に生じた廃液が挙げられるが、これらに限定さ れない。  In one aspect of the present invention, a method for preparing liposuction waste fluid stem cells, comprising: A) a step of obtaining a liposuction waste solution; B) a step of obtaining a cell fraction by centrifuging the liposuction waste solution; C) Subjecting the cell fraction to density gradient centrifugation to separate cells by specific gravity; and D) collecting a cell layer having a specific gravity lower than that of red blood cells. The liposuction waste liquid used in the present method may be a liquid sucked together during liposuction (for example, a force including but not limited to tomescent liquid and blood) or a liquid generated by washing a suction fat with a liquid. Waste liquids include, but are not limited to.
[0114] 脂肪吸引方法は、当業者に周知である技術を用いて、実施することができる。脂肪 吸引方法に用いられる装置としては、例えば、ケイセィ脱脂吸引機サルポンプ SAL7 6— A (ケイセィ医科工業 (株）、東京都文京区本郷 3— 19— 6)が挙げられるが、これら に限定されない。例えばヒトの場合、 0. 0001%アドレナリン入り生理食塩水などの液 体を、予定吸引脂肪量の等量から 2倍量、脂肪組織内に注入し、その後、内径 2— 3 mmの力-ユーレ（金属製吸引管）を用いて、— 250— 900mmHg程度の陰圧で吸引 することによって行う。 [0114] The liposuction method can be performed using techniques well known to those skilled in the art. Examples of the apparatus used for the liposuction method include, but are not limited to, a Keisei degreasing suction device monkey pump SAL76-A (Keisei Medical Industry Co., Ltd., Hongo 3-19-6, Bunkyo-ku, Tokyo). For example, in the case of humans, a fluid such as physiological saline containing 0.0001% adrenaline is injected into adipose tissue in an amount equal to twice the expected aspirated fat mass, and then injected into the adipose tissue. This is performed by suctioning with a negative pressure of about 250-900 mmHg using a mm-force Yule (metal suction tube).
[0115] 吸引した脂肪細胞を洗浄する液体として通常は、生理食塩水が用いられる。しかし 、生理食塩水以外にも、単離すべき幹細胞に悪影響を与えない液体であれば、任意 の液体が使用可能である。具体的には、例えば、リンゲル液、および哺乳動物培養 培地（例えば、 DMEM、 MEM、 M199、および MCDB153など）のような、任意の 等張液が使用可能である。  [0115] Normally, physiological saline is used as a liquid for washing the sucked fat cells. However, any liquid other than physiological saline can be used as long as it does not adversely affect the stem cells to be isolated. Specifically, for example, any isotonic solution such as Ringer's solution and mammalian culture medium (eg, DMEM, MEM, M199, and MCDB153) can be used.
[0116] 脂肪吸引廃液から幹細胞を調製する場合、必要に応じて、脂肪吸引廃液にコラゲ ナーゼなどの酵素処理を行なうことも可能である。しかし、脂肪吸引廃液中に含まれ るコラーゲンの量は、脂肪組織と比較して少ないため、幹細胞調製の原料として脂肪 吸引廃液を用いる場合、コラゲナーゼ処理に必要とされる時間、および Zまたは酵 素処理に必要とされる酵素量が、脂肪組織の場合と比較して、より少ない。  [0116] When preparing stem cells from liposuction waste liquid, the liposuction waste liquid can be treated with an enzyme such as collagenase, if necessary. However, since the amount of collagen contained in the liposuction waste liquid is smaller than that of adipose tissue, when liposuction waste liquid is used as a raw material for preparing stem cells, the time required for collagenase treatment, and the amount of Z or enzyme The amount of enzyme required for the treatment is lower than in the case of adipose tissue.
[0117] 脂肪吸引廃液を遠心分離して細胞画分を得る工程においては、細胞画分と、その 他の成分 (例えば、血漿、手術時に注入した生理食塩水、麻酔薬、止血薬、破壊さ れた脂肪細胞から漏出した脂質成分)を分離する任意の条件を用いることができる。 例えば、 400— 800 X gでの 5— 15分程度の遠心分離を用いることが可能である。  [0117] In the step of obtaining a cell fraction by centrifuging the liposuction waste liquid, the cell fraction and other components (for example, plasma, physiological saline injected at the time of surgery, an anesthetic, a hemostatic agent, and Any conditions for separating the leaked lipid component from the adipocytes) can be used. For example, centrifugation at 400-800 X g for about 5-15 minutes can be used.
[0118] 単離された細胞画分を密度勾配遠心分離にかけて比重による細胞分離を行う工程 では、細胞画分を、例えば、密度勾配遠心分離法によって、その比重によって分離 する。その媒体としては、密度勾配遠心分離法に使用可能な媒体を用いる。好まし い媒体は、 20°Cでの比重が 1. 076—1. 078 gZmlである。また、好ましい媒体の pHは、 4. 5-7. 5である。また、媒体中の好ましいエンドトキシンレベルは 0. 12 E UZml以下である。代表的な媒体としては、スクロース、フイコール (スクロースとェピ クロロヒドリンの共重合物）、およびパーコール (ポリビュルピロリドンの被膜を有するコ ロイド状シリカ製品）が挙げられる力 これらに限定されない。フイコールは、例えば、 Ficoll— Paque PLUS (フアルマシアバイオテク株式会社、東京）、 Histopaque— 1 077 (シグマ アルドリッチ ジャパン株式会社、東京）、およびリンフォプレップ (Lym phoprep) (登録商標）（ニコメッド、オスロ (ノルウェー)）など名称の製品が市販されて いる。パーコールは、 Percoll (シグマ アルドリッチ ジャパン株式会社、東京）という 名称の製品が市販されて!、る。 [0118] In the step of subjecting the isolated cell fraction to density gradient centrifugation to separate cells by specific gravity, the cell fraction is separated by its specific gravity, for example, by density gradient centrifugation. As the medium, a medium usable for density gradient centrifugation is used. A preferred medium has a specific gravity at 20 ° C of 1.076-1.078 gZml. The preferred pH of the medium is 4.5-7.5. Also, the preferred endotoxin level in the medium is 0.12 E UZml or less. Representative media include, but are not limited to, sucrose, ficoll (a copolymer of sucrose and epichlorohydrin), and percoll (a colloidal silica product having a coating of polybulpyrrolidone). Ficoll is, for example, Ficoll-Paque PLUS (Falmacia Biotech Co., Ltd., Tokyo), Histopaque-1077 (Sigma-Aldrich Japan Co., Ltd., Tokyo), and Lym phoprep (registered trademark) (Nicomed, Oslo) (Norway)). Percoll is called Percoll (Sigma Aldrich Japan Co., Ltd., Tokyo) The product with the name is on the market!
[0119] 比重による遠心分離の条件は、 Ficoll— Paque PLUSを媒体として用いる場合、 4 00 X g、 30— 40分であり、ヒストパックを媒体として用いる場合、 370g X g、約 30分 であり、リンフォプレップを媒体として用いる場合、 800 X g、約 20分または 1100 X g 、約 10分である。 [0119] The conditions of centrifugation by specific gravity are 400 X g for 30 to 40 minutes when Ficoll-Paque PLUS is used as a medium, and 370 g X g for about 30 minutes when a Histopak is used as a medium. When using Lymphoprep as the medium, 800 X g for about 20 minutes or 1100 X g for about 10 minutes.
[0120] この比重による遠心分離において、通常最も多い細胞は、赤血球細胞であり、赤色 の細胞層として確認することができる。幹細胞は、赤血球よりも比重が軽いため、幹細 胞を分離する場合には、赤血球よりも軽い細胞層を収集する。好ましくは、比重 1. 0 50-1. 075の範囲の細胞層を収集する。  [0120] In the centrifugation using this specific gravity, the most abundant cells are usually red blood cells, which can be confirmed as a red cell layer. Since stem cells have a lower specific gravity than red blood cells, when separating stem cells, a cell layer lighter than red blood cells is collected. Preferably, a cell layer with a specific gravity in the range of 1.050-1.075 is collected.
[0121] おおまかな細胞の比重は、パーコール、レディグラッドのような密度勾配遠心分離 媒体を塩ィ匕ナトリウム溶液やスクロース溶液で調製し、収集した細胞と共にデンシティ 一マーカービーズ（density marker beads)をカ卩えて遠心し、ビーズによって分け られた 5— 10層のうち、どの層に細胞があるかを確認することで、調べることが可能で ある。  [0121] The approximate specific gravity of the cells is determined by preparing a density gradient centrifugation medium such as Percoll or Ladygrad with a sodium salt solution or a sucrose solution and collecting the density marker beads together with the collected cells. It is possible to investigate by checking which layer contains the cells among the 5-10 layers separated by beads after centrifuging and centrifuging.
[0122] また、層に分離した細胞の回収は、例えば、ピペットを用いて行なうことができる。  [0122] The cells separated into layers can be collected using, for example, a pipette.
[0123] 比重による遠心分離においては、セルセパレーター（例えば、血液成分分離装置 A[0123] In centrifugation using specific gravity, a cell separator (for example, a blood component separation device A
STEC204、（株）アムコ）を使用することも可能である。 STEC204 (Amco) can also be used.
[0124] 収集された細胞を培養する培地としては、例えば、 DMEM、 M199、 MEM、 HBS[0124] As a medium for culturing the collected cells, for example, DMEM, M199, MEM, HBS
S、 Ham' s F12、 BME、 RPMI1640、 MCDB104、 MCDB153 (KGM)が挙げ られる力 これらに限定されない。好ましい培地としては、 DMEM、および M199が 挙げられる。 Powers including, but not limited to, S, Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM). Preferred media include DMEM, and M199.
[0125] 収集された細胞は、ヘテロな細胞の集団であり、 CD13、 29、 44、 49d、 71、 73、 9 9、 105、 151を発現する幹細胞を含み、さらに CD31、 CD34のどちら力、もしくは両 方を発現する幹細胞を含む。  [0125] The collected cells are a heterogeneous population of cells, including stem cells that express CD13, 29, 44, 49d, 71, 73, 99, 105, 151, and furthermore, which of CD31, CD34, Alternatively, it includes a stem cell that expresses both.
[0126] 本発明の 1つの局面において、収集された細胞を、各種条件で培養することによつ て、例えば、血管内皮細胞、神経細胞、平滑筋細胞、心筋細胞、骨格筋細胞、軟骨 細胞、骨細胞、脂肪細胞、靭帯細胞、およびストローマ細胞を得ることが可能である。  [0126] In one aspect of the present invention, by culturing the collected cells under various conditions, for example, vascular endothelial cells, nerve cells, smooth muscle cells, cardiomyocytes, skeletal muscle cells, chondrocytes , Bone cells, adipocytes, ligament cells, and stromal cells.
[0127] さらに、本発明において、以下の方法を用いて、収集された細胞群から、血管内皮 前駆細胞を分離、回収することが可能である。 [0127] Further, in the present invention, vascular endothelium was collected from the collected cell group using the following method. It is possible to separate and collect precursor cells.
[0128] 得られた細胞群を、血管内皮細胞用培地を用いて 1%ゼラチンコート培養皿にて 4 —5日培養する。 0. 25%トリプシンで培養皿から剥がして、 anti— PECAM— 1ビーズ と反応させて、抗体と反応した細胞のみを MACS (磁気細胞分離システム)もしくは F ACS (フローサイトメトリー）を用いて分離する。分離した細胞を、再度、血管内皮細 胞用培地 · 1%ゼラチンコート培養皿にて培養する。わずかに混在する繊維芽細胞な どを除外するため、血管内皮細胞の方がトリプシンで剥がれやすいのを利用して、 0 . 25%トリプシンを 30— 40秒ほど反応させて剥がれた細胞のみを回収し、別の培養 皿で培養することにより、血管内皮前駆細胞に分化した細胞を単離することが可能で め 。 (例 ば、 Hutley LJ, et al: Human adipose tissue endothelial c ells promote preadipocyte proliferation. Am J Physiol Endocrinol Metab. 2001 Nov; 281 (5) : E1037— 44.を参照のこと）。  [0128] The obtained cell group is cultured in a 1% gelatin-coated culture dish for 4 to 5 days using a medium for vascular endothelial cells. 0.25% Trypsin is removed from the culture dish and reacted with anti-PECAM-1 beads. Only the cells that have reacted with the antibody are separated using MACS (magnetic cell separation system) or FACS (flow cytometry). . The separated cells are cultured again in a medium for vascular endothelial cells · 1% gelatin-coated culture dish. Taking advantage of the fact that vascular endothelial cells tend to detach with trypsin in order to exclude slightly mixed fibroblasts, etc., only 0.25% trypsin is reacted for 30-40 seconds to collect only detached cells. By culturing the cells in another culture dish, cells differentiated into vascular endothelial progenitor cells can be isolated. (See, for example, Hutley LJ, et al: Human adipose tissue endothelial cells promote preadipocyte proliferation. Am J Physiol Endocrinol Metab. 2001 Nov; 281 (5): E1037-44.).
[0129] 本発明のさらなる局面において、幹細胞を調製するためのシステムであって: A)脂 肪吸引廃液を得るための手段; B)脂肪吸引廃液を遠心分離して細胞画分を得る手 段;および、 C)細胞画分を密度勾配遠心分離にかけて比重による細胞分離を行う手 段；を含む、システムが提供される。このシステムによって、簡便かつ大量に幹細胞を 、自動的に調製することが可能となる。  [0129] In a further aspect of the present invention, there is provided a system for preparing stem cells, comprising: A) means for obtaining a liposuction waste liquid; B) means for centrifuging the liposuction waste liquid to obtain a cell fraction. And C) means for subjecting the cell fraction to density gradient centrifugation to separate cells by specific gravity. This system makes it possible to easily and automatically prepare large quantities of stem cells.
[0130] 脂肪吸引廃液を得るための手段としては、減圧によって生体内から脂肪を吸引す る手段が挙げられる。例えば、そのような手段としては、脂肪吸引手術 (手動の陰圧 注射器を用いる脂肪吸引手術、および電動の脂肪吸引器を用いる脂肪吸引手術)、 ならびに皮膚を切開することによる除脂術が挙げられるが、これらに限定されない。  [0130] Means for obtaining a liposuction waste liquid include a means for sucking fat from the inside of a living body by decompression. For example, such means include liposuction surgery (liposuction surgery with a manual negative pressure syringe and liposuction surgery with a motorized liposuction device), and delipidation by incising the skin However, it is not limited to these.
[0131] 脂肪吸引廃液を遠心分離して細胞画分を得る手段、および細胞画分を密度勾配 遠心分離にかけて比重による細胞分離を行う手段としては、遠心分離装置が挙げら れる。脂肪吸引廃液を遠心分離して細胞画分を得る場合、その条件としては、 200 一 1200 X g、 3— 60分間の遠心分離力挙げられ、好ましくは 260— 900 X g、 3— 30 分間の遠心分離、より好ましくは 400— 800 X g、 3— 15分間の遠心分離、最も好ま しくは 400 X g、 5— 10分間の遠心分離を行う。また、フイコールを用いて、細胞画分 を密度勾配遠心分離にかけて比重による細胞分離を行う場合、その条件としては、 2 00— 1200 X g、 10— 60分間の遠'、分離力 S挙げられ、好ましくは 260— 900 X g、 1 0— 60分間の遠心分離、好ましくは 370— 1100 X g、 10— 60分間の遠心分離、より 好ましく ίま 400— 800 X g、 10— 40分 f¾の遠'、分離、最も好ましく ίま 400 X g、 30— 40分間の遠心分離を行う。また、このシステムは、必要に応じて、特定の比重の細胞 層を収集する手段を備える。好ましくは、収集される細胞層は、赤血球よりも比重が 軽い細胞層である。そのような手段としては、手動ピぺッター、手動ァスピレーター、 自動ピぺッター、および自動ァスピレーターが挙げられる力 これらに限定されない。 [0131] Examples of a means for obtaining a cell fraction by centrifuging a liposuction waste liquid and a means for subjecting a cell fraction to density gradient centrifugation to separate cells by specific gravity include a centrifugal separator. When the cell fraction is obtained by centrifuging the liposuction waste liquid, the conditions include a centrifugation force of 200 to 1200 X g for 3 to 60 minutes, preferably 260 to 900 X g for 3 to 30 minutes. Perform centrifugation, more preferably 400-800 × g, for 3-15 minutes, most preferably 400 × g, 5-10 minutes. When the cell fraction is subjected to density gradient centrifugation using Ficoll to separate cells by specific gravity, the conditions are as follows: 00—1200 X g, 10-60 min, separation power S, preferably 260-900 X g, 10-60 min centrifugation, preferably 370-1100 X g, 10-60 min Perform centrifugation, more preferably at 400-800 X g for 10-40 min, separation, and most preferably centrifugation at 400 X g for 30-40 min. The system also includes means for collecting a cell layer having a specific gravity as required. Preferably, the cell layer collected is a cell layer having a lower specific gravity than red blood cells. Such means include, but are not limited to, manual pitchers, manual aspirators, automatic pitchers, and automatic aspirators.
[0132] 本発明の幹細胞を用いて、種々の分ィ匕細胞および Zまたは前駆細胞を得ることが できる。具体的には、以下の方法を用いる。  [0132] Using the stem cell of the present invention, various screening cells and Z or progenitor cells can be obtained. Specifically, the following method is used.
[0133] (分化細胞を調製するための方法）  (Method for Preparing Differentiated Cells)
1つの局面において、本発明は、幹細胞から分化細胞を調製するための方法を提 供する。幹細胞力 得られる分ィ匕細胞としては、最終的に分ィ匕した細胞のみならず、 前駆細胞も含まれる。  In one aspect, the present invention provides a method for preparing a differentiated cell from a stem cell. Stem cell force The obtained divided cells include not only cells that have been finally divided but also precursor cells.
[0134] 脂肪吸引廃液由来の幹細胞力 分ィ匕細胞を調製する方法としては、（1)培地中に 分化誘導剤（例えば、デキサメサゾンなど）を添加する方法、および（2)所望の部位 に対応する分化細胞とともに培養する方法が挙げられる。  [0134] Stem cell force derived from liposuction waste fluid can be prepared by (1) a method of adding a differentiation inducer (eg, dexamethasone) to a medium, and (2) a method of preparing a desired site. And a method of culturing with the differentiated cells.
[0135] (培地中に分化誘導剤を添加することにより、幹細胞の分化を誘導する方法）  (Method of Inducing Stem Cell Differentiation by Adding Differentiation Inducing Agent to Medium)
幹細胞の分化を誘導する分化誘導剤としては、デキサメタゾン (dexamethasone) などの副腎皮質ステロイド、インスリン、グルコース、インドメタシン、イソブチルーメチ ルキサンチン（IBMX)、ァスコルべ一トー 2—ホスフェート（ascorbate— 2— phosphate )、ァスコルビン酸およびその誘導体、グリセ口ホスフェート（glycerophosphate)、ェ ストロゲンおよびその誘導体、プロゲステロンおよびその誘導体、アンドロゲンおよび その誘導体、 aFGF - bFGF - EGF - IGF - TGF β · ECGF.BMP· PDGFをはじめと する増殖因子、下垂体エキス、松果体エキス、レチノイン酸、ビタミン D、甲状腺ホル モン、子牛血清、馬血清、ヒト血清、へパリン、炭酸水素ナトリウム、 HEPES、アルブ ミン、トランスフェリン、セレン酸（亜セレン酸ナトリウムなど）、リノレン酸、 3—イソブチル —1ーメチルキサンチン、 5—ァザンシチジンなどの脱メチル化剤、トリコスタチンなどの ヒストン脱ァセチル化剤、ァクチビン、 '1しー2'1し—6などのサィトカィン、へキサメ チレンビスァセトアミド（HMBA)、ジメチルァセトアミド（DMA)、ジブチル cAMP (db cAMP)、ジメチルスルホキシド（DMSO)、ョードデォキシゥリジン（IdU)、ヒドロキシ ゥレア（HU)、シトシンァラビノシド (AmC)、マイトマイシン C (MMC)、酪酸ナトリウム (NaBu)、ポリブレン、セレニウムが挙げられるがそれらに限定されない。 Examples of differentiation inducers that induce stem cell differentiation include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, Proliferation including ascorbic acid and its derivatives, glycerophosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, aFGF-bFGF-EGF-IGF-TGFβECGF.BMPPDGF Factor, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenite (selenium suboxide) Sodium), linolenic acid, 3-iso Kisame Chill -1 over methylxanthine, 5-demethylating agent such Azanshichijin histone de Asechiru agents such as trichostatin, Akuchibin, '1 sea 2'1 teeth such -6 Saitokain to, Tylene bisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (db cAMP), dimethyl sulfoxide (DMSO), ododeoxyperidine (IdU), hydroxyperrea (HU), cytosine Rabinoside (AmC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, but are not limited thereto.
[0136] 使用される培地としては、本発明の細胞混合物は、混合される細胞の維持および 所望の部位に対応する分化細胞への分化を維持する限り、任意の培養液を用いるこ とができる。そのような培養液としては、例えば、 DMEM、 M199、 MEM、 HBSS ( Hanks ' Balanced salt solution)、 Ham， s F12、 BME、 RPMI1640、 MCDB 104、 MCDB153 (KGM)などが挙げられるがそれらに限定されない。このような 培養液には、デキサメタゾン（dexamethasone)などの副腎皮質ステロイド、インスリ ン、グルコース、インドメタシン、イソブチルーメチルキサンチン（IBMX)、ァスコルべ 一トー 2—ホスフェート（ascorbate— 2— phosphate)、ァスコルビン酸およびその誘導 体、グリセ口ホスフェート（glycerophosphate)、エストロゲンおよびその誘導体、プロ ゲステロンおよびその誘導体、アンドロゲンおよびその誘導体、 aFGF'bFGF'EGF •IGF-TGF jS 'ECGF'BMP'PDGFをはじめとする増殖因子、下垂体エキス、松果 体エキス、レチノイン酸、ビタミン D、甲状腺ホルモン、子牛血清、馬血清、ヒト血清、 へパリン、炭酸水素ナトリウム、 HEPES、アルブミン、トランスフェリン、セレン酸（亜セ レン酸ナトリウムなど）、リノレン酸、 3—イソブチルー 1ーメチルキサンチン、 5—ァザンシ チジンなどの脱メチル化剤、トリコスタチンなどのヒストン脱ァセチル化剤、ァクチビン 、 LIF'IL— 2'IL— 6などのサイト力イン、へキサメチレンビスァセトアミド（HMBA)、ジ メチルァセトアミド（DMA)、ジブチル c AMP (dbcAMP)、ジメチルスルホキシド（D MSO)、ョードデォキシゥリジン（IdU)、ヒドロキシゥレア（HU)、シトシンァラビノシド（ AmC)、マイトマイシン C (MMC)、酪酸ナトリウム（NaBu)、ポリプレン、セレニウムな どを 1つまたはその組み合わせとして含ませてぉ 、てもよ!/、。  [0136] As the medium to be used, any culture solution can be used for the cell mixture of the present invention as long as the maintenance of the cells to be mixed and the differentiation into differentiated cells corresponding to the desired site are maintained. . Examples of such a culture medium include, but are not limited to, DMEM, M199, MEM, HBSS (Hanks' Balanced salt solution), Ham, sF12, BME, RPMI1640, MCDB104, MCDB153 (KGM) and the like. . Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, and ascorbate. And its derivatives, glycerophosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, aFGF'bFGF'EGF, IGF-TGF jS 'ECGF'BMP'PDGF and other growth factors , Pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (sodium selenite) Linolenic acid, 3-isobutyl-1-methylxanthi) , Demethylating agents such as 5-azanticidin, histone deacetylating agents such as trichostatin, activin, cytokins such as LIF'IL-2'IL-6, hexamethylene bisacetoamide (HMBA), Dimethylacetoamide (DMA), dibutyl cAMP (dbcAMP), dimethylsulfoxide (DMSO), eododexperidine (IdU), hydroxyperrea (HU), cytosine arabinoside (AmC) , Mitomycin C (MMC), sodium butyrate (NaBu), polypropylene, selenium, etc., as one or a combination thereof.
[0137] 脂肪吸引廃液由来の幹細胞の脂肪細胞への分化誘導は、例えば、 DMEM培地 に、イソブチルーメチルキサンチン、デキサメタゾン、インスリン、およびインドメタシン を添加した培地中で培養することによって行われる力 これに限定されない。  [0137] Induction of differentiation of stem cells derived from liposuction waste fluid into adipocytes is performed, for example, by culturing in a DMEM medium supplemented with isobutyl-methylxanthine, dexamethasone, insulin, and indomethacin. Not limited.
[0138] 脂肪吸引廃液由来の幹細胞の軟骨細胞への分ィヒ誘導は、例えば、 1%の FBSを 含む DMEM培地に、インスリン、ァスコルべ一トー 2—ホスフェート、および TGF—jS 1 を添加した培地中で培養することによって行われる力 これに限定されない。 [0138] Induction of stem cells from liposuction waste fluid into chondrocytes can be performed, for example, by adding 1% FBS. Force performed by culturing in a DMEM medium containing insulin, ascorbate 2-phosphate, and TGF-jS 1 in a medium that is not limited thereto.
[0139] 脂肪吸引廃液由来の幹細胞の骨細胞への分ィ匕誘導は、例えば、 10%の FBSを含 む DMEM培地に、デキサメタゾン、ァスコルべ一トー 2—ホスフェート、 j8—グリセロホ スフエートを添カ卩した培地中で培養することによって行われる力 これに限定されない [0139] Induction of stem cells derived from liposuction waste fluid into bone cells is performed, for example, by adding dexamethasone, ascorbate 2-phosphate and j8-glycerophosphate to a DMEM medium containing 10% FBS. The power produced by culturing in tanned medium, but not limited to
[0140] 脂肪吸引廃液由来の幹細胞の筋細胞への分ィ匕誘導は、例えば、 10%の FBSと 5 %の HS (馬血清）を含む DMEM培地に、デキサメタゾン、ハイド口コルチゾンを添カロ した培地中で培養することによって行われる力 これに限定されない。 [0140] Induction of stem cells derived from liposuction waste fluid into muscle cells was performed, for example, by adding dexamethasone and Hyde-mouth cortisone to a DMEM medium containing 10% FBS and 5% HS (horse serum). The force exerted by culturing in the medium is not limited to this.
[0141] 以下に、実施例に基づいて本発明を説明するが、以下の実施例は、例示の目的の みに提供される。従って、本発明の範囲は、上記発明の詳細な説明にも下記実施例 にも限定されるものではなぐ特許請求の範囲によってのみ限定される。  [0141] Hereinafter, the present invention will be described based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the detailed description of the invention described above nor to the following Examples, and is limited only by the appended claims.
実施例  Example
[0142] 以下に実施例を示して本発明をさらに詳しく説明するが、この発明は以下の例に限 定されるものではない。以下の実施例において用いられる試薬などは、例外を除き、 Sigma (St. Louis, USA)、和光純薬（大阪、 日本)など力も巿販されるものを用い た。幹細胞の供与は、本発明者らが調製した。  [0142] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples. The reagents and the like used in the following examples were, with exceptions, those whose power was also commercially available, such as Sigma (St. Louis, USA) and Wako Pure Chemical (Osaka, Japan). Stem cell donations were prepared by the present inventors.
[0143] (実施例 1：脂肪幹細胞の調製）  (Example 1: Preparation of adipose stem cells)
本実施例では、まず、本実験に対して同意を示したヒトから脂肪幹細胞を脂肪吸引 物から調製した。脂肪吸引物を 1リットル大の分液漏斗を用いて生理食塩水で十分 に洗浄した。上層に脂肪吸引物、下層に生理食塩水が十分に分離したのを確認し、 下層を捨て、肉眼で見て生理食塩水がほぼ透明になるまでこれを繰り返した。この実 施例では、 5回行った。  In this example, first, adipose stem cells were prepared from lipoaspirates from humans who had given consent to this experiment. The lipoaspirate was thoroughly washed with saline using a 1-liter separatory funnel. It was confirmed that the lipoaspirate was sufficiently separated in the upper layer and the physiological saline was sufficiently separated in the lower layer. The lower layer was discarded, and this was repeated until the physiological saline was almost transparent to the naked eye. In this example, the test was performed five times.
[0144] 脂肪吸引物を 10mlと同量 10mlの 0. 075%コラゲナーゼ ZPBS (Gibco)をカロえ、 37°Cでよく攪拌しながら 30分間インキュベートした。この試料に、同量の 10%血清 加 DMEMを加え、 1200 X gで 10分間遠心分離した。  The lipoaspirate was weighed with 10 ml of 0.075% collagenase ZPBS (Gibco) in the same volume as 10 ml and incubated at 37 ° C. for 30 minutes with good stirring. To this sample, the same amount of DMEM supplemented with 10% serum was added, and centrifuged at 1200 × g for 10 minutes.
[0145] 遠心分離により得られたペレットに 0. 16M NH ClZPBS (Gibco)をカ卩えて懸濁  [0145] 0.16M NH ClZPBS (Gibco) was added to the pellet obtained by centrifugation and suspended.
4  Four
し、 25°Cで 10分間インキュベートした。この試料を口径 100 mのメッシュ（Whatma n)を用いて吸引ろ過した。このろ過物を 1200 X gで 5分間遠心分離した。このように して、脂肪細胞由来幹細胞（ASC ; adipose— derived stem cell) (または PLA細 胞）を調製することができる。幹細胞であることは、細胞マーカー（CD4、 CD13、 CD 34、 CD36、 CD49d、 CD71、 CD90、 CD105、 CD117、 CD151)により確認した And incubated at 25 ° C for 10 minutes. This sample was placed on a 100 m mesh (Whatma Suction filtration was performed using n). The filtrate was centrifuged at 1200 × g for 5 minutes. In this way, adipose-derived stem cells (ASCs) (or PLA cells) can be prepared. Stem cells were confirmed by cell markers (CD4, CD13, CD34, CD36, CD49d, CD71, CD90, CD105, CD117, CD151)
[0146] (実施例 2 :脂肪吸引廃液の調製） (Example 2: Preparation of liposuction waste liquid)
脂肪吸引手術前に予定吸引脂肪量以上の、トウメセント液 (0. 0001%アドレナリン を含む生理食塩水）を皮下脂肪層に注入し（トウメセント法 (tumescent法)）、その後 、脂肪吸引のために内径 2— 3mmの力-ユーレ (金属製吸引筒）を使用して、脂肪 吸引手術を行った。脂肪吸引手術は、当該分野において周知であり、例えば、美容 外科手術プラクティス 2、巿田正成 ·谷野隆三郎 ·保阪善昭 共編、文光堂、 P429-469に、その方法が記載される。  Before the liposuction operation, a tomescent solution (a physiological saline solution containing 0.0001% adrenaline), which is equal to or larger than the expected amount of aspirated fat, is injected into the subcutaneous fat layer (tumescent method). Liposuction surgery was performed using a 2-3 mm force-Yure (metal suction tube). Liposuction surgery is well known in the art, and its method is described, for example, in Cosmetic Surgery Practice 2, edited by Masanari Takada, Ryusaburo Tanino, Yoshiaki Hosaka, Bunkodo, P429-469.
[0147] 吸引脂肪を生理食塩水で洗浄した。洗浄によって生じる計 5— 10リットル程度の廃 液のうち、細胞成分の豊富な最初の 1一 2リットルだけを、その後の処理に使用した。  [0147] The suction fat was washed with physiological saline. Of the total 5-10 liters of effluent generated by washing, only the first 1-2 liters, rich in cellular components, were used for further processing.
[0148] (実施例 3 :脂肪吸引廃液力 の、幹細胞懸濁液の調製）  (Example 3: Preparation of Stem Cell Suspension of Liposuction Waste Fluid)
廃液について、以下の 2方法のいずれかを用いて処理することによって、幹細胞懸 濁液を調製した。以下の 2方法のいずれも、コラゲナーゼなどの酵素を用いる処理が 不要であるため、コラゲナーゼなどの酵素の混入がない点においても、従来法で調 製された脂肪組織由来の幹細胞とは異なる。  The waste solution was treated using one of the following two methods to prepare a stem cell suspension. Neither of the following two methods requires a treatment using an enzyme such as collagenase, and therefore differs from adipose tissue-derived stem cells prepared by a conventional method in that there is no contamination with an enzyme such as collagenase.
[0149] (I)調製方法 1  (I) Preparation Method 1
1)脂肪吸引廃液 (通常、 2— 4リットル程度）を 400 X g、 10分遠心した。  1) Liposuction waste liquid (usually about 2 to 4 liters) was centrifuged at 400 X g for 10 minutes.
2)上清を捨てた。ただし、沈殿した細胞は浮遊しやすいため、ァスピレーターを用い て、細胞を失わないよう慎重に吸引した。  2) The supernatant was discarded. However, since the precipitated cells were easily suspended, they were carefully aspirated using an aspirator so as not to lose the cells.
3)沈殿した細胞（ほとんどが赤血球）を 50mlのポリプロピレン製チューブ数本に移し 、再度遠心 (400 X g、 5分)した。  3) The precipitated cells (mostly erythrocytes) were transferred to several 50 ml polypropylene tubes and centrifuged again (400 × g, 5 minutes).
4)上清を吸引し、総量が 15— 20mlとなるように沈殿細胞を集めた。マトリクス成分が 多い場合は 100 mフィルターで濾過'除去した。必要に応じて、再遠心を行なった 5) 50mlのチューブにフイコール（登録商標） 15mlを入れ、その上に層を作るように 極力ゆっくりと細胞液 15— 20mlを加えた。 4) The supernatant was aspirated and the precipitated cells were collected to a total volume of 15-20 ml. If the matrix component was large, it was filtered off with a 100 m filter. Re-centrifuged as needed 5) 15 ml of Ficoll (registered trademark) was placed in a 50 ml tube, and 15-20 ml of the cell solution was added as slowly as possible to form a layer thereon.
6) 400 X g、 30分遠心した。（18— 20。C)  6) Centrifuged at 400 X g for 30 minutes. (18-20.C)
7)遠心後は 4層に分離した。上から (A層）無細胞層 (透明）、（B層）単核球層 (淡赤 色)、（C層)フイコール層 (透明）、（D層)赤血球層 (濃赤色)で、幹細胞を含む接着 細胞群はに含まれていた。 A層を吸引後、 B層および約 3ml程度の C層を細胞懸濁 液として回収し、 50mlのチューブに移した。  7) After centrifugation, it was separated into 4 layers. From the top, (A layer) cell-free layer (transparent), (B layer) mononuclear cell layer (light red), (C layer) ficoll layer (transparent), (D layer) red blood cell layer (dark red), and stem cells The adherent cell group containing was included in. After aspirating the A layer, the B layer and about 3 ml of the C layer were collected as a cell suspension and transferred to a 50 ml tube.
8)回収した細胞懸濁液に、血清加 PBS (10%FBS、もしくは 10%ヒト血清をカ卩えた PBS)を加えて 50mlとし、ピペッティングによる混和後、遠心 (400 X g、 5分）した。 8) To the recovered cell suspension, add serum-added PBS (10% FBS or PBS containing 10% human serum) to make 50 ml, mix by pipetting, and centrifuge (400 xg, 5 minutes) did.
9)上清を吸引し、再度血清加 PBSをカ卩えて 50mlとし、ピペッティングによる混和後、 遠心 (400 X g、 5分)した。 9) The supernatant was aspirated, and serum-added PBS was again calibrated to 50 ml, mixed by pipetting, and centrifuged (400 xg, 5 minutes).
10)上清を吸引し、沈殿した幹細胞を含む細胞群を回収した。  10) The supernatant was aspirated, and a cell group containing precipitated stem cells was collected.
[0150] (Π)調製方法 2 (Π) Preparation method 2
1)クリーンベンチ内で、吸引管を用いて廃液を吸引し、フィルター（ポアサイズ； 120 μ m)付きリザーバーを通して、ろ過液を閉鎖分離バックに封入した。  1) In a clean bench, the waste liquid was sucked using a suction tube, and the filtrate was sealed in a closed separation bag through a reservoir equipped with a filter (pore size: 120 µm).
2)セルセパレーター（血液成分分離装置 ASTEC204、（株）アムコ）で遠心分離法 にて 3回プロセシングを行い、比重の軽い血小板成分、比重の重い赤血球、顆粒球 成分を可及的に除去した。  2) Processing was performed three times by a centrifugal separation method using a cell separator (blood component separator ASTEC204, Amco Co., Ltd.) to remove light-weight platelet components, heavy-weight red blood cells, and granulocyte components as much as possible.
3)幹細胞を高濃度に含む分画 (約 30— 40ml)を採取した。単離した細胞の比重を、 測定したところ、 1. 050-1. 075の範囲であった。  3) Fractions containing high concentrations of stem cells (about 30-40 ml) were collected. The specific gravity of the isolated cells was measured and was in the range of 1.050-1.075.
[0151] おおまかな細胞の比重は、パーコール、レディグラッドのような密度勾配遠心分離 媒体を塩ィ匕ナトリウム溶液やスクロース溶液で調製し、収集した細胞と共にデンシティ 一マーカービーズ（density marker beads)をカ卩えて遠心し、ビーズによって分け られた 5— 10層のうち、どの層に細胞があるかを確認することで、調べることが可能で ある。  [0151] The approximate specific gravity of the cells is determined by preparing a density gradient centrifugation medium such as Percoll or Ladygrad with a sodium chloride solution or a sucrose solution, and collecting the density marker beads together with the collected cells. It is possible to investigate by checking which layer contains the cells among the 5-10 layers separated by beads after centrifuging and centrifuging.
[0152] (実施例 4：回収した幹細胞の特徴付け）  (Example 4: Characterization of collected stem cells)
実施例 3にお ヽて回収した幹細胞を、以下の手順で FACSを用いて特徴付けした [0153] 約 5mlの細胞懸濁液を、染色培地（SM ;0. 5% ゥシ血清アルブミンおよび 0. 05 % NaNを含む PBS)で 2回洗浄した。必要に応じて、細胞を計数した。 The stem cells collected in Example 3 were characterized using FACS by the following procedure. [0153] About 5 ml of the cell suspension was washed twice with a staining medium (SM; PBS containing 0.5% serum albumin and 0.05% NaN). Cells were counted as needed.
3  Three
[0154] 約 1一 10 X 106細胞/ mlの細胞懸濁的に対して、最終濃度として 0. 001—0. 1 [0154] For a cell suspension of approximately 1-10 x 10 6 cells / ml, the final concentration was 0.001-0.1.
gZmlの標識ィ匕抗体 (標識には、フィコエリトリン (PE)、ァロフィコシァニン (APC) および Zまたはフルォレセインイソチオシァネート (FITC)を用いた）を添カ卩した。  gZml was added to a labeled antibody (labeled with phycoerythrin (PE), araphycocynin (APC) and Z or fluorescein isothiocynate (FITC)).
[0155] 氷上で 30分間ほどインキュベーションした後、細胞を洗浄し、 SMを用いて細胞浮 遊液の濃度を 5 X 105細胞/ ml程度に調整した。 After incubation on ice for about 30 minutes, the cells were washed, and the concentration of the cell suspension was adjusted to about 5 × 10 5 cells / ml using SM.
[0156] FACS Vantage (Becton Dickinson社）を使用した。抗体の標識を指標として 、単離した幹細胞における各種 CDタンパク質の発現を解析した。その結果、脂肪吸 引廃液由来の幹細胞は、表 1に示すように、 CD90および CD49dを発現することが 判明した。  [0156] FACS Vantage (Becton Dickinson) was used. Using the label of the antibody as an index, the expression of various CD proteins in the isolated stem cells was analyzed. As a result, as shown in Table 1, it was found that stem cells derived from the fat sucking waste liquid expressed CD90 and CD49d.
[0157] 単離した幹細胞を、 DMEM培地にお!、て 2回継代培養した。継代は、 80%のコン フルエンス時に行った。 2度の継代培養後の細胞を、上記と同様の手順で F ACSに よる分析を行った。その結果を以下の表 1に示す。  [0157] The isolated stem cells were subcultured twice in DMEM medium. Passaging was performed at 80% confluence. The cells after the two subcultures were analyzed by FACS in the same procedure as described above. The results are shown in Table 1 below.
[0158] (表 1： 2回継代培養した後の幹細胞における種々の CDの発現）  (Table 1: Expression of various CDs in stem cells after two subcultures)
CD 発現量  CD expression level
3  Three
4  Four
11c  11c
13 + +  13 + +
14  14
15  Fifteen
16  16
19  19
29 + +  29 ++
31 +  31 +
33  33
34 + 36 + + 34 + 36 ++
38  38
44 +  44 +
45 +  45 +
49d + +  49d ++
54 +  54+
56  56
58 +  58 +
61  61
62E  62E
62P  62P
69  69
71 + +  71 + +
73 + +  73 + +
90 + +  90 ++
104  104
105 + +  105 ++
106  106
117 +  117 +
135  135
144  144
146 +  146 +
151 + +  151 + +
235a - SH3 +  235a-SH3 +
STRO-1 +  STRO-1 +
「一」 =発現検出されず、 "One" = No expression detected,
「 +」 = 20%以下の細胞に検出される 「+ +」= 20%以上の細胞に検出される "+" = Detected in less than 20% of cells "++" = detected in more than 20% of cells
以上の結果から、脂肪吸引廃液から調製された幹細胞には、間葉系幹細胞ではあ るものの、従来法によって調製される脂肪由来幹細胞群と異なり、 CD31、 34陽性細 胞が含まれた。従って、本発明の方法によって調製された幹細胞は、血管内皮への 分ィ匕 (血管新生）が容易かつ高効率で可能な細胞群であることが理解できる。さらに 、今回指標として用いた CD発現は、 2回継代培養した後に確認されていることから、 本発明の幹細胞は、 2回程度の継代培養後もその表現型のほとんどを変化しないこ とが理解される。  From the above results, the stem cells prepared from the liposuction waste liquid contained CD31 and 34 positive cells, although they were mesenchymal stem cells, unlike the adipose-derived stem cell group prepared by the conventional method. Therefore, it can be understood that the stem cells prepared by the method of the present invention are a group of cells that can be easily and highly efficiently transferred to the vascular endothelium (angiogenesis). Furthermore, since the expression of CD, which was used as an index in this study, was confirmed after two subcultures, the stem cells of the present invention did not change most of their phenotypes even after about two subcultures. Is understood.
[0159] (実施例 5：複数の被検体からの廃液より回収した幹細胞の特徴付け）  [0159] (Example 5: Characterization of stem cells recovered from wastewater from multiple specimens)
さらに、複数の被検体からの廃液より幹細胞を回収し、その特徴付を行った。その 結果を以下に示す。  Further, stem cells were collected from waste fluids from a plurality of subjects and characterized. The results are shown below.
[0160] (表 2 :複数の被検体からの廃液より回収した幹細胞の特徴付けの結果）  (Table 2: Results of characterization of stem cells recovered from wastewater from multiple samples)
[0161] [表 2] [0161] [Table 2]
被検体 A B C Subject A B C
継代数 7 1 1  Passage 7 1 1
細胞数 10000 10000 30000  Cell count 10000 10000 30000
培地 DMEM Μ1 Θ9 M199  Medium DMEM Μ1 Θ9 M199
CD4 5.1 N. T.  CD4 5.1 N.T.
CD13 + 100.0 99.6  CD13 + 100.0 99.6
CD16 N. T. 1.9 1.1  CD16 N.T.1.9 1.1
CD29 + 99.9 98.9  CD29 + 99.9 98.9
CD31 - 8.0 1.7  CD31-8.0 1.7
CD34 80.3 80.6  CD34 80.3 80.6
CD36 + 27.6 1 5.6  CD36 + 27.6 1 5.6
CD44 + 100.0 99.4  CD44 + 100.0 99.4
GD45 - 8.1 0.9  GD45-8.1 0.9
CD49d + 78.0 79.4  CD49d + 78.0 79.4
CD54 N. T. N- T. 95.6  CD54 N.T.N-T.95.6
CD56 N. T. 2.1 9.0  CD56 N.T. 2.1 9.0
GD57 N. T. N. T. 0-1  GD57 N.T.N.T. 0-1
CD69 - 0.0 0.0  CD69-0.0 0.0
CD71 + 95.4 53.5  CD71 + 95.4 53.5
CD73 N. T. 89.5 98.5  CD73 N.T. 89.5 98.5
CD90 + 100.0 N. T.  CD90 + 100.0 N.T.
CD105 + 99.8 92.4  CD105 + 99.8 92.4
CD106 0.6 1 -2  CD106 0.6 1 -2
CD1 17 - 10.4 7.1  CD1 17-10.4 7.1
CD135 - 0.5 0.0  CD135-0.5 0.0
CD151 + 98.7 99.4  CD151 + 98.7 99.4
CD235a - 4.5 N. T.  CD235a-4.5 N.T.
STRO 1 N. T. 4.1 5.7 数字は、細胞集団中で、各タンパク質を発現する幹細胞の割合 (％)  STRO 1 N.T. 4.1 5.7 Number indicates percentage of stem cells expressing each protein in cell population (%)
「-」 =発現検出されず、「 +」=発現検出された、 N. T. =試験せず。  "-" = Expression not detected, "+" = expression detected, NT = not tested.
収集された幹細胞は、その集団のほとんどの細胞が、 CD13、 CD29、 CD34、 CD 36、 CD44、 CD49d、 CD54、 CD58、 CD71、 CD73、 CD90、 CD105、 CD106 、 CD151、および SH3について陽性であった。従って、本発明の脂肪幹細胞は、 C D13、 CD29、 CD34、 CD36、 CD44、 CD49d、 CD54、 CD58、 CD71、 CD73、 CD90、 CD105、 CD106、 CD151、および SH3力らなる群より選択される少なくと も 1つのタンパク質を発現する細胞である。 CD106を発現する幹細胞であることが、 その特徴の 1つである。また、 CD31、 CD45、 CD117、および CD146については、 その幹細胞の集団の一部が陽性であり、一部は陰性であった。 [0163] さらに、その幹細胞の集団は、 CD3、 CD4、 CD14、 CD15、 CD16、 CD19、 CD 33、 CD38、 CD56、 CD61、 CD62e、 CD62p、 CD69、 CD104、 CD135、および CD144については、陰性であった。従って、本発明の脂肪幹細胞は、 CD3、 CD4、 CD14、 CD15、 CD16、 CD19、 CD33、 CD38、 CD56、 CD61、 CD62e、 CD62 p、 CD69、 CD104、 CD135、および CD144の少なくとも 1つを発現しない細胞で ある。 The collected stem cells showed that most cells in the population were positive for CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151, and SH3 . Thus, the adipose stem cell of the present invention comprises at least one selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151, and SH3. Are also cells that express one protein. One of its features is that it is a stem cell that expresses CD106. For CD31, CD45, CD117, and CD146, some of the stem cell populations were positive and some were negative. [0163] Furthermore, the stem cell population was negative for CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144. Was. Thus, the adipose stem cell of the present invention is a cell that does not express at least one of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144. It is.
[0164] この幹細胞の集団を、分化誘導培地で培養する場合、 2— 3週間で骨、軟骨、脂肪 など臓器特異的なタンパク質の発現が認められた。この幹細胞の集団は、ヒト真皮由 来培養線維芽細胞とは異なり、線維芽細胞の多くの細胞で発現する CD56を、発現 しな力つた。逆に、この幹細胞の集団が発現する CD105の発現は、線維芽細胞で は、通常は見られな力つた。また、この幹細胞の集団が発現する CD49dの発現は、 骨髄由来間葉系幹細胞では通常は見られな力 た。  [0164] When this population of stem cells was cultured in a differentiation-inducing medium, expression of organ-specific proteins such as bone, cartilage, and fat was observed in a few weeks. This population of stem cells, unlike cultured human fibroblasts derived from human dermis, did not express CD56, which is expressed on many cells of fibroblasts. Conversely, expression of CD105, which is expressed by this population of stem cells, was unusual in fibroblasts. In addition, the expression of CD49d expressed by this population of stem cells was not normally observed in bone marrow-derived mesenchymal stem cells.
[0165] また、 CD31、 CD34、 CD36、 CD45、 CD106、および CD117は培養期間が長く なると発現が無くなる傾向が見られた。そのため、継代培養を続けた場合、継代培養 前に見られた CD106発現が見られなくなる場合があった。  [0165] In addition, CD31, CD34, CD36, CD45, CD106, and CD117 tended to lose their expression as the culture period became longer. Therefore, when subculture was continued, CD106 expression observed before subculture was sometimes not observed.
[0166] (実施例 6 :幹細胞の増殖）  (Example 6: Proliferation of stem cells)
上記実施例のように調製した幹細胞を、以下の表 3に示すようなコーティングを施し たディッシュ上で培養した。培養条件は、以下のとおりであった。  The stem cells prepared as in the above example were cultured on a coated dish as shown in Table 3 below. The culture conditions were as follows.
[0167] 細胞密度： 862細胞 Zcm2 [0167] Cell density: 862 cells Zcm 2
培地： M199 (酸性 FGF 2ngZml+へパリン 5 μ gZml+抗生物質含有） 温度： 37度  Medium: M199 (acidic FGF 2 ng Zml + heparin 5 μg Zml + antibiotics) Temperature: 37 degrees
継代数： 3 (脂肪細胞由来幹細胞）  Passage number: 3 (adipocyte-derived stem cells)
CO濃度： 5. 0%。  CO concentration: 5.0%.
2  2
[0168] [表 3] べクトン ディッキンソンバイオコート製品一 g [0168] [Table 3] Becton Dickinson Bio Coat Product 1 g
Figure imgf000044_0001
Figure imgf000044_0001
フイブリンのりは、以下のようにして調製した。  Fibrin glue was prepared as follows.
[0169] (A液）  [0169] (Solution A)
ACD加血 (テルモ 4連バッグ）またはその等価物（4時間以内のものを使用する琴 が好ましいようである）を使用した。この血液を、 4°C 3000rpm lOmin (あるいは 4000rpm 6分)で処理した。バフィ一コート、赤血球を取り除き血漿を使用した。こ の血漿は、 80°Cで急速冷凍するか、あるいは、 40°Cでエアキャップなどを巻いて ゆっくり凍結させた。（フイブリンの収量を出来るだけ多くするため）。ここで、静置した 状態で凍結'解凍を 2度繰り返すと収量は 1. 5倍になる。あるいは、— 20度で振盪し ながら緩速凍結し、 4°C水槽で緩速解凍したほうが、収量が多力つた。 20°Cで保存 する。解凍は、 4°Cで 16— 18— 24時間程度行った。  ACD bleeding (Terumo quadruple bags) or its equivalent (a koto using less than 4 hours seems to be preferred) was used. The blood was processed at 3000 rpm 10 min at 4 ° C (or 4000 rpm for 6 minutes). Buffy coat, red blood cells were removed and plasma was used. The plasma was snap frozen at 80 ° C or slowly frozen at 40 ° C with an air cap or the like. (To maximize the yield of fibrin). Here, if freeze-thaw is repeated twice while still standing, the yield will be 1.5 times. Alternatively, slower freezing with shaking at -20 ° C and slow thawing in a 4 ° C water bath yielded more yield. Store at 20 ° C. Thawing was performed at 4 ° C for 16-18-24 hours.
[0170] 80°Cで凍結保存されていたものを 4°Cで解凍して用いた。 （24時間） [0170] Those frozen and stored at 80 ° C were thawed at 4 ° C and used. (24hours)
(B液）  (B liquid)
トロンビン末 5000単位；0. 5M塩化カルシウム液 lml；ァプロチュン 3500単位；蒸 留水 7. 5mlで調製した B液を、蒸留水にて 50倍、 500倍に希釈 '調製した。  Thrombin powder 5000 units; 0.5 M calcium chloride solution 1 ml; aprotune 3500 units; distilled water 7.5 ml of solution B was diluted 50 times and 500 times with distilled water.
[0171] このフイブリンのりは、市販される日本臓器 (製品名：ティシール)または藤沢薬品（ 製品名：ボルヒール)またはへキスト (製品名：ベリプラスト）力も入手して用いることも できる。 [0171] This fibrin glue can also be obtained from commercially available Japanese organs (product name: Tissile) or Fujisawa Pharmaceutical (product name: Bolhir) or Hoechst (product name: beriplast).
[0172] 上述のフイブリンのりを、以下のようにして培養容器にコーティングした。培養容器と して 10cmディッシュ（FALCON 35—3003 Tissue Culture Dish, Becton Dickinson)の容¾： 用いた。 [0173] コーティングの方法は以下のとおりである。 [0172] The above-mentioned fibrin glue was coated on a culture vessel as follows. A 10 cm dish (FALCON 35-3003 Tissue Culture Dish, Becton Dickinson) was used as a culture vessel. [0173] The coating method is as follows.
10cm dishに A液 3mlを加えてプレート前面が浸るように振盪したのち、不要な A液 を吸引'回収した。それから、調製した B液 3mlをカ卩えてやはり同様に振盪したのち余 分は吸引除去して風乾した。（30分)。  3 ml of solution A was added to a 10 cm dish, and the plate was shaken so that the front of the plate was immersed. Then, unnecessary solution A was suctioned and collected. Then, 3 ml of the prepared solution B was dried and shaken in the same manner, and the remainder was removed by suction and air-dried. (Half an hour).
[0174] このようにして調製したディッシュを用いて、以下のような条件で幹細胞を培養した。 [0174] Using the dish thus prepared, stem cells were cultured under the following conditions.
その手順を以下に示す。  The procedure is described below.
[0175] 細胞密度： 862細胞 Zcm2 [0175] Cell density: 862 cells Zcm 2
培地： M199 (酸性 FGF 2ngZml+へパリン 5 μ gZml+抗生物質）  Medium: M199 (acidic FGF 2ngZml + heparin 5μgZml + antibiotic)
温度： 37°C  Temperature: 37 ° C
継代数： 3 (脂肪細胞由来幹細胞）  Passage number: 3 (adipocyte-derived stem cells)
CO濃度： 5. 0%  CO concentration: 5.0%
2  2
(結果）  (Result)
培養した結果を以下の表および図 1に示す。  The results of the culture are shown in the following table and FIG.
[0176] [表 4] [0176] [Table 4]
0曰 2曰 0 said 2 said
コ^イングなし 50000 73333  No coining 50000 73333
コラーゲン I 50000 122222  Collagen I 50000 122222
コラーゲン IV 50000 97778  Collagen IV 50000 97778
ラミニン 50000 122222  Laminin 50000 122222
フイブリンのリ X 50 50000 171111  Fibrin Refill X 50 50000 171111
フイブリンのリ X 500 50000 146667  Fibrin Lis X 500 50000 146667
ゼラチン 50000 122222  Gelatin 50000 122222
(数は、細胞数である） (The number is the number of cells)
図 1および上記表 4からも明らかなように、いずれのコーティング剤を用いても、コー ティングなしよりも顕著に幹細胞の増殖が上昇していることが明らかになった。このよう な細胞の多分ィ匕能を調べたところ、維持されていることも明らかになった。  As is clear from FIG. 1 and Table 4 above, it was found that the use of any of the coating agents significantly increased the proliferation of stem cells as compared to the case without the coating. Examination of the probable ability of such cells revealed that they were maintained.
(実施例 7 :別の幹細胞）  (Example 7: Another stem cell)
実施例 6に準じて、他の幹細胞 (骨髄由来の間葉系幹細胞など）についても、増殖 促進活性があるかどうかを確認する。骨髄由来の間葉系幹細胞は、骨髄から、 FAC Sにてソートすることによって調製する。セルソーターを用いて、 CD34+, CD117 (c kit) +, Sea— 1+を未分ィ匕の指標として用いて、必要に応じて SH— 2 (CD73) , SHAccording to Example 6, it is confirmed whether or not other stem cells (such as mesenchymal stem cells derived from bone marrow) also have a proliferation promoting activity. Bone marrow-derived mesenchymal stem cells are Prepared by sorting in S. Using a cell sorter, using CD34 +, CD117 (c kit) +, Sea-1 + as an indicator of undivided, SH-2 (CD73), SH
-3 (CD105) , STRO-1, CD13, CD45などを参酌して選抜する。 -3 (CD105), STRO-1, CD13, CD45, etc.
[0178] 実施例 6において用いたディッシュを用いる。その結果、骨髄由来の幹細胞でも、 同様に増殖が上昇することが明らかになる。 [0178] The dish used in Example 6 is used. As a result, it becomes clear that proliferation of bone marrow-derived stem cells also increases.
[0179] (実施例 8 :多分化能の確認） (Example 8: Confirmation of pluripotency)
実施例 6および 7で調製される細胞の多分化能を確認する。当該分野にお!ヽて周 知の方法によって、本発明の幹細胞を分化させたところ、分ィ匕能が保持されているこ とが明らかになる。  The pluripotency of the cells prepared in Examples 6 and 7 is confirmed. When the stem cell of the present invention is differentiated by a known method in this field, it becomes clear that the differentiation ability is maintained.
[0180] (実施例 9 :別の幹細胞） (Example 9: Another stem cell)
実施例 6に準じて、他の幹細胞 (ES細胞など）についても、増殖促進活性があるか どうかを確認する。 ES細胞は当該分野において公知の手法により確立する。  According to Example 6, it is confirmed whether other stem cells (such as ES cells) also have a growth promoting activity. ES cells are established by techniques known in the art.
[0181] 実施例 6において用いたディッシュを用いる。その結果、骨髄由来の幹細胞でも、 同様に増殖が上昇することが明らかになる。 [0181] The dish used in Example 6 is used. As a result, it becomes clear that proliferation of bone marrow-derived stem cells also increases.
[0182] (実施例 7： PRP (血小板含有血漿)を用いた細胞増殖比較実験） (Example 7: Comparative experiment of cell proliferation using PRP (platelet-containing plasma))
(実験材料及び実験方法）  (Experimental materials and methods)
•細胞：ヒト脂肪糸且織由来間質細胞 (脂肪細胞由来幹細胞）  • Cells: Human fatty fibroblast-derived stromal cells (fat cell-derived stem cells)
•培地： 10% FBS (ゥシ胎仔血清） ZM199 (Medium 199、ペニシリン Zストレプ トマイシン，酸性 FGF,へパリン添加）  • Medium: 10% FBS (ゥ fetal serum) ZM199 (Medium 199, penicillin Z streptomycin, acid FGF, heparin added)
•PRPコーティングディッシュ： A液（トロンビン 5000U、 0. 5M CaCl 1ml,ァプロ  • PRP coating dish: Solution A (Thrombin 5000U, 0.5M CaCl 1ml, apro
2  2
チュン 35000U)を滅菌水で 50倍希釈する。希釈後の A液をディッシュに広げ、その 上にヒト PRPを全体に広げた後乾燥させる。  Dilute 35000U) 50 times with sterile water. Spread the diluted solution A on a dish, spread human PRP on it, and dry.
[0183] (PRPコートディッシュを用いた増殖実験） [0183] (Expansion experiment using PRP coated dish)
FBS10%添加した M199を用いて、ヒト PRPコート 6ゥエル一プレート，非コーティン グ 6ゥエループレートにヒト脂肪細胞由来幹細胞 (A)を 2 X 104細胞 Zゥエルにて播種 した。 4日後に培地交換、 7日後に細胞数を測定した。 Using M199 supplemented with 10% FBS, human adipocyte-derived stem cells (A) were seeded on a human PRP-coated 6-well plate and a non-coated 6-well plate at 2 × 10 4 cells. After 4 days, the medium was replaced, and after 7 days, the number of cells was measured.
[0184] (結果） [0184] (Result)
10%FBS添加 M199培地を用いた脂肪細胞由来幹細胞の増殖には、 PRPコート が効果的であった。 For growth of adipocyte-derived stem cells using M199 medium supplemented with 10% FBS, PRP-coated Was effective.
[0185] 以上のように、本発明の好ましい実施形態を用いて本発明を例示してきた力 本発 明は、この実施形態に限定して解釈されるべきものではない。本発明は、特許請求 の範囲によってのみその範囲が解釈されるべきであることが理解される。当業者は、 本発明の具体的な好ましい実施形態の記載から、本発明の記載および技術常識に 基づいて等価な範囲を実施することができることが理解される。本明細書において引 用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載さ れているのと同様にその内容が本明細書に対する参考として援用されるべきであるこ とが理解される。  [0185] As described above, the present invention which has exemplified the present invention using the preferred embodiment of the present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the appended claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and common technical knowledge from the description of the specific preferred embodiments of the present invention. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety, as if the content itself were specifically described herein. Is understood.
産業上の利用可能性  Industrial applicability
[0186] 本発明は、幹細胞 (特に、脂肪由来の幹細胞）に関し簡便に増殖速度を上昇させる ことを可能にする。従って、本発明は、幹細胞を利用する医療、医薬、農学分野など において有用性を有する。 [0186] The present invention makes it possible to easily increase the proliferation rate of stem cells (particularly, stem cells derived from fat). Therefore, the present invention has utility in medical, pharmaceutical, agricultural, etc. fields utilizing stem cells.

Claims

請求の範囲 The scope of the claims
[I] フイブリノゲンまたはその誘導体を含む、幹細胞を培養するための培養容器。  [I] A culture vessel for culturing stem cells, comprising fibrinogen or a derivative thereof.
[2] 前記フイブリノゲンまたはその誘導体は、前記培養容器にコーティングされる、請求 項 1に記載の培養容器。  [2] The culture container according to claim 1, wherein the fibrinogen or a derivative thereof is coated on the culture container.
[3] 前記フイブリノゲンまたはその誘導体は、前記培養容器に 50 μ m— 300 μ mの厚さ でコーティングされる、請求項 1に記載の培養容器。 [3] The culture vessel according to claim 1, wherein the fibrinogen or a derivative thereof is coated on the culture vessel with a thickness of 50 µm to 300 µm.
[4] 前記フイブリノゲンまたはその誘導体は、フイブリンを含む、請求項 1に記載の培養容  [4] The culture volume according to claim 1, wherein the fibrinogen or a derivative thereof includes fibrin.
[5] 前記フイブリンは、フイブリンモノマー、フイブリンポリマーおよびフィブリン塊からなる 群より選択される形態を含む、請求項 1に記載の培養容器。 [5] The culture container according to claim 1, wherein the fibrin has a form selected from the group consisting of a fibrin monomer, a fibrin polymer, and a fibrin clot.
[6] 前記フイブリノゲンまたはその誘導体は、プロテアーゼを含む、請求項 1に記載の培 養容器。 [6] The culture container according to claim 1, wherein the fibrinogen or a derivative thereof contains a protease.
[7] 前記フイブリノゲンまたはその誘導体は、トロンビンまたはノ トロキソビンを含む、請求 項 1に記載の培養容器。  [7] The culture container according to claim 1, wherein the fibrinogen or a derivative thereof includes thrombin or notroxobin.
[8] 前記フイブリノゲンまたはその誘導体は、 exトロンビンを含む、請求項 1に記載の培養 谷器。 [8] The culture device according to claim 1, wherein the fibrinogen or a derivative thereof includes ex-thrombin.
[9] 前記フイブリノゲンまたはその誘導体は、プロテアーゼ阻害剤を含む、請求項 1に記 載の培養容器。  [9] The culture vessel according to claim 1, wherein the fibrinogen or a derivative thereof contains a protease inhibitor.
[10] 前記プロテアーゼ阻害剤は、ァプロチュンおよび Zまたは 4 (アミノメチル)シクロへ キサンカルボン酸を含む、請求項 9に記載の培養容器。  [10] The culture vessel according to claim 9, wherein the protease inhibitor comprises aprotune and Z or 4 (aminomethyl) cyclohexanecarboxylic acid.
[I I] 前記フイブリノゲンまたはその誘導体は、フイブリンのりである、請求項 1に記載の培 養容器。  [II] The culture container according to claim 1, wherein the fibrinogen or a derivative thereof is fibrin glue.
[12] 前記フイブリンのりは、フイブリンモノマー、フイブリンポリマー、フイブリノゲンまたは血 小板の全部もしくは一部を成分として含む、請求項 11に記載の培養容器。  12. The culture container according to claim 11, wherein the fibrin glue contains, as a component, all or a part of a fibrin monomer, a fibrin polymer, a fibrinogen, or a platelet.
[13] 前記フイブリンのりは、該フイブリンのり中に含まれる可溶性のフイブリノゲン (I因子） 力 トロンビンによって不溶性のフイブリン (フイブリンモノマー）に転換される力 フィ ブリンモノマーは互いの N末端と C末端が重合して、フイブリンポリマーとなり、フイブリ ン塊を形成して!/ヽくと!ヽぅ接着性を有する、請求項 11に記載の培養容器。 [13] The fibrin glue has the ability to convert soluble fibrinogen (factor I) contained in the fibrin glue into insoluble fibrin (fibrin monomer) by thrombin. 12. The culture vessel according to claim 11, wherein the culture vessel becomes a fibrin polymer to form a fibrin mass and has an adhesive property.
[14] 前記フイブリンのりは、クリオプレシピテート、血漿または血小板含有血漿 (PRP)を 5[14] The fibrin glue is used for cryoprecipitate, plasma or platelet-containing plasma (PRP).
00倍以下に希釈したものである、請求項 11に記載の培養容器。 12. The culture vessel according to claim 11, wherein the culture vessel has been diluted to 00 times or less.
[15] 前記フイブリンのりは、クリオプレシピテート、血漿または血小板含有血漿 (PRP)を 5[15] The fibrin glue is used for cryoprecipitate, plasma or platelet-containing plasma (PRP).
0倍一 500倍に希釈したものである、請求項 11に記載の培養容器。 12. The culture vessel according to claim 11, wherein the culture vessel is diluted 0-fold to 500-fold.
[16] 前記幹細胞は、脂肪組織由来である、請求項 1に記載の培養容器。 [16] The culture container according to claim 1, wherein the stem cells are derived from adipose tissue.
[17] 前記幹細胞は、間葉系幹細胞である、請求項 1に記載の培養容器。 [17] The culture vessel according to claim 1, wherein the stem cells are mesenchymal stem cells.
[18] 前記幹細胞は、脂肪吸引物に由来する、請求項 1に記載の培養容器。 [18] The culture container according to claim 1, wherein the stem cells are derived from a lipoaspirate.
[19] 前記幹細胞は、脂肪細胞由来幹細胞 (ASC ; adipose— derived stem cell)であ る、請求項 1に記載の培養容器。 [19] The culture container according to claim 1, wherein the stem cell is an adipose-derived stem cell (ASC).
[20] 前記フイブリンまたはその分解物は、 0. 1— lOOmg/cm2の濃度で含まれることを特 徴とする、請求項 1に記載の培養容器。 [20] The culture vessel according to claim 1, wherein the fibrin or a degradation product thereof is contained at a concentration of 0.1 to 100 mg / cm 2 .
[21] 前記培養容器の材質は、ガラス、シリカ、シリコン、セラミック、二酸化珪素、プラスチ ック、金属、天然ポリマーおよび合成ポリマー力 なる群より選択される材料を含む、 請求項 1に記載の培養容器。 [21] The culture according to claim 1, wherein the material of the culture container includes a material selected from the group consisting of glass, silica, silicon, ceramic, silicon dioxide, plastic, metal, natural polymer, and synthetic polymer. container.
[22] 幹細胞を調製するための方法であって、 [22] A method for preparing a stem cell, comprising:
A)幹細胞を提供する工程；および  A) providing stem cells; and
B)該幹細胞を、培地ならびにフイブリノゲンまたはその誘導体を含む培養容器中で 培養する工程、  B) culturing the stem cells in a culture vessel containing a medium and fibrinogen or a derivative thereof,
を包含する、方法。  A method comprising:
[23] 前記培養は、 pH7. 2-7. 4、温度 37°C、および CO濃度 5%という条件下で培養  [23] The culture was performed under the conditions of pH 7.2-7.4, temperature of 37 ° C, and CO concentration of 5%.
2  2
される、請求項 22に記載の方法。  23. The method of claim 22, wherein the method is performed.
[24] 前記培養は、 DMEM、 P199、 MEM、 Hanks平衡化塩溶液（HBSS)、 Ham' s F 12、 BME、 RPMI1640、 MCDB104および MCDB153 (KGM)からなる群より選 択される培地を用いて行われる、請求項 22に記載の方法。  [24] The culture is performed using a medium selected from the group consisting of DMEM, P199, MEM, Hanks' balanced salt solution (HBSS), Ham's F12, BME, RPMI1640, MCDB104 and MCDB153 (KGM). 23. The method of claim 22, wherein the method is performed.
[25] 前記培養は、副腎皮質ステロイド、インスリン、グルコース、インドメタシン、イソブチル ーメチルキサンチン（IBMX)、ァスコルビン酸およびその誘導体、グリセ口ホスフェート 、エストロゲンおよびその誘導体、プロゲステロンおよびその誘導体、アンドロゲンお よびその誘導体、増殖因子、下垂体エキス、松果体エキス、レチノイン酸、ビタミン D 、甲状腺ホルモン、子牛血清、馬血清、ヒト血清、へパリン、炭酸水素ナトリウム、 HE PES,アルブミン、トランスフェリン、セレン酸塩、リノレン酸、 3 イソブチルー 1 メチル キサンチン、脱メチル化剤、ヒストン脱ァセチル化剤、ァクチビン、サイト力イン、へキ サメチレンビスァセトアミド（HMBA)、ジメチルァセトアミド（DMA)、ジブチル cAMP (dbcAMP)、ジメチルスルホキシド（DMSO)、ョードデォキシゥリジン（IdU)、ヒドロ キシゥレア（HU)、シトシンァラビノシド（AraC)、マイトマイシン C (MMC)、酪酸ナト リウム (NaBu)、ポリプレンおよびセレニウム力もなる群より選択される成分のうち少な くとも 1つを含む培養液中で行われる、請求項 22に記載の方法。 [25] The culture is performed using corticosteroids, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbic acid and its derivatives, glycerol phosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives. , Growth factor, pituitary extract, pineal extract, retinoic acid, vitamin D , Thyroid hormone, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HE PES, albumin, transferrin, selenate, linolenic acid, 3-isobutyl-1-methyl xanthine, demethylating agent, histone deacetylation Agent, activin, cytoforce, hexamethylenebisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethylsulfoxide (DMSO), ododexperidine (IdU ), Hydroxyurea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polyprene and at least one component selected from the group consisting of selenium powers 23. The method according to claim 22, which is performed in a culture solution.
[26] さらに、前記幹細胞を入手する工程を包含する、請求項 22に記載の方法。 26. The method according to claim 22, further comprising a step of obtaining said stem cells.
[27] さらに、前記幹細胞を分化させる工程を包含する、請求項 22に記載の方法。 [27] The method according to claim 22, further comprising a step of differentiating the stem cell.
[28] 前記フイブリノゲンまたはその誘導体は、前記培養容器にコーティングされる、請求 項 22に記載の方法。 [28] The method according to claim 22, wherein the fibrinogen or a derivative thereof is coated on the culture vessel.
[29] 幹細胞を培養するための、フイブリノゲンまたはその誘導体がコーティングされた培養 容器を生産するための方法であって、  [29] A method for producing a culture vessel coated with fibrinogen or a derivative thereof for culturing stem cells, comprising:
A)培養容器を提供する工程；  A) providing a culture vessel;
B)該培養容器にフイブリノゲンまたはその誘導体を提供する工程；および B) providing fibrinogen or a derivative thereof to the culture vessel;
C)該フイブリノゲンまたはその誘導体を該培養容器の表面に固定する工程、 を包含する、方法。 C) fixing the fibrinogen or a derivative thereof on the surface of the culture vessel.
[30] 前記フイブリノゲンまたはその誘導体の提供は、無菌操作にて行われる、請求項 29 に記載の方法。  [30] The method according to claim 29, wherein the provision of the fibrinogen or a derivative thereof is performed by an aseptic operation.
[31] 前記固定は、乾燥状態で一定時間放置すること、または紫外線照射という特徴を有 する、請求項 29に記載の方法。  31. The method according to claim 29, wherein the fixing is characterized by being left in a dry state for a predetermined time, or by irradiating with ultraviolet rays.
[32] 幹細胞を培養するための、組織接着剤がコーティングされた培養容器。 [32] A culture vessel coated with a tissue adhesive for culturing stem cells.
[33] 前記組織接着剤は、シァノアクリレート系接着剤、ゼラチン アルデヒド系接着剤およ びフイブリングルー系接着剤力もなる群より選択される、請求項 32に記載の培養容器 33. The culture container according to claim 32, wherein the tissue adhesive is selected from the group consisting of a cyanoacrylate adhesive, a gelatin aldehyde adhesive, and a fibrin glue adhesive.
[34] 幹細胞を培養するための、細胞外マトリクスがコーティングされた培養容器。 [34] A culture vessel coated with an extracellular matrix for culturing stem cells.
[35] 前記幹細胞は、脂肪組織由来である、請求項 34に記載の培養容器。 35. The culture container according to claim 34, wherein the stem cells are derived from adipose tissue.
[36] 前記幹細胞は、間葉系幹細胞である、請求項 34に記載の培養容器。 36. The culture container according to claim 34, wherein the stem cells are mesenchymal stem cells.
[37] 前記幹細胞は、脂肪細胞由来幹細胞である、請求項 34に記載の培養容器。 [37] The culture container according to claim 34, wherein the stem cells are fat cell-derived stem cells.
[38] 前記幹細胞は、脂肪吸引物に由来する、請求項 34に記載の培養容器。 38. The culture container according to claim 34, wherein the stem cells are derived from a lipoaspirate.
[39] 前記細胞外マトリクスは、コラーゲン、ラミニン、フイブロネクチン、テネイシン、サイトタ クチン、へキサブラキオン、ェンタクチン、ナイトジェン、ビトロネクチン、ゼラチンおよ びプロテオダリカン力 なる群より選択される、請求項 34に記載の培養容器。 [39] The extracellular matrix according to claim 34, wherein the extracellular matrix is selected from the group consisting of collagen, laminin, fibronectin, tenascin, cytotactin, hexabrachion, entactin, nightgen, vitronectin, gelatin and proteodarican. The culture container according to any one of the preceding claims.
[40] 前記細胞外マトリクスは、 3 μ g/cm2— 20 μ g/cm2の濃度で含まれることを特徴と する、請求項 34に記載の培養容器。 [40] The culture container according to claim 34, wherein the extracellular matrix is contained at a concentration of 3 µg / cm 2 to 20 µg / cm 2 .
[41] 幹細胞を培養するための、ゼラチンがコーティングされた培養容器。 [41] A culture vessel coated with gelatin for culturing stem cells.
[42] 幹細胞を調製するための方法であって、 [42] A method for preparing a stem cell, comprising:
A)培地、ならびに細胞外マトリクスおよび Zまたはゼラチンを含む培養容器を提供 する工程；  A) providing a medium and a culture vessel containing the extracellular matrix and Z or gelatin;
B)幹細胞を該培養容器に配置する工程；  B) arranging stem cells in the culture vessel;
C)該幹細胞を所望の期間培養する工程、  C) culturing the stem cells for a desired period,
を包含する、方法。  A method comprising:
[43] 幹細胞を培養するための、細胞外マトリクスおよび Zまたはゼラチンがコーティングさ れた培養容器を生産するための方法であって、  [43] A method for producing a culture vessel coated with an extracellular matrix and Z or gelatin for culturing stem cells,
A)培養容器を提供する工程；  A) providing a culture vessel;
B)該培養容器に細胞外マトリクスおよび Zまたはゼラチンを提供する工程；および c)該細胞外マトリクスおよび Zまたはゼラチンを該培養容器の表面に固定するェ 程、  B) providing the extracellular matrix and Z or gelatin to the culture vessel; and c) immobilizing the extracellular matrix and Z or gelatin on the surface of the culture vessel.
を包含する、方法。  A method comprising:
[44] フイブリノゲンまたはその誘導体の、幹細胞を調製するための使用。  [44] Use of fibrinogen or a derivative thereof for preparing a stem cell.
[45] 細胞外マトリクスおよび Zまたはゼラチンの、幹細胞を調製するための使用。  [45] Use of extracellular matrix and Z or gelatin for preparing stem cells.
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