WO2005090550A1 - Procédé de prolifération de cellule souche - Google Patents

Procédé de prolifération de cellule souche 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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Prior art keywords
stem cells
culture
cells
fibrinogen
culture vessel
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PCT/JP2005/004831
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English (en)
Japanese (ja)
Inventor
Kotaro Yoshimura
Daisuke Matsumoto
Emiko Aiba
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Biomaster, Inc.
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Priority to JP2006511226A priority Critical patent/JPWO2005090550A1/ja
Publication of WO2005090550A1 publication Critical patent/WO2005090550A1/fr

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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

Procédé d'amplification de cellules souches (par exemple des cellules souches de tissus telles que des cellules souches mésenchymateuses (en particulier dérivées de matière grasse)). Il est fourni une cuve de culture recouverte de fibrinogène dont la colle de fibrine est un exemple typique ou de son dérivé ou d'une matrice extracellulaire (par exemple du collagène (par exemple de type I, de type IV, etc.), de la laminine, de la fibronectine, etc.) ou de gélatine ou similaire. En plus, il est fourni un procédé de préparation de cellules souches, comprenant les étapes consistant à (A) fournir des cellules souches et (B) faire croître les cellules souches dans une cuve de culture contenant un milieu de culture et un fibrinogène ou son dérivé.
PCT/JP2005/004831 2004-03-18 2005-03-17 Procédé de prolifération de cellule souche WO2005090550A1 (fr)

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WO2007037561A1 (fr) * 2005-09-30 2007-04-05 Tobishi Pharmaceutical Co., Ltd. Agent d'activation des cellules souches et/ou des cellules progeniteurs
JP2007289076A (ja) * 2006-04-25 2007-11-08 Kaneka Corp 脂肪組織から幹細胞を採取するのに適した細胞分離装置、およびその方法
JP2008228629A (ja) * 2007-03-19 2008-10-02 Hajime Inoue 上皮幹細胞の分化誘導方法
JP2009524425A (ja) * 2006-01-27 2009-07-02 プロスティミクス カンパニー リミテッド 脂肪由来幹細胞を利用した成長因子の大量生産方法
JP2011004705A (ja) * 2009-06-29 2011-01-13 Olympus Corp 細胞分離装置および細胞分離方法
TWI382090B (zh) * 2005-09-30 2013-01-11 Tobishi Pharmaceutical Co 幹細胞及／或前驅細胞的賦活劑
JP2013063088A (ja) * 2012-12-14 2013-04-11 Nippon Institute For Biological Science 脂肪組織間質細胞の神経細胞への分化誘導方法
CN106754364A (zh) * 2017-03-14 2017-05-31 南京九寿堂医药科技有限公司 一种提高肺癌干细胞富集效率的细胞培养容器
JP2019081713A (ja) * 2017-10-27 2019-05-30 日本メナード化粧品株式会社 毛包幹細胞の未分化状態維持剤
KR102056391B1 (ko) 2017-03-14 2019-12-16 울산과학기술원 하이드로젤 패치
CN110709504A (zh) * 2017-06-05 2020-01-17 梅约医学教育与研究基金会 培养、增殖和分化干细胞的方法和材料
WO2022050282A1 (fr) * 2020-09-01 2022-03-10 株式会社セルファイバ Échafaudage, procédé de production d'échafaudage, culture cellulaire, procédé de culture cellulaire
US11679180B2 (en) 2016-12-07 2023-06-20 Mayo Foundation For Medical Education And Research Methods and materials for using fibrin supports for retinal pigment epithelium transplantation

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JP2002315567A (ja) * 2001-04-18 2002-10-29 Yasuhiko Tabata 細胞接着活性物質を含有してなる幹細胞培養用基材

Patent Citations (1)

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JP2002315567A (ja) * 2001-04-18 2002-10-29 Yasuhiko Tabata 細胞接着活性物質を含有してなる幹細胞培養用基材

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TWI382090B (zh) * 2005-09-30 2013-01-11 Tobishi Pharmaceutical Co 幹細胞及／或前驅細胞的賦活劑
WO2007037561A1 (fr) * 2005-09-30 2007-04-05 Tobishi Pharmaceutical Co., Ltd. Agent d'activation des cellules souches et/ou des cellules progeniteurs
JP2009509914A (ja) * 2005-09-30 2009-03-12 東菱薬品工業株式会社 幹細胞及び／又は前駆細胞の賦活剤
EA014296B1 (ru) * 2005-09-30 2010-10-29 Тобиси Фармасьютикал Ко., Лтд. Средство, активирующее стволовые клетки и/или клетки-предшественники
AU2006295607B2 (en) * 2005-09-30 2010-12-16 Tobishi Pharmaceutical Co., Ltd. Activating agent of stem cells and/or progenitor cells
JP2009524425A (ja) * 2006-01-27 2009-07-02 プロスティミクス カンパニー リミテッド 脂肪由来幹細胞を利用した成長因子の大量生産方法
JP2007289076A (ja) * 2006-04-25 2007-11-08 Kaneka Corp 脂肪組織から幹細胞を採取するのに適した細胞分離装置、およびその方法
JP2008228629A (ja) * 2007-03-19 2008-10-02 Hajime Inoue 上皮幹細胞の分化誘導方法
JP2011004705A (ja) * 2009-06-29 2011-01-13 Olympus Corp 細胞分離装置および細胞分離方法
JP2013063088A (ja) * 2012-12-14 2013-04-11 Nippon Institute For Biological Science 脂肪組織間質細胞の神経細胞への分化誘導方法
US11679180B2 (en) 2016-12-07 2023-06-20 Mayo Foundation For Medical Education And Research Methods and materials for using fibrin supports for retinal pigment epithelium transplantation
CN106754364A (zh) * 2017-03-14 2017-05-31 南京九寿堂医药科技有限公司 一种提高肺癌干细胞富集效率的细胞培养容器
KR102056391B1 (ko) 2017-03-14 2019-12-16 울산과학기술원 하이드로젤 패치
KR102624998B1 (ko) * 2017-06-05 2024-01-12 메이오 파운데이션 포 메디칼 에쥬케이션 앤드 리써치 줄기 세포의 배양, 증식 및 분화를 위한 방법 및 물질
KR20200016240A (ko) * 2017-06-05 2020-02-14 메이오 파운데이션 포 메디칼 에쥬케이션 앤드 리써치 줄기 세포의 배양, 증식 및 분화를 위한 방법 및 물질
JP2020522264A (ja) * 2017-06-05 2020-07-30 メイヨ・ファウンデーション・フォー・メディカル・エデュケーション・アンド・リサーチ 幹細胞を培養し、増殖させ、分化させるための方法及び材料
EP3635094A4 (fr) * 2017-06-05 2021-03-17 Mayo Foundation for Medical Education and Research Procédés et matériels pour la culture, la prolifération et la différenciation de cellules souches
CN110709504A (zh) * 2017-06-05 2020-01-17 梅约医学教育与研究基金会 培养、增殖和分化干细胞的方法和材料
JP7311433B2 (ja) 2017-06-05 2023-07-19 メイヨ・ファウンデーション・フォー・メディカル・エデュケーション・アンド・リサーチ 幹細胞を培養し、増殖させ、分化させるための方法及び材料
JP7015518B2 (ja) 2017-10-27 2022-02-03 日本メナード化粧品株式会社 毛包幹細胞の未分化状態維持剤
JP2019081713A (ja) * 2017-10-27 2019-05-30 日本メナード化粧品株式会社 毛包幹細胞の未分化状態維持剤
WO2022050282A1 (fr) * 2020-09-01 2022-03-10 株式会社セルファイバ Échafaudage, procédé de production d'échafaudage, culture cellulaire, procédé de culture cellulaire

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