WO2006022091A1 - Method of culturing human bone marrow-origin mesenchymal stem cells using human serum medium - Google Patents

Method of culturing human bone marrow-origin mesenchymal stem cells using human serum medium Download PDF

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WO2006022091A1
WO2006022091A1 PCT/JP2005/013020 JP2005013020W WO2006022091A1 WO 2006022091 A1 WO2006022091 A1 WO 2006022091A1 JP 2005013020 W JP2005013020 W JP 2005013020W WO 2006022091 A1 WO2006022091 A1 WO 2006022091A1
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mesenchymal stem
stem cells
cells
cell
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PCT/JP2005/013020
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French (fr)
Japanese (ja)
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Hajime Ohgushi
Akira Fujisawa
Hiroko Machida
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National Institute Of Advanced Industrial Scienceand Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure

Definitions

  • the present invention relates to a cell culture method for efficiently and safely culturing large amounts of human thread and tissue cells for use in regenerative medicine, cell therapy, and the like.
  • Patent Document 1 is an invention relating to a culture solution obtained by adding growth factors and the like together with human serum to a culture solution of human mature hepatocytes
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2002-78484
  • JP 2003-52360 Patent Document 3 describes the presence of a basement membrane extracellular matrix.
  • the invention relates to a method in which 2-10% human serum is added when culturing mesenchymal stem cells under the circumstances, and JP 2003-235548 (Patent Document 4) describes the growth of human serum and human serum in a culture medium for human cells. It is a patent application characterized by including a factor.
  • Patent Document 5 cell culture as part of a therapeutic drug test method for osteoporosis patients, which is treated with continuous enzyme treatment using biopsy sections of patient iliac bones.
  • the content is characterized by using 1 to 12% range of added serum such as urine fetal serum, human serum, urine serum albumin or Ultrocell.
  • the object and contents of the present invention are clearly different.
  • Non-Patent Document 1 Hiroyuki Funaoka
  • Hajime Ogushi Bone Regenerative Medicine, Rheumatology, 30: 430-435, 2003
  • Non-Patent Document 2 Yonsei Medical Journal, vol. 45, p61-67, 2004 (Non-patent Document 2) published a paper on basic research results using urchin fetal serum.
  • Patent Document 1 WO2002 / 024875
  • Patent Document 2 JP 2002-78484 A
  • Patent Document 3 JP 2003-52360
  • Patent Document 4 JP-A 2003-235548
  • Patent Document 5 Special Tables 11-506010
  • Non-Patent Document 1 Hiroyuki Funaoka, Ogushi Hajime: Bone Regenerative Medicine, Rheumatology, 30: 430— 435, 20 03
  • Non-Patent Document 2 Yonsei Medical Journal, vol. 45, p61-67, 2004
  • An object of the present invention is to provide a method for culturing mesenchymal stem cells for transplantation into humans safely and efficiently from human bone marrow fluid.
  • the present inventor has examined the optimum conditions for culturing mesenchymal stem cells derived from bone marrow, the proliferation rate of stem cells, the removal of unnecessary cells other than stem cells, and the activity of stem cells (proliferation ability, A culture method excellent in differentiation induction ability, high survival rate, etc.) was established.
  • the present invention relates to the following method.
  • GO step (Bone marrow fluid obtained by adding heparin Z buffer obtained in 0 is cultured in a-MEM medium containing human serum 10-20% v Zv, and the medium is changed at least twice during the culture. Removing blood cell components floating in the culture medium; and
  • a cell group mainly composed of mesenchymal stem cells using a cell dissociating agent is selectively peeled off, and adherent cells other than mesenchymal stem cells are separated from the peeled cells and separated into another container. In the step of selectively culturing mesenchymal stem cells.
  • heparin Z buffer solution is a heparin ZPBS (Phosphate buffered saline) solution.
  • a-MEM comprises the following components at the following concentrations (mg / L):
  • KCL About 320-480
  • NaCl about 5440-8160
  • D-Glucose about 800-1200
  • Lipoic Acid About 0.16-0.24
  • L-Alanine About 20-30
  • L-Aspartic Acid about 24-36
  • L-Glutamic Acid about 60-90
  • L-Tyrosine (disodium salt) about 41.6— 62.4
  • Biotin about 0.08- 0.12
  • Niacinamide about 0.8—1.2
  • Vitamin B 12 About 1.12— 1.68
  • Adenosine about 8-12
  • Thymidine about 8-12
  • step (iii) use a 0.05% trypsin / 0.53mM EDTA solution or an enzyme such as protease with the same action as a cell dissociator:! ⁇ For 10 minutes in the culture vessel.
  • Item 5 The method according to any one of Items 1 to 4, wherein the mesenchymal stem cells adhered to the cell are selectively detached.
  • the cell concentration of mesenchymal stem cells isolated and purified after detachment in step (m) is set to 1 X ⁇ 4 to ⁇ X 10 6 cellsZmL, seeded on scaffold, 8-12mM j8-Glycerophosphate, 16-24 ⁇ g ZmL Vitamin C and 80-120nM dexamethasone supplemented with human serum ex- Item 6.
  • Item 7 Treat the subcultured cells with trypsin or a cell dissociator such as protease with the same action to adjust the cell concentration to 1 X 10 6 to 1 X 10 8 cells ZmL.
  • Item 7 The method according to any one of Items 1 to 6, wherein the method is seeded on a scaffold such as cultivated and cultured for 1 to 24 hours.
  • the scaffold is porous hydroxyapatite, porous calcium phosphate ceramics such as porous calcium triphosphate, porous calcium carbonate, and V of alumina having a surface porous structure.
  • porous calcium phosphate ceramics such as porous calcium triphosphate, porous calcium carbonate, and V of alumina having a surface porous structure.
  • a scaffold material a biomaterial or a material coated with an inorganic material based on calcium phosphate, such as bioactive glass or hydroxyapatite, on the surface of a biomaterial based on titanium.
  • an inorganic material based on calcium phosphate such as bioactive glass or hydroxyapatite
  • the cells cultured by this method have a survival rate of 90% or more in an environment other than the culture environment at least 24 hours, and can be stored for a long time in the environment other than the culture environment. Or can be transported.
  • FIG. 1 shows a 100-fold magnified image of cell morphology after 9 days of culturing stem cells derived from human bone marrow
  • Fig. Lb is also a 40-fold magnified image
  • Fig. Lc is the same species.
  • the human cells were cultured in a medium supplemented with urchin fetal serum, and a 100-fold magnified image of the morphology of the cells after 9 days was shown.
  • FIG. 1d also shows a 40-fold magnified image. It can be seen that human bone marrow-derived mesenchymal stem cells are more active when cultured in autologous serum medium than when cultured in urine fetal serum medium.
  • FIG. 2a shows isolated human mesenchymal stem cells
  • FIG. 2b shows adherent cells remaining after detachment of human mesenchymal stem cells by trypsin treatment.
  • FIG. 3 shows a porous structure formed by coating a large number of alumina beads with a diameter of 0.8 mm on the surface of an alumina ceramic.
  • Bone tissue is formed by seeding and culturing mesenchymal stem cells in such a porous structure on the surface of the artificial joint in contact with the bone tissue.
  • Fig. 4 shows the cell surface antigen pattern (FACS analysis) of human mesenchymal cells cultured in autologous serum
  • Fig. 4a shows the pattern of CD34
  • Fig. 4b shows the blood cell marker
  • Fig. 4c shows the pattern of CD44, which is a marker including mesenchymal cells
  • Fig. 4d shows the pattern of CD90. This suggests that cells cultured with autoserum are non-hemocytic and mesenchymal stem cells.
  • FIG. 5 shows the cell viability of human mesenchymal cells cultured in autoserum for 24 hours outside the carbon dioxide incubator.
  • the cultured cells were stored in physiological saline (mouth), physiological saline (PBS: A) containing phosphate buffer, and culture medium ( ⁇ ), and all showed cell viability of 90% or more.
  • the human serum is not particularly limited, but it is desirable to use autologous serum when bone marrow fluid is also collected from a patient and transplanted into the patient. Safety that can deny virus infections from healthy relatives such as relatives if autologous serum cannot be used or the necessary amount of autologous serum cannot be obtained due to the severity of the patient's disease, general condition, etc. It is desirable to use high-quality human serum.
  • mesenchymal stem cells cultured by the method of the present invention when bone tissue is repaired / regenerated, the mesenchymal stem cells obtained before transplantation are induced into bone tissue or bone tissue. It is desirable to differentiate into cells that can be produced (eg, osteoblasts or precursors thereof). Alternatively, the mesenchymal stem cells obtained by the method of the present invention can be directly transplanted to the affected area depending on the transplant target (for example, muscle, organ, etc.) and differentiated in the same manner as surrounding cells at the transplant site. is there.
  • Human serum (preferably autologous serum) can be prepared, for example, by centrifuging (patient) blood 250-400 ml under the condition of 3000 rpm (centrifugal gravity 1580 g).
  • the human serum is preferably supplemented with ⁇ -20% medium supplemented with 10-20% vZv.
  • a -MEM medium with the following concentration range (mgZL) is preferred.
  • KCL About 320-480
  • NaCl about 5440-8160
  • D-Glucose about 800-1200
  • Lipoic Acid About 0.16-0.24
  • L-Alanine About 20-30
  • L-Aspartic Acid about 24-36
  • L-Glutamic Acid about 60-90
  • L-Tyrosine (disodium salt) about 41.6— 62.4
  • Biotin about 0.08- 0.12
  • i-Inositol About 1.6-2.4
  • Niacinamide about 0.8—1.2
  • Vitamin B About 1.12-1.68
  • Thymidine approx. 8-12
  • a cell culture medium having an about 15% vZv autoserum concentration.
  • the autoserum concentration is about 10% or more and about 20% or less, and a desirable concentration is about 15% for safe, effective and efficient cell culture.
  • the culture solution can be a constituent element of the present invention as long as it is a substantially equivalent culture solution for culturing the component system of the a-MEM medium and the mesenchymal stem cells.
  • heparin buffer solution preferably heparin phosphate buffer solution (hemoline ZPBS solution) in an amount of about 80% or more and about 120% or less with respect to the bone marrow fluid is usually 5 to 15 UZmL.
  • hemoline ZPBS solution heparin phosphate buffer solution
  • Centrifugation is performed at about 500 rpm (centrifugal gravity 40 g) or more and about 1500 rpm (centrifugal gravity 390 g) or less, preferably about lOOOrpm (centrifugal gravity 140 g) for 1 to 20 minutes.
  • adherent cells selectively grow on the bottom surface of the culture vessel.
  • adherent cells cells other than mesenchymal stem cells sometimes appear.
  • Adherent cells other than mesenchymal stem cells have a completely different appearance from mesenchymal stem cells and can be easily distinguished. Furthermore, this cell has been confirmed to be CD34, 45, positive by cell surface antigen analysis, and this force has also been confirmed to be a cell derived from the blood cell lineage.
  • Adhesion using trypsin or a cell dissociation agent such as protease having the same action Using a cell separator (cell sorter) in a floating state in which all cells are detached from the bottom surface and adhesion between cells is released.
  • a cell separator cell sorter
  • the stem cells attached to the bottom surface of the culture dish are separated from the bottom surface using trypsin or a protease having an equivalent action, and the like. The operation is performed in a floating state where mutual adhesion has been resolved.
  • porous hydroxyapatite and porous tricalcium phosphate are used as the scaffold, and the bone tissue is differentiated in the pores of these ceramics or in the vicinity of the surface of the pores. And used for transplantation into the bone defect of the patient.
  • mesenchymal stem cells are seeded at the bone contact portion of the artificial joint, and are distributed to the bone tissue to transplant the force.
  • a porous structure is created by baking a large number of alumina bead balls with a diameter of 0.8 mm as shown in Fig.
  • a mesenchymal stem cell is seeded on the surface of the scaffolding material, differentiated into bone tissue, and then transplanted Get good treatment results with the law.
  • Stem cells to be seeded on these scaffolds are seeded uniformly by adjusting the cell concentration to 1 ⁇ 10 4 to 1 ⁇ 10 6 cells / mL. Then, it culture
  • the concentration in the autoserum medium is about 8-12 mM for j8-Glycerophosphate, desirably about 10 mM, about 16-24 ⁇ g / mL for vitamin C, desirably about 20 ⁇ g ZmL,
  • the cell concentration is adjusted to a relatively high concentration of 1 ⁇ 10 6 to 1 ⁇ 10 8 cells / mL by subculture, etc., and this is seeded on scaffolds such as ceramics and polymers, and 1 to 24:00 After being attached to the scaffold at high density by culturing for a short period of time, it is directly transplanted to the bone, and then it is separated into bone tissue at the site where the mesenchymal stem cells are transplanted.
  • This method has the advantage that when the mesenchymal stem cells are obtained in a relatively large amount, the culture time until transplantation is short.
  • mesenchymal stem cells In the treatment of ischemic myocardial recovery or dilated cardiomyopathy, or chronic peripheral arterial occlusive disease (diabetic necrosis, arteriosclerotic obstruction, Birja's disease, etc.), mesenchymal stem cells When injecting itself into the affected area, the mesenchymal stem cells are used as they are.
  • Serum was obtained by collecting 250-400 ml of blood from a patient with a bone defect and centrifuging for 10 minutes at 3000 rpm.
  • As the autoserum 500 mL of ⁇ - ⁇ EM medium having the above-mentioned specific composition and 88 mL of autoserum were added to obtain a cell culture medium having a 15% vZv autoserum concentration.
  • heparin phosphate buffer was added in an amount equivalent to bone marrow fluid (corresponding to 100%).
  • the heparinized bone marrow was centrifuged at lOOOrpm for 10 minutes. After centrifugation, separate from the bottom into three layers: red blood cell, nucleated cell layer, and plasma. After confirming that the plasma was removed, the plasma was removed. Incubate in 30 mL autologous serum medium per 2 to 3 mL in total of two layers of red blood cells and nucleated cell layers, and replace the ⁇ -MEM medium supplemented with autologous serum approximately every 2 days. In addition, floating blood cell components were removed. This medium exchange was performed 4 times. When the medium was changed 4 times, blood cell components were completely removed.
  • FIG. 2a shows the form of mesenchymal stem cells that were selectively detached from mesenchymal stem cells by trypsin treatment and cultured in a separate container.
  • Fig. 2b shows adherent cells that remain on the bottom of the culture vessel after trypsinization and detachment of mesenchymal stem cells.
  • mesenchymal stem cells cultured using autoserum were analyzed. As shown in Fig. 4, these cells were negative for blood cell markers CD34 and CD45, and mesenchymal cells were also The included markers CD44 and CD90 were positive, indicating that the cultured cells were non-hemocytic and mesenchymal stem cell lines.
  • the cells cultured by this method have a survival rate of 90% or more in an environment other than the culture environment for at least 24 hours, and can be stored for a long time outside the culture environment, or It became possible to carry.
  • a 1 ⁇ 10 4 to 1 ⁇ 10 6 cells ZmL culture solution was prepared from the mesenchymal stem cells obtained in Example 1, and this was prepared by coating a large number of 0.8 mm diameter alumina beads shown in FIG. 7-28 in autologous serum a MEM medium seeded on a scaffold material that has a porous structure and also has the power of alumina ceramics and supplemented with ⁇ -Glycerop hosphate (10 mM), Vitamin C (20 i ug / mL) and dexamethasone (lOOnM). Cultured for days.
  • the present invention greatly contributes to the application of cell * tissue engineering to regenerative medicine, and has an important role in the practical application and industrialization of cell * tissue engineering.

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Abstract

A method of preparing human mesenchymal stem cells to be transplanted into humans by collecting human bone marrow fluid and culturing mesenchymal stem cells originating in the bone marrow, characterized by involving the following steps: (i) the step of adding 80 to 120% v/v (based on the amount of the collected bone marrow fluid) of a heparin/buffer solution having a concentration of about 5 to 15 U/mL; (ii) the step of culturing the bone marrow fluid containing the heparin/buffer solution obtained in the step (i) in an α-MEM medium containing 10 to 20% v/v of human serum and exchanging the medium at least twice during the culture to thereby remove blood cell components suspending in the liquid culture; and (iii) the step of selectively peeling off cells mainly comprising mesenchymal stem cells from the culture container with the use of a cell-dissociating agent, separating adhesive cells other than the mesenchymal stem cells from the cells thus peeled off, and selectively culturing the mesenchymal stem cells in another container.

Description

明 細 書  Specification
ヒト血清培地を用いるヒト骨髄由来間葉系幹細胞培養法  Human bone marrow-derived mesenchymal stem cell culture method using human serum medium
技術分野  Technical field
[0001] 本発明は、再生医療、細胞治療等に用いるためのヒトの糸且織 ·細胞を効率的、かつ 安全に大量に培養するための細胞培養法に関するものである。  [0001] The present invention relates to a cell culture method for efficiently and safely culturing large amounts of human thread and tissue cells for use in regenerative medicine, cell therapy, and the like.
背景技術  Background art
[0002] ヒトの細胞を清潔な細胞培養室などで培養し、これをヒトの体に移植することによつ てけがや病気を治す再生医療が研究開発されつつあり、実用ィ匕も始まっている。この 場合、効率的かつ安全に細胞を培養することが求められており、従来より培地にはゥ シ胎児血清が用いられてきた。ゥシ胎児血清は、種々の成長因子を含め極めて豊富 な栄養素を含むといわれているものの、異種動物の血清を用いる培養法では、種の 違 ヽに関しての安全性を保証することが難 、と 、う側面がある。このような懸念にも かかわらず、他に適当な材料が見あたらないことから、実際にはゥシ胎児血清がヒト の細胞培養に用いられてきた。  [0002] Regenerative medicine to cure injuries and diseases by culturing human cells in a clean cell culture room and transplanting them into the human body has been researched and developed, and practical use has started. Yes. In this case, it is required to culture the cells efficiently and safely, and conventionally, rabbit fetal serum has been used as the medium. Although fetal bovine serum is said to contain extremely abundant nutrients, including various growth factors, it is difficult to guarantee the safety of species differences in culture methods using serum from different species. There are other aspects. Despite these concerns, ushi fetal serum has actually been used for human cell culture because no other suitable material has been found.
[0003] しかしながら、数年前から BSE (Bovine Spongiform Encephalopathy) の発生に伴 い、ゥシ胎児血清の安全性の確保が緊急の課題となり、ゥシ胎児血清を用いない無 血清培地の開発も進められて 、るが、未だ細胞増殖率などの面で満足できる成果に は至っていない。  [0003] However, with the occurrence of BSE (Bovine Spongiform Encephalopathy) for several years, ensuring the safety of urchin fetal serum has become an urgent issue, and the development of a serum-free medium that does not use urinary fetal serum has also been promoted. However, it has not yet achieved satisfactory results in terms of cell proliferation rate.
[0004] 近年、ヒト血清を培地に添加する技術力^、くつか特許出願されている。  [0004] In recent years, several patents have been filed for technical capabilities to add human serum to a culture medium.
[0005] 例えば WO2002/024875 (特許文献 1)は、ヒト成熟肝細胞の培養液にヒト血清ととも に増殖因子等を添加した培養液に関する発明であり、特開 2002-78484 (特許文献 2 )は培養軟骨細胞の製造法にぉ 、てヒト血清と共に軟骨形成性成長因子などの成長 因子を添加する方法に関する発明であり、特開 2003-52360 (特許文献 3)は基底膜 細胞外基質の存在下で間葉系幹細胞を培養するに際して 2— 10%のヒト血清を添 加するという方法に関する発明であり、特開 2003-235548 (特許文献 4)はヒト細胞の 培養用培地にヒト血清と増殖因子を含むことを特長とする特許出願である。 [0005] For example, WO2002 / 024875 (Patent Document 1) is an invention relating to a culture solution obtained by adding growth factors and the like together with human serum to a culture solution of human mature hepatocytes, and Japanese Patent Application Laid-Open No. 2002-78484 (Patent Document 2). Is an invention relating to a method for producing cultured chondrocytes, and a method for adding a growth factor such as chondrogenic growth factor together with human serum. JP 2003-52360 (Patent Document 3) describes the presence of a basement membrane extracellular matrix. The invention relates to a method in which 2-10% human serum is added when culturing mesenchymal stem cells under the circumstances, and JP 2003-235548 (Patent Document 4) describes the growth of human serum and human serum in a culture medium for human cells. It is a patent application characterized by including a factor.
[0006] すなわち、従来技術においては、培地にヒト血清を用いるに際しては増殖因子を含 むことを条件として 、る力、もしくは特殊な基質の存在下での培養を条件として 、る。 これらの増殖因子は、細胞に対する作用機序がある程度明らかになっているものの、 これらの影響を受けた細胞がヒト体内で中 ·長期的にどのような挙動をするの力、未だ 解明の途上にある。これらの細胞の移植対象がヒトであるだけに安全の上にも安全を 期する必要がある。 [0006] That is, in the prior art, when human serum is used as a medium, it contains a growth factor. It is necessary to cultivate in the presence of a special substrate or the presence of a special substrate. Although the mechanism of action of these growth factors has been clarified to some extent, the power of how these affected cells behave in the medium to long term in the human body is still in the process of elucidation. is there. Since these cells are transplanted by humans, it is necessary to be safe and secure.
[0007] さらに特表平 11-506010 (特許文献 5)においては、骨粗しょう症患者の治療薬試験 法の一環としての細胞培養で、患者腸骨の生検切片を用いてこれを連続酵素処理し て分離し、骨芽細胞前駆細胞を培養する際に、 1〜12%の範囲のゥシ胎児血清、ヒ ト血清、ゥシ血清アルブミンまたはウルトロセルなどの添加血清を用いることを特長と する内容であり、本発明の目的及び内容とは明らかに異なる。  [0007] Furthermore, in Japanese translation of PCT National Publication No. 11-506010 (Patent Document 5), cell culture as part of a therapeutic drug test method for osteoporosis patients, which is treated with continuous enzyme treatment using biopsy sections of patient iliac bones. In addition, when culturing osteoblast progenitor cells, the content is characterized by using 1 to 12% range of added serum such as urine fetal serum, human serum, urine serum albumin or Ultrocell. The object and contents of the present invention are clearly different.
[0008] なお、発明者の一人である大串は、舟岡宏幸、大串 始:骨の再生医学、リウマチ 科、 30 : 430— 435, 2003 (非特許文献 1)の論文にて本発明の基礎となる研究成 果を発表している。同じく Yonsei Medical Journal,vol.45,p61-67,2004 (非特許文献 2 )にもゥシ胎児血清を用いた基礎的な研究成果を論文発表して ヽる。  [0008] It should be noted that one of the inventors, Ogushi is Hiroyuki Funaoka, Hajime Ogushi: Bone Regenerative Medicine, Rheumatology, 30: 430-435, 2003 (Non-Patent Document 1) and the basis of the present invention. The results of this research are announced. Similarly, Yonsei Medical Journal, vol. 45, p61-67, 2004 (Non-patent Document 2) published a paper on basic research results using urchin fetal serum.
特許文献 1： WO2002/024875  Patent Document 1: WO2002 / 024875
特許文献 2：特開 2002-78484  Patent Document 2: JP 2002-78484 A
特許文献 3：特開 2003-52360  Patent Document 3: JP 2003-52360
特許文献 4：特開 2003- 235548  Patent Document 4: JP-A 2003-235548
特許文献 5：特表平 11-506010  Patent Document 5: Special Tables 11-506010
非特許文献 1 :舟岡宏幸、大串 始:骨の再生医学、リウマチ科、 30 : 430— 435, 20 03  Non-Patent Document 1: Hiroyuki Funaoka, Ogushi Hajime: Bone Regenerative Medicine, Rheumatology, 30: 430— 435, 20 03
非特許文献 2 : Yonsei Medical Journal,vol.45,p61-67,2004  Non-Patent Document 2: Yonsei Medical Journal, vol. 45, p61-67, 2004
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、ヒトに移植するための間葉系幹細胞を、ヒト骨髄液から安全且つ効率的 に培養する方法を提供することを目的とする。 [0009] An object of the present invention is to provide a method for culturing mesenchymal stem cells for transplantation into humans safely and efficiently from human bone marrow fluid.
課題を解決するための手段  Means for solving the problem
[0010] 患者本人の骨髄細胞を採取'培養し、これを元の患者に移植する場合、異種の動 物の血清を一切用いず、可能な限り患者本人の血清、すなわち自己血清を含む培 地を用いることが理想的である。患者の症状により、培養に必要とする量の血清を採 取できない場合は、患者の近親者などの健常人の血清を用いるということも、ゥシ胎 児血清を用いるよりは安全性が高い。また、骨髄由来の間葉系幹細胞を培養する場 合、治療効果を上げるためには、目的とする組織 (例えば骨組織)まで分化させること も重要であり、この細胞の分ィ匕を目的とする培養においても、ヒト血清、特に可能な限 り自己血清を用いた培地を使用することの意義は大きい。 [0010] When a patient's own bone marrow cells are collected and cultured and transplanted to the original patient, It is ideal to use as much of the patient's serum as possible, ie, a medium containing autologous serum, without using any serum from the product. When the amount of serum required for culture cannot be collected due to patient symptoms, it is safer to use serum from healthy individuals such as the patient's close relatives than using urine fetal serum. In addition, when culturing mesenchymal stem cells derived from bone marrow, it is important to differentiate to the target tissue (for example, bone tissue) in order to increase the therapeutic effect. In this culture, it is significant to use a medium using human serum, especially autologous serum as much as possible.
[0011] 本発明者は、骨髄由来の間葉系幹細胞を培養するための最適条件を検討し、幹 細胞の増殖速度、幹細胞以外の不要な細胞の除去、さらには幹細胞の活性 (増殖 能、分化誘導能、高い生存率など）において優れた培養方法を確立した。  [0011] The present inventor has examined the optimum conditions for culturing mesenchymal stem cells derived from bone marrow, the proliferation rate of stem cells, the removal of unnecessary cells other than stem cells, and the activity of stem cells (proliferation ability, A culture method excellent in differentiation induction ability, high survival rate, etc.) was established.
[0012] 即ち、本発明は、以下の方法に関する。  [0012] That is, the present invention relates to the following method.
1. ヒト骨髄液を採取し、骨髄由来の間葉系幹細胞を培養して、ヒトに移植するため のヒト間葉系幹細胞を調製する方法であって、以下の工程を含むことを特徴とする方 法：  1. A method of preparing human mesenchymal stem cells for harvesting human bone marrow fluid, culturing mesenchymal stem cells derived from bone marrow, and transplanting them to humans, comprising the following steps: Method:
(0 採取した骨髄液の分量に対して、濃度 5〜15UZmL程度のへパリン Z緩衝液を 80〜 120%vZv添加する工程、  (0 Step of adding 80-120% vZv of heparin Z buffer solution with a concentration of about 5-15 UZmL to the collected amount of bone marrow fluid,
GO 工程 (0で得られたへパリン Z緩衝液を添加した骨髄液を、ヒト血清を 10〜20%v Zv含む a -MEM培地で培養し、該培養中に少なくとも 2回培地交換することにより培 養液中に浮遊する血球成分を取り除く工程、および  GO step (Bone marrow fluid obtained by adding heparin Z buffer obtained in 0 is cultured in a-MEM medium containing human serum 10-20% v Zv, and the medium is changed at least twice during the culture. Removing blood cell components floating in the culture medium; and
(iii) 細胞解離剤を用いて間葉系幹細胞を主とする細胞群を培養容器力 選択的に 剥離し、剥離された細胞群から間葉系幹細胞以外の接着細胞を分離し、別の容器で 選択的に間葉系幹細胞を培養する工程。  (iii) A cell group mainly composed of mesenchymal stem cells using a cell dissociating agent is selectively peeled off, and adherent cells other than mesenchymal stem cells are separated from the peeled cells and separated into another container. In the step of selectively culturing mesenchymal stem cells.
2. 工程 (0で得られたへパリン Z緩衝液を添加した骨髄液を回転数 500〜1500rp mの遠心分離 (遠心重力で 40〜390g)を行い、下層から赤血球層、有核細胞層及 び血漿層の 3層に分離し、血漿層を除!ヽた赤血球層及び有核細胞層の混合物を、 工程 (ii)に供することを特徴とする項 1に記載の方法。  2. Process (centrifuge the bone marrow fluid added with heparin Z buffer obtained in step 0 at a rotational speed of 500-1500 rpm (40-390 g by centrifugal gravity), from the lower layer to the erythrocyte layer, nucleated cell layer and Item 2. The method according to Item 1, wherein the mixture of the erythrocyte layer and the nucleated cell layer separated into three layers, and the plasma layer, is subjected to step (ii).
3. へパリン Z緩衝液が、へパリン ZPBS(Phosphate buffered saline)液である項 1ま たは 2に記載の方法。 4. a -MEMが以下の成分を以下の濃度 (mg/L)で含むものである項 1〜3のいずれ かに記載の方法： 3. The method according to item 1 or 2, wherein the heparin Z buffer solution is a heparin ZPBS (Phosphate buffered saline) solution. 4. The method according to any one of Items 1 to 3, wherein the a-MEM comprises the following components at the following concentrations (mg / L):
CaCl (anhyd.):約 160— 240  CaCl (anhyd.): About 160-240
2  2
KCL:約 320- 480  KCL: About 320-480
MgSO (anhyd.):約 78.4-117.6  MgSO (anhyd.): About 78.4-117.6
4  Four
NaCl:約 5440- 8160  NaCl: about 5440-8160
NaHCO：約 1760-2640  NaHCO: approx. 1760-2640
3  Three
NaH Ρ〇 ·Η Ο:約 112 - 168  NaH Ρ〇ΗΗ: Approximately 112-168
2 4 2  2 4 2
D- Glucose:約 800- 1200  D-Glucose: about 800-1200
Lipoic Acid:約 0.16- 0.24  Lipoic Acid: About 0.16-0.24
Sodium Pyruvate:約 88- 132  Sodium Pyruvate: About 88-132
L- Alanine:約 20- 30  L-Alanine: About 20-30
レ\^010½'1"^：1:約101.6-152.4  Les \ ^ 010½'1 "^: 1: approx. 101.6-152.4
L-Asparagine · H O：約 40— 60  L-Asparagine · H O: approx. 40-60
2  2
L-Aspartic Acid:約 24- 36  L-Aspartic Acid: about 24-36
L—Cystine'2HCl:約 24.8— 37.2  L—Cystine'2HCl: approx.24.8— 37.2
L- Cysteine'HCl'H O:約 80-120  L-Cysteine'HCl'H O: approx. 80-120
2  2
L- Glutamic Acid:約 60- 90  L-Glutamic Acid: about 60-90
L-Glutamine：約 233.6 - 350.4  L-Glutamine: Approximately 233.6-350.4
Glycine:約 40- 60  Glycine: about 40-60
L-Histidine HCl-H O:約 33.6 - 50.4  L-Histidine HCl-H 2 O: About 33.6-50.4
2  2
L-Isoleucine: 41.6-62.4  L-Isoleucine: 41.6-62.4
L- Leucine:約 41.6- 62.4  L-Leucine: About 41.6-62.4
L- Lysine · HCl：約 58.4- 87.6  L-Lysine · HCl: approx. 58.4-87.6
L- Methionine:約 12- 18  L- Methionine: About 12-18
L- Phenylalanine：約 25.6- 38.4  L-Phenylalanine: Approximately 25.6-38.4
L- Proline:約 32- 48  L-Proline: approx. 32-48
L— Serine:約 20 - 30 L- Threonine：約 38.4- 57.6 L— Serine: approx. 20-30 L-Threonine: About 38.4-57.6
L- Tryprophan:約 8- 12  L- Tryprophan: About 8-12
L-Tyrosine(disodium salt):約 41.6— 62.4  L-Tyrosine (disodium salt): about 41.6— 62.4
L- Valine:約 36.8- 55.2  L-Valine: approx. 36.8-55.2
L- Ascorbic Acid:約 40- 60  L- Ascorbic Acid: approx. 40-60
Biotin:約 0.08- 0.12  Biotin: about 0.08- 0.12
D-Ca Pantothenate:約 0.8— 1.2  D-Ca Pantothenate: About 0.8—1.2
Choline Chloride:約 0.8- 1.2  Choline Chloride: about 0.8-1.2
Folic Acid:約 0.8- 1.2  Folic Acid: about 0.8-1.2
i- Inositol:約 1.6 - 2.4  i-Inositol: approx.1.6-2.4
Niacinamide：約 0.8— 1.2  Niacinamide: about 0.8—1.2
Pyridoxal HC1:約 0.8- 1.2  Pyridoxal HC1: about 0.8-1.2
Riboflavin:約 0.08- 0.12  Riboflavin: about 0.08- 0.12
Thiamine HC1:約 0.8- 1.2  Thiamine HC1: about 0.8-1.2
Vitamin B 12：約 1.12— 1.68  Vitamin B 12: About 1.12— 1.68
Adenosine:約 8 - 12  Adenosine: about 8-12
Cytidine:約 8- 12  Cytidine: About 8-12
Guanosine：約 8- 12  Guanosine: About 8-12
Uridine:約 8- 12  Uridine: approx. 8-12
2 ' Deoxyadenosine：約 8- 12  2 'Deoxyadenosine: approx. 8-12
2'Deoxycytidine HC1:約 8.8- 13.2  2'Deoxycytidine HC1: approx.8.8-13.2
2 ' Deoxyguanosine：約 8- 12  2 'Deoxyguanosine: approx. 8-12
Thymidine:約 8- 12  Thymidine: about 8-12
5. 工程（iii)において、 0.05%トリプシン/ 0.53mM EDTA溶液、もしくは同様の 作用を持つプロテアーゼなどの酵素を細胞解離剤として用いて、：!〜 10分間培養容 器で反応させることにより培養容器に接着した間葉系幹細胞を選択的に剥離するこ とを特徴とする、項 1〜4のいずれかに記載の方法。  5. In step (iii), use a 0.05% trypsin / 0.53mM EDTA solution or an enzyme such as protease with the same action as a cell dissociator:! ~ For 10 minutes in the culture vessel. Item 5. The method according to any one of Items 1 to 4, wherein the mesenchymal stem cells adhered to the cell are selectively detached.
6. 工程 (m)で剥離後に分離精製された間葉系幹細胞の細胞濃度を 1 X ιο4〜ι X 106 cellsZmLに調整し、これを足場材に播種し、 8〜12mMの j8 - Glycerophosphat e、 16〜24 μ gZmLの Vitamin C及び 80〜120nMのデキサメサゾンを添カ卩した、ヒ ト血清含有 ex -MEM培地で 7日〜28日間培養し、骨芽細胞もしくはこの前駆細胞に 分化させることを特徴とする項 1〜5のいずれかに記載の方法。 6. The cell concentration of mesenchymal stem cells isolated and purified after detachment in step (m) is set to 1 X ιο 4 to ι X 10 6 cellsZmL, seeded on scaffold, 8-12mM j8-Glycerophosphate, 16-24μg ZmL Vitamin C and 80-120nM dexamethasone supplemented with human serum ex- Item 6. The method according to any one of Items 1 to 5, wherein the method is cultured in a MEM medium for 7 to 28 days and differentiated into osteoblasts or progenitor cells thereof.
7. 継代培養した細胞をトリプシンもしくは同様の作用を持つプロテアーゼ等の細胞 解離剤で処理を行い、細胞濃度を 1 X 106〜1 X 108 cellsZmLに調整し、これをセ ラミックス、ポリマーなどの足場材に播種し、 1〜24時間培養することを特長とする項 1〜6のいずれかに記載の方法。 7. Treat the subcultured cells with trypsin or a cell dissociator such as protease with the same action to adjust the cell concentration to 1 X 10 6 to 1 X 10 8 cells ZmL. Item 7. The method according to any one of Items 1 to 6, wherein the method is seeded on a scaffold such as cultivated and cultured for 1 to 24 hours.
8. 足場材が多孔質の水酸アパタイト、多孔質の三燐酸カルシウム等の燐酸カルシ ゥム系セラミックス、多孔体の炭酸カルシウム及び表面多孔体構造を持つアルミナの V、ずれかである項 6または 7に記載の方法。  Item 6 or 6, wherein the scaffold is porous hydroxyapatite, porous calcium phosphate ceramics such as porous calcium triphosphate, porous calcium carbonate, and V of alumina having a surface porous structure. The method according to 7.
9. 足場材として生体用金属材料、もしくはチタンを主成分とする生体用金属の表 面に、生体活性ガラス、あるいは水酸アパタイトなどの燐酸カルシウムを主成分とする 無機材料をコーティングした材料を用いることを特徴とする項 6または 7に記載の方法  9. Use as a scaffold material a biomaterial or a material coated with an inorganic material based on calcium phosphate, such as bioactive glass or hydroxyapatite, on the surface of a biomaterial based on titanium. The method according to item 6 or 7, characterized in that
10. ヒト血清として、骨髄液と同一のヒトの血清、すなわち自己血清を用いることを 特徴とする項 1〜9のいずれかに記載の方法。 10. The method according to any one of Items 1 to 9, wherein the human serum is the same human serum as bone marrow fluid, ie, autoserum.
発明の効果  The invention's effect
[0013] 本発明により、自己の細胞培養を行うに際して、胎児ゥシ血清に代えて自己血清を 用いた培地を用いても、細胞生存率 90%以上を得ることが可能となり、安全かつ効 率的に細胞培養を行うことができるようになり、種の異なる血清を用いることの懸念や ゥシ由来の感染症の懸念が完全に払拭され、再生医療の安全性の確保、患者の安 心感の保持など、著しい効果を発揮している。  [0013] According to the present invention, when autologous cell culture is performed, it is possible to obtain a cell viability of 90% or more even when using a medium using autologous serum instead of fetal urchin serum, which is safe and effective. Cell culture can be performed, and the concerns of using different types of sera and the infectious diseases of ushi are completely eliminated, ensuring the safety of regenerative medicine, and the patient's peace of mind. Remarkable effects such as retention of
[0014] また、図 5に示すように、この方法で培養した細胞は、培養環境以外の環境で生存 する生存率が、少なくとも 24時間で 90%以上あり、培養環境以外で長期の保存、あ るいは運搬することが可能となった。  In addition, as shown in FIG. 5, the cells cultured by this method have a survival rate of 90% or more in an environment other than the culture environment at least 24 hours, and can be stored for a long time in the environment other than the culture environment. Or can be transported.
[0015] さらに、骨髄由来の間葉系幹細胞を体外において安全かつ効率的に骨糸且織に分 ィ匕させる培養方法が確立され、人工骨や人工関節などを足場材として用いる方法が 開発され、これらインプラントと周囲骨とのより早期な密着が可能となり、治療成績の 向上に大きく貢献している。 [0015] Furthermore, a culture method for safely and efficiently dissociating bone marrow-derived mesenchymal stem cells into bone and tissue outside the body has been established, and a method using an artificial bone, an artificial joint or the like as a scaffolding material has been established. It has been developed to enable early contact between these implants and the surrounding bone, greatly contributing to improved treatment results.
図面の簡単な説明  Brief Description of Drawings
[0016] [図 1]図 laはヒト骨髄由来の幹細胞を自己血清培地で培養し、 9日後の細胞の形態 の 100倍拡大像を示し、図 lbは同じく 40倍拡大像、図 lcは同種のヒト細胞を、ゥシ 胎児血清を加えた培地で培養した 9日後の細胞の形態の 100倍拡大像を表し、図 1 dは同じく 40倍拡大像を示す。ヒト骨髄由来間葉系幹細胞は、自己血清培地で培養 した方が、ゥシ胎児血清培地で培養するよりも活性度が高 、ことが伺える。  [0016] [Fig. 1] Fig. La shows a 100-fold magnified image of cell morphology after 9 days of culturing stem cells derived from human bone marrow, Fig. Lb is also a 40-fold magnified image, and Fig. Lc is the same species. The human cells were cultured in a medium supplemented with urchin fetal serum, and a 100-fold magnified image of the morphology of the cells after 9 days was shown. FIG. 1d also shows a 40-fold magnified image. It can be seen that human bone marrow-derived mesenchymal stem cells are more active when cultured in autologous serum medium than when cultured in urine fetal serum medium.
[図 2]図 2aは分離されたヒト間葉系幹細胞、図 2bはトリプシン処理によりヒト間葉系幹 細胞を剥離した後に残留した接着細胞を示す。  [FIG. 2] FIG. 2a shows isolated human mesenchymal stem cells, and FIG. 2b shows adherent cells remaining after detachment of human mesenchymal stem cells by trypsin treatment.
[図 3]図 3は、アルミナセラミックス表面に、直径 0.8mmのアルミナビーズを多数コート することにより作られた多孔構造を示す。人工関節の骨組織と接する面のこのような 多孔構造に間葉系幹細胞を播種し、培養することによって骨組織を形成する。  [FIG. 3] FIG. 3 shows a porous structure formed by coating a large number of alumina beads with a diameter of 0.8 mm on the surface of an alumina ceramic. Bone tissue is formed by seeding and culturing mesenchymal stem cells in such a porous structure on the surface of the artificial joint in contact with the bone tissue.
[図 4]図 4は、自己血清で培養されたヒト間葉系細胞の細胞表面抗原パターン (FACS 分析）で、図 4aは血球系のマーカーである CD34のパターン，図 4bは同じく血球系 マーカーの CD45のパターン，図 4cは間葉系細胞も含まれるマーカーである CD44 のパターンであり、図 4dは同じく CD90のパターンである。これによつて、自己血清を 用いて培養した細胞は、非血球系で、間葉系幹細胞である可能性を示唆している。  [Fig. 4] Fig. 4 shows the cell surface antigen pattern (FACS analysis) of human mesenchymal cells cultured in autologous serum, Fig. 4a shows the pattern of CD34, a blood cell marker, and Fig. 4b shows the blood cell marker. Fig. 4c shows the pattern of CD44, which is a marker including mesenchymal cells, and Fig. 4d shows the pattern of CD90. This suggests that cells cultured with autoserum are non-hemocytic and mesenchymal stem cells.
[図 5]図 5は、自己血清で培養されたヒト間葉系細胞の炭酸ガスインキュベータ一外 での 24時間の細胞生存率を示す。 培養細胞は、生理的食塩水（口）、燐酸緩衝液 を含む生理的食塩水 (PBS : A)及び培養液 (參）中で保存され、いずれも 90%以上 の細胞生存率を示した。  [FIG. 5] FIG. 5 shows the cell viability of human mesenchymal cells cultured in autoserum for 24 hours outside the carbon dioxide incubator. The cultured cells were stored in physiological saline (mouth), physiological saline (PBS: A) containing phosphate buffer, and culture medium (參), and all showed cell viability of 90% or more.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0017] 本発明において、ヒト血清としては、特に限定されないが、骨髄液を患者力も採取し 、当該患者に移植する場合には、自己血清を使用するのが望ましい。患者の疾患の 重篤度、全身の状態などにより自己血清を使用できないか、必要な量の自己血清を 得ることができない場合には、近親者などの健常人由来のウィルス感染を否定出来 る安全性の高 ヽヒト血清を用いるのが望ま 、。 [0018] また、本発明の方法で培養した間葉系幹細胞は、骨組織の修復 ·再生を行う場合 には、移植前に得られた間葉系幹細胞を骨組織或 ヽは骨組織を誘導できる細胞 (例 えば骨芽細胞またはその前駆体）に分化させるのが望ましい。或いは、本発明の方 法で得られた間葉系幹細胞は、移植の対象 (例えば筋肉、臓器等）によっては患部 に直接移植し、移植部位で周囲の細胞と同様に分化させることも可能である。 In the present invention, the human serum is not particularly limited, but it is desirable to use autologous serum when bone marrow fluid is also collected from a patient and transplanted into the patient. Safety that can deny virus infections from healthy relatives such as relatives if autologous serum cannot be used or the necessary amount of autologous serum cannot be obtained due to the severity of the patient's disease, general condition, etc. It is desirable to use high-quality human serum. [0018] In the case of mesenchymal stem cells cultured by the method of the present invention, when bone tissue is repaired / regenerated, the mesenchymal stem cells obtained before transplantation are induced into bone tissue or bone tissue. It is desirable to differentiate into cells that can be produced (eg, osteoblasts or precursors thereof). Alternatively, the mesenchymal stem cells obtained by the method of the present invention can be directly transplanted to the affected area depending on the transplant target (for example, muscle, organ, etc.) and differentiated in the same manner as surrounding cells at the transplant site. is there.
[0019] ヒト血清 (好ましくは自己血清）の調製は、例えば (患者)血液 250〜400mlについ て、 3000rpm (遠心重力 1580g)の条件にて遠心分離を行うことにより行うことができ る。該ヒト血清は、 α-ΜΕΜ培地に、好ましくは 10〜20%vZv添カ卩される。 a -MEM 培地は、次の濃度範囲 (mgZL)を持つものが好ま 、。  [0019] Human serum (preferably autologous serum) can be prepared, for example, by centrifuging (patient) blood 250-400 ml under the condition of 3000 rpm (centrifugal gravity 1580 g). The human serum is preferably supplemented with α-20% medium supplemented with 10-20% vZv. a -MEM medium with the following concentration range (mgZL) is preferred.
[0020] α-ΜΕΜ培地の成分と濃度 (mgZL)  [0020] Components and concentration of α- α medium (mgZL)
CaCl (anhyd.):約 160- 240  CaCl (anhyd.): About 160-240
2  2
KCL:約 320-480  KCL: About 320-480
MgSO (anhyd.) :78.4-117.6  MgSO (anhyd.): 78.4-117.6
4  Four
NaCl:約 5440- 8160  NaCl: about 5440-8160
NaHCO：約 1760- 2640  NaHCO: approx. 1760-2640
3  Three
NaH PO -H O:約 112— 168  NaH PO -H O: approx. 112—168
2 4 2  2 4 2
D- Glucose:約 800- 1200  D-Glucose: about 800-1200
Lipoic Acid:約 0.16- 0.24  Lipoic Acid: About 0.16-0.24
Sodium Pyruvate:約 88- 132  Sodium Pyruvate: About 88-132
L- Alanine:約 20- 30  L-Alanine: About 20-30
し- ^1"^1:約101.6-152.4  Shi- ^ 1 "^ 1: About 101.6-152.4
L-Asparagine · H O：約 40— 60  L-Asparagine · H O: approx. 40-60
2  2
L-Aspartic Acid:約 24- 36  L-Aspartic Acid: about 24-36
L- Cystine · 2HC1：約 24.8- 37.2  L-Cystine · 2HC1: Approximately 24.8-37.2
L- Cysteine'HCl'H O:約 80- 120  L-Cysteine'HCl'H O: approx. 80-120
2  2
L- Glutamic Acid:約 60- 90  L-Glutamic Acid: about 60-90
L-Glutamine：約 233.6- 350.4  L-Glutamine: Approximately 233.6- 350.4
Glycine:約 40— 60 L-Histidine HCl-H O:約 33.6— 50.4Glycine: about 40-60 L-Histidine HCl-H 2 O: approx. 33.6-50.4
22
L-Isoleucine:約41.6-62.4 L-Isoleucine: About 41.6-62.4
L-Leucine: 41.6-62.4  L-Leucine: 41.6-62.4
L- Lysine · HCl：約 58.4- 87.6  L-Lysine · HCl: approx. 58.4-87.6
L- Methionine:約 12 - 18  L- Methionine: About 12-18
L - Phenylalanine：約 25.6 - 38.4  L-Phenylalanine: About 25.6-38.4
L- Proline:約 32- 48  L-Proline: approx. 32-48
L- Serine:約 20- 30  L- Serine: About 20-30
L- Threonine：約 38.4- 57.6  L-Threonine: About 38.4-57.6
L- Tryprophan:約 8 - 12  L- Tryprophan: approx. 8-12
L-Tyrosine(disodium salt):約 41.6— 62.4 L-Tyrosine (disodium salt): about 41.6— 62.4
L- Valine:約 36.8- 55.2 L-Valine: approx. 36.8-55.2
L- Ascorbic Acid:約 40- 60  L- Ascorbic Acid: approx. 40-60
Biotin:約 0.08- 0.12  Biotin: about 0.08- 0.12
D-Ca Pantothenate:約 0.8— 1.2 D-Ca Pantothenate: About 0.8—1.2
Choline Chloride:約 0.8- 1.2 Choline Chloride: about 0.8-1.2
Folic Acid:約 0.8- 1.2  Folic Acid: about 0.8-1.2
i- Inositol:約 1.6- 2.4 i-Inositol: About 1.6-2.4
Niacinamide:約 0.8— 1.2  Niacinamide: about 0.8—1.2
Pyridoxal HCl:約 0.8- 1.2  Pyridoxal HCl: About 0.8-1.2
Riboflavin:約 0.08- 0.12  Riboflavin: about 0.08- 0.12
Thiamine HCl:約 0.8- 1.2  Thiamine HCl: About 0.8-1.2
Vitamin B ：約 1.12 - 1.68  Vitamin B: About 1.12-1.68
12  12
Adenosine:約 8- 12  Adenosine: approx. 8-12
Cytidine:約 8- 12 Cytidine: About 8-12
Guanosine：約 8- 12 Guanosine: About 8-12
Uridine:約 8- 12 Uridine: approx. 8-12
2 ' Deoxyadenosine：約 8— 12 2 ' Deoxycytidine HC1 :約 8.8- 13.2 2 'Deoxyadenosine: approx. 8—12 2 'Deoxycytidine HC1: approx.8.8-13.2
2 ' Deoxyguanosine：約 8- 12  2 'Deoxyguanosine: approx. 8-12
Thymidine :約 8- 12  Thymidine: approx. 8-12
本発明の特に好ましい実施形態では、 a -MEM培地 500mLに、自己血清約 88m Lを加え、約 15%vZv自己血清濃度を有する細胞培地とする。なお、この自己血清 濃度は、細胞培養を安全かつ効果的、効率的に行うためには約 10%以上、約 20% 以下、望ましい濃度としては約 15%である。  In a particularly preferred embodiment of the present invention, about 88 mL of autologous serum is added to 500 mL of a-MEM medium to give a cell culture medium having an about 15% vZv autoserum concentration. The autoserum concentration is about 10% or more and about 20% or less, and a desirable concentration is about 15% for safe, effective and efficient cell culture.
[0021] なお、 a -MEM培地の成分系と間葉系幹細胞の培養に関し実質的に同等な培養 液であれば、その培養液も本発明の構成要素となり得る。  [0021] It should be noted that the culture solution can be a constituent element of the present invention as long as it is a substantially equivalent culture solution for culturing the component system of the a-MEM medium and the mesenchymal stem cells.
[0022] 骨髄液を採取後、骨髄液に対して約 80%以上、約 120%以下の量のへパリン緩衝 液、好ましくはへパリン燐酸緩衝液 (へノ リン ZPBS液)を通常 5〜15UZmL程度、 好ましくは lOUZmL程度カ卩え、保存あるいは運搬の用に供する。このへノ リン添カロ 骨髄液を約 500rpm (遠心重力 40g)以上約 1500rpm (遠心重力 390g)以下、望ま しくは約 lOOOrpm (遠心重力 140g)で 1〜20分間遠心分離を行う。遠心分離後、下 から赤血球、有核細胞層、及び血漿の 3層に分離していることを確認後、血漿を取り 除く。赤血球、有核細胞層の 2層の合計で 2〜3mL当たり 30mL程度の自己血清培 地で培養し、ほぼ 2日おきに血清培地 (好ましくは自己血清を添加した a MEM培 地)の交換を行う。  [0022] After collecting the bone marrow fluid, heparin buffer solution, preferably heparin phosphate buffer solution (hemoline ZPBS solution) in an amount of about 80% or more and about 120% or less with respect to the bone marrow fluid is usually 5 to 15 UZmL. Prepare about 1OUZmL for storage or transportation. Centrifugation is performed at about 500 rpm (centrifugal gravity 40 g) or more and about 1500 rpm (centrifugal gravity 390 g) or less, preferably about lOOOrpm (centrifugal gravity 140 g) for 1 to 20 minutes. After centrifugation, confirm that the cells are separated into three layers of red blood cells, nucleated cell layers, and plasma from the bottom, and then remove the plasma. Cultivate in about 30 mL of autologous serum medium for 2 to 3 mL of erythrocytes and nucleated cell layer in total, and replace serum medium (preferably a MEM medium supplemented with autoserum) approximately every 2 days. Do.
[0023] なお、遠心分離しないで骨髄そのものを培養することによつても、効率は落ちるが 接着系の間葉系幹細胞の培養は可能である。さらにへパリン Z緩衝液を調整するの に PBSを通常用いる力 PBSに限らず生理的食塩水を用いても良ぐさらに MEMを含 む種々の培養液に 5〜 15UZmlのへパリンを添カ卩しても良!、。  [0023] It is also possible to culture adherent mesenchymal stem cells by culturing bone marrow itself without centrifugation, although the efficiency is lowered. Furthermore, the ability to normally use PBS to adjust the heparin Z buffer solution is not limited to PBS. Physiological saline may be used. 5-15 UZml of heparin is added to various culture media including MEM. OK!
[0024] この血清培地交換の際に、浮遊している血球成分を取り除く。この培地交換を繰り 返し行うことにより、血球成分が逐次除去され、少なくとも 2回、好ましくは少なくとも 3 回または少なくとも 4回の培地交換により完全に血球成分が除去され、培養皿の底面 に骨髄由来の間葉系幹細胞を中心とする接着細胞 (すなわち間葉系細胞)のみが選 択的に増殖する。  [0024] During the replacement of the serum medium, floating blood cell components are removed. By repeating this medium exchange, blood cell components are sequentially removed, and the blood cell components are completely removed by medium exchange at least twice, preferably at least 3 times or at least 4 times. Only adherent cells (ie, mesenchymal cells) centering on mesenchymal stem cells selectively proliferate.
[0025] 上記の操作により、培養容器の底面では接着細胞のみが選択的に増殖するが、接 着細胞の中には、間葉系幹細胞以外の細胞が時として現れることがある。間葉系幹 細胞以外の接着細胞は、間葉系幹細胞とは外観が全く異なっているため、容易に区 別可能である。さらにこの細胞は、細胞表面抗原解析により CD34, 45,陽性である ことを確認しており、このこと力も血球系由来の細胞であることを確認している。間葉 系幹細胞とそれ以外の接着細胞を効率的に分離する方法として、細胞解離剤による 剥離効果の差、すなわち培養容器底面への細胞接着力の差を利用し、 0.05%トリプ シン Z0.53mM EDTA溶液、もしくは同様の効果を有するプロテアーゼを、 1〜10 分間、平均的には 3分間培養容器で反応させることにより、間葉系幹細胞が剥離し、 それ以外の接着細胞は底面に付着'残留させることができる（図 2b)。剥離した間葉 系幹細胞を主集団とする細胞群は、別の培養容器で培養する。これを 1から 2度繰り 返すことにより、間葉系幹細胞を高純度で分離精製し、増殖させることができる（図 2a[0025] By the above operation, only adherent cells selectively grow on the bottom surface of the culture vessel. Among the adherent cells, cells other than mesenchymal stem cells sometimes appear. Adherent cells other than mesenchymal stem cells have a completely different appearance from mesenchymal stem cells and can be easily distinguished. Furthermore, this cell has been confirmed to be CD34, 45, positive by cell surface antigen analysis, and this force has also been confirmed to be a cell derived from the blood cell lineage. 0.05% trypsin Z0.53mM as a method for efficiently separating mesenchymal stem cells and other adherent cells using the difference in the detachment effect of the cell dissociator, that is, the difference in cell adhesion to the bottom of the culture vessel By reacting the EDTA solution or a protease with the same effect in a culture vessel for 1 to 10 minutes, on average for 3 minutes, the mesenchymal stem cells are detached, and other adherent cells adhere to the bottom surface. (Figure 2b). A cell group mainly composed of exfoliated mesenchymal stem cells is cultured in a separate culture vessel. By repeating this once or twice, mesenchymal stem cells can be separated and purified with high purity and proliferated (Fig. 2a).
) o ) o
[0026] トリプシンあるいは同様の作用を持つプロテアーゼ等の細胞解離剤を用いて接着 細胞を底面から全て剥離し、細胞相互の凝着も解かれた浮遊状態で細胞分離器 (セ ルソーター）を用いて間葉系幹細胞以外の細胞を分離し、別の培養容器にて間葉系 幹細胞を再び培養することによって、間葉系幹細胞を主集団とする細胞群の培養を 行うことも可能である。  [0026] Adhesion using trypsin or a cell dissociation agent such as protease having the same action Using a cell separator (cell sorter) in a floating state in which all cells are detached from the bottom surface and adhesion between cells is released. By separating cells other than mesenchymal stem cells and culturing the mesenchymal stem cells again in another culture vessel, it is also possible to culture a cell group mainly composed of mesenchymal stem cells.
[0027] 骨髄由来の間葉系幹細胞をさらに骨組織に分化させる場合、培養皿底面に付着し た幹細胞に対して、トリプシン、あるいは同等の作用を持つプロテアーゼなどを用い て底面から分離し、細胞相互の凝着も解かれた浮遊状態で操作を行う。  [0027] When the bone marrow-derived mesenchymal stem cells are further differentiated into bone tissue, the stem cells attached to the bottom surface of the culture dish are separated from the bottom surface using trypsin or a protease having an equivalent action, and the like. The operation is performed in a floating state where mutual adhesion has been resolved.
[0028] 骨組織に分化させる場合、その足場材として多孔性の水酸アパタイト、多孔性の燐 酸三カルシウムを用いてこれらのセラミックスの細孔内、あるいは細孔の表面近傍に 骨組織を分化させ、患者の骨欠損部への移植に用いる。さらに、人工関節とそれを 支える周囲骨との生着を早める目的で、人工関節の骨接触部に間葉系幹細胞を播 種し、骨組織に分ィ匕させて力もこれを移植する。人工関節への応用例として、アルミ ナセラミックス製の人工足関節の骨組織との接触部表面に、図 3に示すような直径 0. 8mmの多数のアルミナ製のビーズ球を焼き付けることによりポーラス構造を成してい る足場材の表面に間葉系幹細胞を播種し、骨組織に分化させて、その後移植する方 法で良好な治療成績を得て 、る。 [0028] When differentiating into bone tissue, porous hydroxyapatite and porous tricalcium phosphate are used as the scaffold, and the bone tissue is differentiated in the pores of these ceramics or in the vicinity of the surface of the pores. And used for transplantation into the bone defect of the patient. In addition, for the purpose of accelerating the engraftment between the artificial joint and the surrounding bone that supports it, mesenchymal stem cells are seeded at the bone contact portion of the artificial joint, and are distributed to the bone tissue to transplant the force. As an example of application to an artificial joint, a porous structure is created by baking a large number of alumina bead balls with a diameter of 0.8 mm as shown in Fig. 3 on the surface of the contact area of an artificial ankle joint made of alumina ceramics with the bone tissue. A mesenchymal stem cell is seeded on the surface of the scaffolding material, differentiated into bone tissue, and then transplanted Get good treatment results with the law.
[0029] これらの足場材に播種する幹細胞は、細胞濃度を 1 X 104〜1 X 106 cells/mLに 調整し、一様に播種する。その後、次の培地を用いて培養する。すなわち自己血清 培地中の濃度を、 j8 -Glycerophosphateが約 8〜12mM、望ましくは約 10mM, Vita min Cが約 16〜24 μ g/mL,望ましくは約 20 μ gZmL及びデキサメサゾンが約 80 〜120nM、望ましくは約 ΙΟΟηΜとなるように添加'調整した自己血清培地にて定期 的に培地交換を行い、骨組織への分ィ匕の進行を定期的に観察 '検査しつつ、 7日以 上 28日未満の間培養し、所定の骨細胞への分ィ匕が確認された段階で、患者本人に 移植する。また、継代培養などにより細胞の濃度を 1 X 106〜1 X 108 cells/mLの比 較的高濃度に調整し、これをセラミックス、ポリマーなどの足場材に播種し、 1〜24時 間の短期間培養することによって足場材に高密度で付着せしめた後、直接骨に移植 することにより、間葉系幹細胞が移植された部位で骨組織に分ィ匕する。この方法は、 比較的大量に間葉系幹細胞が得られる場合に、移植までの培養時間が短くてすむ という利点がある。 [0029] Stem cells to be seeded on these scaffolds are seeded uniformly by adjusting the cell concentration to 1 × 10 4 to 1 × 10 6 cells / mL. Then, it culture | cultivates using the following culture medium. That is, the concentration in the autoserum medium is about 8-12 mM for j8-Glycerophosphate, desirably about 10 mM, about 16-24 μg / mL for vitamin C, desirably about 20 μg ZmL, and about 80-120 nM for dexamethasone, Desirably about 培 地 η 交換 is added to the conditioned autologous serum medium, and the medium is periodically changed, and the progress of separation into the bone tissue is regularly observed. Incubate for less than the period of time, and transplant to the patient when the distribution to the bone cells is confirmed. In addition, the cell concentration is adjusted to a relatively high concentration of 1 × 10 6 to 1 × 10 8 cells / mL by subculture, etc., and this is seeded on scaffolds such as ceramics and polymers, and 1 to 24:00 After being attached to the scaffold at high density by culturing for a short period of time, it is directly transplanted to the bone, and then it is separated into bone tissue at the site where the mesenchymal stem cells are transplanted. This method has the advantage that when the mesenchymal stem cells are obtained in a relatively large amount, the culture time until transplantation is short.
[0030] 虚血性の心筋の回復や拡張型心筋症の治療、もしくは慢性末梢性動脈閉塞性疾 患 (糖尿病性壊死、動脈硬化性閉塞症、バージャ一病など）において、間葉系幹細 胞そのものを患部に注入する場合は、間葉系幹細胞のまま用いる。  [0030] In the treatment of ischemic myocardial recovery or dilated cardiomyopathy, or chronic peripheral arterial occlusive disease (diabetic necrosis, arteriosclerotic obstruction, Birja's disease, etc.), mesenchymal stem cells When injecting itself into the affected area, the mesenchymal stem cells are used as they are.
実施例  Example
[0031] 以下、本発明を実施例を用いてより詳細に説明するが、本発明がこれら実施例に 限定されな ヽことは ヽうまでもな!/ヽ。  [0031] Hereinafter, the present invention will be described in more detail with reference to Examples, but it is needless to say that the present invention is not limited to these Examples!
実施例 1：間葉系幹細胞の培養  Example 1: Mesenchymal stem cell culture
骨欠損部を有する患者から血液 250〜400mlを採血し、 3000rpmの条件にて 10 分間遠心分離を行い、血清を得た。該自己血清は、前記の特定組成を有する α -Μ EM培地 500mL〖こ、自己血清 88mLを加え、 15%vZv自己血清濃度を有する細胞 培地を得た。  Serum was obtained by collecting 250-400 ml of blood from a patient with a bone defect and centrifuging for 10 minutes at 3000 rpm. As the autoserum, 500 mL of α-Μ EM medium having the above-mentioned specific composition and 88 mL of autoserum were added to obtain a cell culture medium having a 15% vZv autoserum concentration.
[0032] 次に、前記患者から骨髄液を採取後、 lOUZmLのへパリン燐酸緩衝液を骨髄液 と等量（100%に相当）加えた。このへパリン添加骨髄液を lOOOrpmで 10分間遠心 分離を行った。遠心分離後、下から赤血球、有核細胞層、及び血漿の 3層に分離し ていることを確認後、血漿を取り除いた。赤血球、有核細胞層の 2層の合計で 2〜3m L当たり 30mLの自己血清培地で培養し、ほぼ 2日おきに自己血清を添加した α— MEM培地の交換を行い、血清培地交換の際に、浮遊している血球成分を取り除い た。この培地交換を 4回行った。培地交換を 4回行った時点で、完全に血球成分が除 去された。 [0032] Next, after collecting bone marrow fluid from the patient, lOUZmL of heparin phosphate buffer was added in an amount equivalent to bone marrow fluid (corresponding to 100%). The heparinized bone marrow was centrifuged at lOOOrpm for 10 minutes. After centrifugation, separate from the bottom into three layers: red blood cell, nucleated cell layer, and plasma. After confirming that the plasma was removed, the plasma was removed. Incubate in 30 mL autologous serum medium per 2 to 3 mL in total of two layers of red blood cells and nucleated cell layers, and replace the α-MEM medium supplemented with autologous serum approximately every 2 days. In addition, floating blood cell components were removed. This medium exchange was performed 4 times. When the medium was changed 4 times, blood cell components were completely removed.
[0033] 培養皿の底面に接着した細胞を 0.05%トリプシン Z0.53mM EDTA溶液で 3分間 処理することにより間葉系幹細胞を剥離し、これを別の容器に移して、前記自己血清 含有 α—MEM培地を用いてさらに培養した。 0.05%トリプシン Z0.53mM EDTA 溶液を用いる細胞剥離処理を 2回繰り返し、間葉系幹細胞を分離精製した。  [0033] By treating the cells adhering to the bottom of the culture dish with 0.05% trypsin Z0.53mM EDTA solution for 3 minutes, the mesenchymal stem cells were detached, transferred to another container, and the autoserum-containing α- Further culturing was performed using MEM medium. Cell detachment treatment using 0.05% trypsin Z0.53mM EDTA solution was repeated twice to separate and purify mesenchymal stem cells.
[0034] ヒト骨髄由来の細胞を自己血清含有 a MEM培地で培養した場合の細胞写真（ 図 l -a、 b)とゥシ胎児血清含有 α— MEM培地で培養した場合の写真細胞（図 l -c、 d)を比較したところ、自己血清含有 a—MEM培地での培養が、活性 (増殖速度、 分化誘導能、生存度など)及び増殖された細胞の数にぉ ヽて明らかに優れて ヽるこ とが確認された。  [0034] Cellular photographs of human bone marrow-derived cells cultured in autoserum-containing a MEM medium (Figures l-a and b) and photographic cells cultured in ushi fetal serum-containing α-MEM medium (Figure l) -c, d) was compared, and culture in autoserum-containing a-MEM medium was clearly superior in terms of activity (growth rate, differentiation-inducing ability, viability, etc.) and the number of proliferated cells. It was confirmed to speak.
[0035] また、表 1に示すように、実際の症例 13例において自己血清とゥシ胎児血清の培 養細胞数を培養日数で除して細胞増殖度を求め比較したところ、 13例中 11例で自 己血清の使用の方が細胞の増殖が同等以上の成績を示した。  [0035] Further, as shown in Table 1, in 13 actual cases, the number of cultured cells of autologous serum and rabbit fetal serum was divided by the number of culture days to determine the degree of cell proliferation. In the example, the use of autologous serum showed the same or better cell growth.
[0036] [表 1]  [0036] [Table 1]
ヒト間葉系幹細胞の増殖度  Proliferation of human mesenchymal stem cells
増殖度：（細胞数ノ培養日数 )x1 06 (+：自己血清の増殖度が同等以上） 症例番号 自己血清 牛胎児血清 特記事項 Proliferation: (Number of cells in culture days) x 1 0 6 (+: Autoserum growth rate is equal or higher) Case number Autologous serum Fetal bovine serum Remarks
1 0.54 0.51 +  1 0.54 0.51 +
2 0.18 0.15 +  2 0.18 0.15 +
3 0.13 0.12 +  3 0.13 0.12 +
4 0.48 0.57  4 0.48 0.57
5 0.66 0.14 +  5 0.66 0.14 +
6 0.31 0.25 +  6 0.31 0.25 +
7 0.33 0.33 +  7 0.33 0.33 +
8 0.41 0.26 +  8 0.41 0.26 +
9 0.26 0.2  9 0.26 0.2
10 0.14 0.14 +  10 0.14 0.14 +
11 0.48 0.29 +  11 0.48 0.29 +
12 0.46 0.58  12 0.46 0.58
13 0.8 0.62 + [0037] 図 2aは、トリプシン処理を行うことにより間葉系幹細胞の選択的な付着細胞剥離 を行い、この剥離した細胞を別容器にて培養した細胞、すなわち間葉系幹細胞の形 態を表し、図 2bはトリプシン処理を行って間葉系幹細胞を剥離後に培養容器の底面 になお残留 ·付着した状態の接着細胞を示す。 13 0.8 0.62 + [0037] FIG. 2a shows the form of mesenchymal stem cells that were selectively detached from mesenchymal stem cells by trypsin treatment and cultured in a separate container. Fig. 2b shows adherent cells that remain on the bottom of the culture vessel after trypsinization and detachment of mesenchymal stem cells.
[0038] さらに、自己血清を用いて培養した間葉系幹細胞の表面抗原の解析を行い、図 4 に示すようにこれらの細胞は血球系マーカーである CD34及び CD45が陰性、間葉 系細胞も含まれるマーカーである CD44及び CD90が陽性であり、培養した細胞が 非血球系で間葉系幹細胞系であることが示された。  [0038] Further, surface antigens of mesenchymal stem cells cultured using autoserum were analyzed. As shown in Fig. 4, these cells were negative for blood cell markers CD34 and CD45, and mesenchymal cells were also The included markers CD44 and CD90 were positive, indicating that the cultured cells were non-hemocytic and mesenchymal stem cell lines.
[0039] さらに、図 5に示すように、この方法で培養した細胞は、培養環境以外の環境で生 存する生存率が、少なくとも 24時間で 90%以上あり、培養環境以外で長期の保存、 あるいは運搬することが可能となった。  [0039] Furthermore, as shown in FIG. 5, the cells cultured by this method have a survival rate of 90% or more in an environment other than the culture environment for at least 24 hours, and can be stored for a long time outside the culture environment, or It became possible to carry.
実施例 2：骨組織への分化誘導  Example 2: Induction of differentiation into bone tissue
実施例 1で得られた間葉系幹細胞から 1 X 104〜1 X 106 cellsZmLの培養液を調 整し、これを図 3に示す直径 0.8mmのアルミナビーズを多数コートすることにより作ら れた多孔構造を有するアルミナセラミックス力もなる足場材料に播種し、 β -Glycerop hosphate ( 10mM)、 Vitamin C (20 iu g/mL)及びデキサメサゾン（lOOnM)を添カロ した自己血清 a MEM培地で 7〜28日間培養した。 A 1 × 10 4 to 1 × 10 6 cells ZmL culture solution was prepared from the mesenchymal stem cells obtained in Example 1, and this was prepared by coating a large number of 0.8 mm diameter alumina beads shown in FIG. 7-28 in autologous serum a MEM medium seeded on a scaffold material that has a porous structure and also has the power of alumina ceramics and supplemented with β-Glycerop hosphate (10 mM), Vitamin C (20 i ug / mL) and dexamethasone (lOOnM). Cultured for days.
[0040] この培養により、所定の骨細胞への分化が確認され、患者本人の骨欠損部に移植 するための移植材料が得られることを確認した。  [0040] By this culture, it was confirmed that differentiation into predetermined bone cells was confirmed, and a transplant material for transplantation into the bone defect site of the patient himself was obtained.
産業上の利用の可能性  Industrial applicability
[0041] 本発明は、細胞 *組織工学の再生医療への応用に大きく貢献し、細胞 *組織工学を 実用化、産業化する上でも重要な意味'役割を有する発明である。 [0041] The present invention greatly contributes to the application of cell * tissue engineering to regenerative medicine, and has an important role in the practical application and industrialization of cell * tissue engineering.

Claims

請求の範囲 The scope of the claims
[1] ヒト骨髄液を採取し、骨髄由来の間葉系幹細胞を培養して、ヒトに移植するためのヒト 間葉系幹細胞を調製する方法であって、以下の工程を含むことを特徴とする方法： [1] A method of preparing human mesenchymal stem cells for harvesting human bone marrow fluid, culturing mesenchymal stem cells derived from bone marrow, and transplanting to humans, comprising the following steps: how to:
(0 採取した骨髄液の分量に対して、濃度 5〜15UZmL程度のへパリン Z緩衝液を 80〜 120%vZv添加する工程、 (0 Step of adding 80-120% vZv of heparin Z buffer solution with a concentration of about 5-15 UZmL to the collected amount of bone marrow fluid,
GO 工程 (0で得られたへパリン Z緩衝液を添加した骨髄液を、ヒト血清を 10〜20%v Zv含む a -MEM培地で培養し、該培養中に少なくとも 2回培地交換することにより培 養液中に浮遊する血球成分を取り除く工程、および  GO step (Bone marrow fluid obtained by adding heparin Z buffer obtained in 0 is cultured in a-MEM medium containing human serum 10-20% v Zv, and the medium is changed at least twice during the culture. Removing blood cell components floating in the culture medium; and
(iii) 細胞解離剤を用いて間葉系幹細胞を主とする細胞群を培養容器力 選択的に 剥離し、剥離された細胞群から間葉系幹細胞以外の接着細胞を分離し、別の容器で 選択的に間葉系幹細胞を培養する工程。  (iii) A cell group mainly composed of mesenchymal stem cells using a cell dissociating agent is selectively peeled off, and adherent cells other than mesenchymal stem cells are separated from the peeled cells and separated into another container. In the step of selectively culturing mesenchymal stem cells.
[2] 工程 (0で得られたへノ リン Z緩衝液を添加した骨髄液を回転数 500〜 1500rpmの 遠心分離 (遠心重力で 40〜390g)を行い、下層から赤血球層、有核細胞層及び血 漿層の 3層に分離し、血漿層を除いた赤血球層及び有核細胞層の混合物を、工程 (i i)に供することを特徴とする請求項 1に記載の方法。 [2] Step (centrifuge the bone marrow fluid with the addition of the Heline Z buffer solution obtained in 0 at a rotation speed of 500 to 1500 rpm (40 to 390 g by centrifugal gravity). 2. The method according to claim 1, wherein the mixture of the red blood cell layer and the nucleated cell layer separated into three plasma layers and excluding the plasma layer is subjected to step (ii).
[3] へパリン Z緩衝液が、へパリン ZPBS(Phosphate buffered saline)液である請求項 1ま たは 2に記載の方法。 [3] The method according to claim 1 or 2, wherein the heparin Z buffer solution is a heparin ZPBS (Phosphate buffered saline) solution.
[4] a -MEMが以下の成分を以下の濃度 (mg/L)で含むものである請求項 1〜3のいずれ かに記載の方法：  [4] The method according to any one of claims 1 to 3, wherein the a-MEM contains the following components at the following concentrations (mg / L):
CaCl (anhyd.) :約 160— 240  CaCl (anhyd.): Approx. 160-240
2  2
KCL:約 320-480  KCL: About 320-480
MgSO (anhyd.) :約 78.4- 117.6  MgSO (anhyd.): About 78.4-117.6
4  Four
NaCl :約 5440- 8160  NaCl: Approx. 5440-8160
NaHCO：約 1760- 2640  NaHCO: approx. 1760-2640
3  Three
NaH ΡΟ · Η Ο :約 112— 168  NaH ΡΟ · Η Ο: Approximately 112-168
2 4 2  2 4 2
D- Glucose :約 800- 1200  D-Glucose: about 800-1200
Lipoic Acid :約 0.16- 0.24  Lipoic Acid: About 0.16-0.24
Sodium Pyruvate :約 88- 132 L- Alanine:約 20- 30 Sodium Pyruvate: About 88-132 L-Alanine: About 20-30
L-Arginine · HCl：約 101.6- 152.4 L-Asparagine · H O：約 40一 60  L-Arginine · HCl: approx. 101.6-152.4 L-Asparagine · H 2 O: approx. 40 1 60
2  2
L-Aspartic Acid:約 24- 36  L-Aspartic Acid: about 24-36
L— Cystine · 2HC1：約 24.8— 37.2 L- Cysteine'HCト H O:約 80- 120  L—Cystine · 2HC1: About 24.8— 37.2 L-Cysteine'HC H H: About 80-120
2  2
L- Glutamic Acid:約 60- 90  L-Glutamic Acid: about 60-90
L-Glutamine：約 233.6— 350.4 L-Glutamine: approx. 233.6- 350.4
Glycine:約 40- 60 Glycine: about 40-60
L-Histidine HCl-H O:約 33.6— 50.4  L-Histidine HCl-H 2 O: approx. 33.6-50.4
2 2
L-Isoleucine：約 41.6- 62.4 L-Isoleucine: About 41.6-62.4
L- Leucine:約 41.6- 62.4  L-Leucine: About 41.6-62.4
L- Lysine · HCl：約 58.4- 87.6  L-Lysine · HCl: approx. 58.4-87.6
L- Methionine：約 12- 18  L- Methionine: About 12-18
L— Phenylalanine：約 25.6— 38.4  L—Phenylalanine: approx. 25.6— 38.4
L- Proline:約 32- 48  L-Proline: approx. 32-48
L- Serine:約 20- 30  L- Serine: About 20-30
L- Threonine：約 38.4- 57.6  L-Threonine: About 38.4-57.6
L- Tryprophan:約 8- 12  L- Tryprophan: About 8-12
L-Tyrosine(disodium salt):約 41.6- 62.4 L-Tyrosine (disodium salt): about 41.6-62.4
L- Valine:約 36.8- 55.2 L-Valine: approx. 36.8-55.2
L- Ascorbic Acid:約 40- 60  L- Ascorbic Acid: approx. 40-60
Biotin:約 0.08- 0.12  Biotin: about 0.08- 0.12
D-Ca Pantothenate:約 0.8 - 1.2 D-Ca Pantothenate: about 0.8-1.2
Choline Chloride:約 0.8- 1.2 Choline Chloride: about 0.8-1.2
Folic Acid:約 0.8- 1.2  Folic Acid: about 0.8-1.2
i- Inositol:約 1.6- 2.4 i-Inositol: About 1.6-2.4
Niacinamide:約 0.8— 1.2 Pyridoxal HC1 :約 0.8- 1.2 Niacinamide: about 0.8—1.2 Pyridoxal HC1: about 0.8-1.2
Riboflavin :約 0.08- 0.12  Riboflavin: about 0.08- 0.12
Thiamine HC1 :約 0.8- 1.2  Thiamine HC1: about 0.8-1.2
Vitamin B ：約 1.12- 1.68  Vitamin B: About 1.12- 1.68
12  12
Adenosine :約 8- 12  Adenosine: approx. 8-12
Cytidine :約 8- 12  Cytidine: About 8-12
Guanosine：約 8- 12  Guanosine: About 8-12
Uridine :約 8- 12  Uridine: approx. 8-12
2 ' Deoxyadenosine：約 8- 12  2 'Deoxyadenosine: approx. 8-12
2 ' Deoxycytidine HC1 :約 8.8- 13.2  2 'Deoxycytidine HC1: approx.8.8-13.2
2 ' Deoxyguanosine：約 8- 12  2 'Deoxyguanosine: approx. 8-12
Thymidine :約 8- 12  Thymidine: approx. 8-12
[5] 工程（iii)において、 0.05%トリプシン Z0.53mM EDTA溶液、もしくは同様の作用を 持つプロテアーゼなどの酵素を細胞解離剤として用いて、 1〜10分間培養容器で反 応させることにより培養容器に接着した間葉系幹細胞を選択的に剥離することを特徴 とする、請求項 1〜4のいずれかに記載の方法。  [5] In step (iii), use a 0.05% trypsin Z0.53mM EDTA solution or an enzyme such as protease having the same action as a cell dissociator to react in the culture vessel for 1 to 10 minutes. The method according to any one of claims 1 to 4, wherein the mesenchymal stem cells adhered to the cell are selectively detached.
[6] 工程 (iii)で剥離後に分離精製された間葉系幹細胞の細胞濃度を 1 X 104〜1 X 106 cellsZmLに調整し、これを足場材に播種し、 8〜12mMの 13 - Glycerophosphate、 1 6〜24 gZmLの Vitamin C及び 80〜120nMのデキサメサゾンを添カ卩した、ヒト血 清含有 oc -MEM培地で 7日〜28日間培養し、骨芽細胞もしくはこの前駆細胞に分化 させることを特徴とする請求項 1〜5のいずれかに記載の方法。 [6] The cell concentration of the mesenchymal stem cells separated and purified after detachment in step (iii) is adjusted to 1 X 10 4 to 1 X 10 6 cells ZmL, seeded on the scaffold, and 8-12 mM 13- Glycerophosphate, cultured in human serum-containing oc-MEM medium supplemented with 16-24 gZmL Vitamin C and 80-120 nM dexamethasone for 7-28 days to differentiate into osteoblasts or progenitor cells The method according to any one of claims 1 to 5, wherein:
[7] 継代培養した細胞をトリプシンもしくは同様の作用を持つプロテアーゼ等の細胞解離 剤で処理を行い、細胞濃度を 1 X 106〜1 X 108 cells/mLに調整し、これをセラミツ タス、ポリマーなどの足場材に播種し、 1〜24時間培養することを特長とする請求項 1 〜6の!、ずれかに記載の方法。 [7] Treat the subcultured cells with trypsin or a cell dissociator such as protease with the same action to adjust the cell concentration to 1 X 10 6 to 1 X 10 8 cells / mL. The method according to any one of claims 1 to 6, characterized by seeding on a scaffold such as a polymer and culturing for 1 to 24 hours.
[8] 足場材が多孔質の水酸アパタイト、多孔質の三燐酸カルシウム等の燐酸カルシウム 系セラミックス及び多孔体の炭酸カルシウム、表面多孔体構造を持つアルミナの 、ず れかである請求項 6または 7に記載の方法。 [8] The scaffold according to claim 6 or 6, wherein the scaffold is any one of porous hydroxyapatite, porous calcium phosphate ceramics such as porous calcium triphosphate, porous calcium carbonate, and alumina having a surface porous structure. The method according to 7.
[9] 足場材が生体用金属材料、もしくはチタンを主成分とする生体用金属の表面に、生 体活性ガラス、あるいは水酸アパタイトなどの燐酸カルシウムを主成分とする無機材 料をコーティングした材料を用いることを特徴とする請求項 6または 7に記載の方法。 [9] A material in which the scaffold is a metal material for living organisms or a surface of a metal for living organisms containing titanium as a main component, coated with an inorganic material mainly containing calcium phosphate such as bioactive glass or hydroxyapatite The method according to claim 6 or 7, wherein:
[10] ヒト血清として、骨髄液と同一のヒトの血清、すなわち自己血清を用いることを特徴と する請求項 1〜9のいずれかに記載の方法。  [10] The method according to any one of [1] to [9], wherein the human serum is the same human serum as bone marrow fluid, that is, autoserum.
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