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WO2017164467A1 - Culture de cellules souches mésenchymateuses pour la prévention ou le traitement d'une maladie immunitaire ou d'une maladie inflammatoire et procédé de préparation associé - Google Patents

Culture de cellules souches mésenchymateuses pour la prévention ou le traitement d'une maladie immunitaire ou d'une maladie inflammatoire et procédé de préparation associé Download PDF

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WO2017164467A1
WO2017164467A1 PCT/KR2016/009112 KR2016009112W WO2017164467A1 WO 2017164467 A1 WO2017164467 A1 WO 2017164467A1 KR 2016009112 W KR2016009112 W KR 2016009112W WO 2017164467 A1 WO2017164467 A1 WO 2017164467A1
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mesenchymal stem
stem cell
cell culture
disease
inflammatory
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Korean (ko)
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이성구
김미형
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Anterogen Co Ltd
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Anterogen Co Ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the present invention relates to a mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases having significant immunosuppression and anti-inflammatory function by a three-dimensional culture method and a method for preparing the same.
  • mesenchymal stem cells are known to exist in most organs of the body, including bone marrow, fat, liver, and muscle. Mesenchymal stem cells have autologous proliferation, osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon-ligament fibroblasts, adipocytes ( adipocytes).
  • stem cell culture solution can be used for clinical purposes, such as pharmaceuticals and cosmetic compositions.
  • bovine serum is commonly used to culture mesenchymal stem cells, and the culture solution containing bovine serum is inappropriate for clinical application. That is, since the use of animal-derived proteins such as bovine serum is restricted for clinical application of cell culture, it is preferable to exclude bovine serum from the culture of stem cells.
  • bovine serum proteins are necessary for the proliferation and maintenance of stem cells, a method for culturing mesenchymal stem cells in a serum-free medium and obtaining a culture solution containing a high concentration of a bioactive substance is required.
  • Korean Patent Publication No. 10-2009-0001163 (2009. 01. 08) is a stem cell cultured in serum-free medium after culturing mesenchymal stem cells obtained from human bone marrow, umbilical cord blood or placental tissue, cultured stem A method for producing a large amount of bFGF or TGF- ⁇ from mesenchymal stem cells by applying physical stimulation of hypoxic culture to cells is disclosed.
  • WO 2007/086637 discloses adult stem cells extracted from adipose cells from mammals and cultured in serum medium and then subcultured in serum-free medium, followed by hypoxic culture in cultured stem cells.
  • a method for producing bFGF, VEGF or TGF- ⁇ in large quantities from mesenchymal stem cells by applying physical irritation such as ultraviolet irradiation, nutrient deficiency, or mechanical friction, and adding vitamin A, B, C, or D to the culture Doing.
  • stem cells are simply two-dimensionally cultured, or two-dimensional cultures are performed at low oxygen conditions in order to increase the content of VEGF, etc., considering that it is difficult to stably maintain the stem cells in a serum-free medium. There was a problem that the physiological activity of the stem cell culture medium is lowered.
  • immune diseases are diseases in which the components of the mammalian immune system cause, mediate, or contribute to the pathology of mammals.
  • inflammatory diseases are diseases that are attracting worldwide attention. It is an urgent disease. Inflammation is generally a localized protective response of body tissues to host invasion by foreign substances or harmful stimuli.
  • causes of inflammation include infectious causes such as bacteria, viruses and parasites; Physical causes such as burns or irradiation; Chemicals such as toxins, drugs or industrial preparations; Immune conditions such as allergies and autoimmune responses, or conditions associated with oxidative stress.
  • Inflammation is characterized by pain, redness, swelling, fever and ultimate loss of function of the infected area. These symptoms are the result of a series of complex interactions that occur between cells of the immune system.
  • the response of the cell results in an interactive network of different groups of inflammatory mediators: proteins (eg cytokines, enzymes (eg proteases, peroxidases), major basic proteins, adhesion molecules (ICAM, VCAM), lipid mediators (e.g. eicosanoids, prostaglandins, leukotriene, platelet activating factor (PAF)), reactive oxygen species (e.g.
  • autoimmune disease one of immune diseases, is characterized by the immune system attacking its own organs and causing a spontaneous reaction. These responses are due to the recognition of auto-antigens by T lymphocytes, leading to humoral (autoantibody production) and cellular (increased lymphocyte and macrophage cytotoxic activity) immune responses.
  • Autoimmune diseases include rheumatoid disease, psoriasis, systemic dermatitis, multiple sclerosis, lupus erythematosus, or worsening of immune response by antigen, ie asthma, drug or food allergy. These diseases are all limited and chronic diseases, and in some cases fatal, to date there is no effective treatment method for treating the diseases. Therefore, any drug, medicine or medium that can alleviate or alleviate the disease during the course of the disease will be an important solution for the health of the patient.
  • the present inventors have made efforts to develop a stem cell culture medium that can be used for clinical use for the application of therapeutic agents for immune diseases or inflammatory diseases.
  • the biocompatible scaffolds in serum-free medium are compared by comparing the culture medium obtained according to the existing two-dimensional culture.
  • Mesenchymal stem cell culture obtained according to the three-dimensional culture by using the inflammatory cytokine TNF- ⁇ and IFN- ⁇ secretion that does not induce an immune response, and increases the immune response, and immunomodulatory factor By confirming high secretion of phosphorus PGE2, the present invention was completed by revealing that the culture solution obtained according to the above method can be used for the prevention or treatment of immune diseases or inflammatory diseases.
  • An object of the present invention is to provide a method for producing mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases.
  • Another object of the present invention to provide a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases prepared by the above method.
  • Another object of the present invention is to provide a composition for producing mesenchymal stem cell culture for treating immune diseases or inflammatory diseases, which contains a biocompatible scaffold and a serum-free medium comprising any one or more of bEGF or EGF as an active ingredient. .
  • Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases containing the mesenchymal stem cell culture medium prepared by the above production method as an active ingredient.
  • Another object of the present invention is to provide a method for preventing or ameliorating an immune disease or inflammatory disease, comprising administering to the individual a mesenchymal stem cell culture prepared by the method.
  • Another object of the present invention is to provide a method for treating an immune disease or inflammatory disease, comprising the step of administering a mesenchymal stem cell culture prepared by the above method to an individual suffering from an immune disease or an inflammatory disease.
  • Another object of the present invention is to provide a use of the mesenchymal stem cell culture prepared by the above method for use as a composition for the prevention or treatment of immune diseases or inflammatory diseases.
  • Another object of the present invention is to provide a biocompatible scaffold for use as a composition for preparing mesenchymal stem cell culture for the treatment of immune diseases or inflammatory diseases, and the use of serum-free medium containing any one or more of bEGF or EGF. .
  • It provides a method for producing a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases comprising the step of (b) collecting the culture medium.
  • the present invention provides a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases prepared by the above method.
  • the present invention provides a composition for producing mesenchymal stem cell culture for the treatment of immune diseases or inflammatory diseases containing a biocompatible scaffold and a serum-free medium comprising any one or more of bEGF or EGF as an active ingredient.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases containing the mesenchymal stem cell culture medium prepared by the above method as an active ingredient.
  • the present invention provides a method for preventing or ameliorating an immune disease or inflammatory disease, comprising administering to the individual a mesenchymal stem cell culture prepared by the method.
  • the present invention provides a method for treating an immune disease or inflammatory disease comprising administering a mesenchymal stem cell culture prepared by the above method to an individual suffering from an immune disease or an inflammatory disease.
  • the present invention also provides a use of the mesenchymal stem cell culture prepared by the above method for use as a composition for preventing or treating immune diseases or inflammatory diseases.
  • the present invention also provides a biocompatible scaffold for use as a composition for preparing mesenchymal stem cell culture for the treatment of immune diseases or inflammatory diseases, and a serum-free medium comprising any one or more of bEGF or EGF.
  • Stem cell cultures obtained by three-dimensional culture of mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the method of the present invention are more immunomodulatory than stem cell cultures obtained by two-dimensional culture according to a conventional method. Since PGE2 is secreted in high concentration and excellent in suppressing immune response and inflammatory cytokine secretion, it can be usefully used as a composition for preventing or treating immune diseases or inflammatory diseases.
  • Figure 1 is treated with mesenchymal stem cell culture medium obtained by the two-dimensional culture method (2D CM) or mesenchymal stem cell culture medium (3D CM) obtained by the three-dimensional culture method and the rate of inhibition of peripheral blood mononuclear cell proliferation The figure shown.
  • 2D CM two-dimensional culture method
  • 3D CM mesenchymal stem cell culture medium
  • Figure 2 is treated with mesenchymal stem cell culture obtained by the two-dimensional culture method (2D CM) or mesenchymal stem cell culture obtained by the three-dimensional culture method (3D CM) by concentration and secreted from peripheral blood mononuclear cells Figure showing the inhibition rate of TNF- ⁇ secretion.
  • 2D CM two-dimensional culture method
  • 3D CM three-dimensional culture method
  • Figure 3 is treated with mesenchymal stem cell culture obtained by the two-dimensional culture method (2D CM) or mesenchymal stem cell culture obtained by the three-dimensional culture method (3D CM) by concentration and secreted from peripheral blood mononuclear cells Figure showing IFN- ⁇ secretion inhibition rate.
  • 2D CM two-dimensional culture method
  • 3D CM three-dimensional culture method
  • Figure 4 is a diagram showing the secretion amount of PGE2 (prostaglandin E2) in mesenchymal stem cell culture obtained by the two-dimensional culture method (2D CM) or mesenchymal stem cell culture obtained by the three-dimensional culture method (3D CM) to be.
  • PGE2 prostaglandin E2
  • Mesenchymal stem cells (mesenchymal stem cells)" herein is self-propagation is possible, and it has a multipotent, CD73 +, CD90 +, CD105 +, CD14 - the cellular phenotype -, CD20 -, CD34 -, CD45 It refers to a cell that represents, and can be isolated from, but not limited to, bone marrow, adipose tissue, umbilical cord blood and the like.
  • physiologically active substance refers collectively to cytokines, cell growth factors, and immunomodulators that may affect the function of cells or the body.
  • bioactive substances include VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), TGF-beta (Tumor growth factor-beta), IGF (Insulin growth factor), etc. It is not limited to this.
  • stem cell culture liquid refers to a cell culture supernatant cultured stem cells.
  • Stem cell culture medium contains various bioactive substances secreted from cells during stem cell culture.
  • a biocompatible scaffold means a support made of a material having affinity with cells and a so-called cell adhesive surface and capable of attaching and culturing cells three-dimensionally.
  • naturally occurring supports include, but are not limited to, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, and the like.
  • synthetic polymer include, but are not limited to, poly (alpha-hydroxy acid) series, poly (vinyl alcohol), polyanhydride, and the like.
  • the present invention is a.
  • It provides a method for producing a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases comprising the step of (b) collecting the culture medium.
  • the mesenchymal stem cells in step (a) is preferably isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.
  • the mesenchymal stem cells in step (a) is preferably cultured in a substrate medium and proliferation medium and then subcultured.
  • the three-dimensional culture is preferably obtained at least three times the stem cell culture medium exchange medium at intervals of three days.
  • step (a) it is preferable to add and culture any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) to the serum-free medium.
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • the biocompatible scaffold in step (b) is preferably a natural or synthetic polymer as a cell support having a cell-adhesive surface, for example alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydr). Hydroxy acid) series, poly (vinyl alcohol), polyanhydride and the like, but is not limited thereto.
  • the immune diseases include Behcet's disease, polymyositis, cutaneous myositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatitis, Goodfiction syndrome, autoimmune meningitis, Sjogren's syndrome, systemic erythritis Reflection lupus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, dystrophic epidermal detachment, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic Anemia, multiple sclerosis, myasthenia gravis, vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scler
  • the inflammatory diseases include gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascia disease, viral infection, Bacterial infections, fungal infections, burns, wounds from surgical or dental surgery, PGE hyperplasia, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iris, scleritis, uveitis or acute and chronic inflammatory diseases It is not limited to this.
  • the mesenchymal stem cell culture medium preferably inhibits the secretion of TNF- ⁇ or IFN- ⁇ by an immune response and immune response, but is not limited thereto.
  • the mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
  • the mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 Pg / ml to 10000 pg / ml of PGE2, and 7900 pg / ml to 9800 pg / ml of PGE2. Even more preferred.
  • PGE2 prostaglandin E2
  • the present inventors treated the mesenchymal stem cell culture obtained according to the three-dimensional culture method to the peripheral blood mononuclear cells that caused the immune response, the cell proliferation of the peripheral blood mononuclear cells is suppressed It confirmed that it became (refer FIG. 1).
  • the present inventors treated the mesenchymal stem cell culture medium obtained according to the three-dimensional culture method to the peripheral blood mononuclear cells that caused the immune response, and thus suppressed TNF- ⁇ and IFN- ⁇ secretion from the peripheral blood mononuclear cells. It was confirmed that (see Fig. 2 and 3).
  • the present inventors confirmed that in the mesenchymal stem cell culture obtained according to the three-dimensional culture method, PGE2, an immunomodulator, is significantly increased than the mesenchymal stem cell culture obtained according to the two-dimensional culture method (FIG. 4). Reference).
  • stem cells culture medium containing high concentrations of PGE2 by three-dimensional culture of mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the method of the present invention excellent in immune response and inhibitory effect of inflammatory cytokines
  • the present invention can be usefully used for the preparation of mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases.
  • Cultivation of mesenchymal stem cells according to the present invention can be carried out according to the following procedure, the medium used for cell culture is not limited thereto.
  • Mesenchymal stem cells obtained from the tissues are suspended in the substrate medium and inoculated in a culture vessel at a concentration of 10,000-40,000 cells / cm2 and then cultured.
  • the substrate medium is incubated in DMEM or DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth) medium containing 10% bovine serum for about 24 hours.
  • DMEM or DMEM / F12 Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth
  • the growth medium is DMEM or DMEM / F12 containing 10% bovine serum, an epidermal growth factor (EGF) at 0.1-100 ng / ml and / or a basic fibroblast growth factor (bFGF) at 0.1-100 ng / ml, Rapidly proliferates the adherent mesenchymal stem cells to increase the amount of cells in a short period of time.
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • the growth medium is removed and cells are removed from the culture vessel by trypsin treatment.
  • cells are diluted 1: 3 to 1: 4 and incubated with proliferation medium in a new culture vessel. In this way, additional subcultures are possible.
  • the cultured cells are washed three or more times with PBS to remove FBS, and cultured in a serum-free medium attached to a biocompatible scaffold.
  • Cell culture in the scaffold does not require a conventional cell culture container, there is an advantage that can be cultured in a large amount in a sterile bottle or culture bag can be conveniently cultured at a lower cost.
  • the present invention also provides a composition containing a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases prepared according to the method of the present invention.
  • the mesenchymal stem cell culture medium preferably inhibits the secretion of TNF- ⁇ or IFN- ⁇ by an immune response and immune response, but is not limited thereto.
  • the mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
  • the mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 Pg / ml to 10000 pg / ml of PGE2, and 7900 pg / ml to 9800 pg / ml of PGE2. Even more preferred.
  • PGE2 prostaglandin E2
  • Mesenchymal stem cell cultures obtained by three-dimensional culture of mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the preparation method of the present invention are excellent in suppressing the immune response and secretion of inflammatory cytokines. Or it may be usefully used as a composition for the prevention or treatment of inflammatory diseases.
  • the present invention provides a composition for producing mesenchymal stem cell culture for the treatment of immune diseases or inflammatory diseases containing a biocompatible scaffold and a serum-free medium comprising any one or more of bEGF or EGF as an active ingredient.
  • the biocompatible scaffold is a cell support having a cell adhesive surface, preferably a natural or synthetic polymer, for example, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) series, Poly (vinyl alcohol), polyanhydride, and the like, but is not limited thereto.
  • a natural or synthetic polymer for example, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) series, Poly (vinyl alcohol), polyanhydride, and the like, but is not limited thereto.
  • the serum-free medium can be used without limitation a basic medium known in the art, but using a serum is added.
  • the basal medium may be prepared by artificially synthesizing, or a commercially prepared medium may be used.
  • commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, ⁇ -MEM ( ⁇ -Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium, but are not limited thereto.
  • DMEM or DMEM / F12 may be used.
  • bFGF is preferably added in an amount of 0.5 to 2 ng / ml, more preferably in an amount of 0.7 to 1.5 ng / ml, and even more preferably 1 ng / ml.
  • EGF is preferably added 3-7 ng / ml, more preferably 4-6 ng / ml, and even more preferably 5 ng / ml.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases containing mesenchymal stem cell culture prepared according to the method of the present invention as an active ingredient.
  • the mesenchymal stem cell culture medium preferably inhibits the secretion of TNF- ⁇ or IFN- ⁇ by an immune response and immune response, but is not limited thereto.
  • the mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
  • the mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 Pg / ml to 10000 pg / ml of PGE2, and 7900 pg / ml to 9800 pg / ml of PGE2. Even more preferred.
  • PGE2 prostaglandin E2
  • Mesenchymal stem cell cultures obtained by three-dimensional culture of mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the preparation method of the present invention are excellent in suppressing the immune response and secretion of inflammatory cytokines. Or it may be usefully used as a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
  • the present invention provides a method for preventing or ameliorating an immune disease or inflammatory disease, comprising administering to a subject a mesenchymal stem cell culture prepared according to the method of the present invention.
  • the mesenchymal stem cell culture medium preferably inhibits the secretion of TNF- ⁇ or IFN- ⁇ by an immune response and immune response, but is not limited thereto.
  • the mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
  • the mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 Pg / ml to 10000 pg / ml of PGE2, and 7900 pg / ml to 9800 pg / ml of PGE2. Even more preferred.
  • PGE2 prostaglandin E2
  • the mesenchymal stem cell culture obtained by three-dimensional culture of mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the preparation method of the present invention is excellent in suppressing the immune response and secretion of inflammatory cytokines. It can be usefully used as a method for preventing or ameliorating immune diseases or inflammatory diseases by administration.
  • the present invention provides a method for treating an immune disease or inflammatory disease comprising administering a mesenchymal stem cell culture prepared according to the method of the present invention to an individual suffering from an immune disease or an inflammatory disease.
  • the mesenchymal stem cell culture medium preferably inhibits the secretion of TNF- ⁇ or IFN- ⁇ by an immune response and immune response, but is not limited thereto.
  • the mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
  • the mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 Pg / ml to 10000 pg / ml of PGE2, and 7900 pg / ml to 9800 pg / ml of PGE2. Even more preferred.
  • PGE2 prostaglandin E2
  • Mesenchymal stem cell cultures obtained by three-dimensional culture of mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the preparation method of the present invention are excellent in suppressing the immune response and secretion of inflammatory cytokines. Or it may be usefully used as a method for treating an immune disease or an inflammatory disease by administering to an individual suffering from an inflammatory disease.
  • the present invention also provides the use of mesenchymal stem cell culture prepared according to the method of the present invention for use as a composition for the prevention or treatment of immune diseases or inflammatory diseases.
  • the mesenchymal stem cell culture medium preferably inhibits the secretion of TNF- ⁇ or IFN- ⁇ by an immune response and immune response, but is not limited thereto.
  • the mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
  • the mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 Pg / ml to 10000 pg / ml of PGE2, and 7900 pg / ml to 9800 pg / ml of PGE2. Even more preferred.
  • PGE2 prostaglandin E2
  • Mesenchymal stem cell cultures obtained by three-dimensional culture of mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the preparation method of the present invention are excellent in suppressing the immune response and secretion of inflammatory cytokines. Or it may be usefully used as a composition for preventing or treating inflammatory diseases.
  • the present invention also provides a biocompatible scaffold for use as a composition for preparing mesenchymal stem cell culture for the treatment of immune diseases or inflammatory diseases, and a serum-free medium comprising any one or more of bEGF or EGF.
  • the biocompatible scaffold is a cell support having a cell adhesive surface, preferably a natural or synthetic polymer, for example, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) series, Poly (vinyl alcohol), polyanhydride, and the like, but is not limited thereto.
  • a natural or synthetic polymer for example, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) series, Poly (vinyl alcohol), polyanhydride, and the like, but is not limited thereto.
  • the serum-free medium can be used without limitation a basic medium known in the art, but using a serum is added.
  • the basal medium may be prepared by artificially synthesizing, or a commercially prepared medium may be used.
  • commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, ⁇ -MEM ( ⁇ -Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium, but are not limited thereto.
  • DMEM or DMEM / F12 may be used.
  • bFGF is preferably added in an amount of 0.5 to 2 ng / ml, more preferably in an amount of 0.7 to 1.5 ng / ml, and even more preferably 1 ng / ml.
  • EGF is preferably added 3-7 ng / ml, more preferably 4-6 ng / ml, and even more preferably 5 ng / ml.
  • Adipose tissue is usually obtained by liposuction, but is not limited thereto.
  • Adipose-derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction as follows: Adipose tissue was washed 3-4 times with the same volume of KRB solution to remove blood. Then, the same volume of collagenase solution as adipose tissue was added and reacted in a 37 ° C. water bath. It was transferred to a centrifuge tube and centrifuged at 20, 1500 rpm for 10 minutes . After centrifugation, the fat layer as a supernatant was removed and the collagenase solution as a lower layer was separated carefully so as not to shake.
  • the substrate medium was suspended and centrifuged for 5 minutes at 20 °C, 1500 rpm. At this time, the subsidence was the stromal-vascular fraction, and the supernatant was removed.
  • the stromal-vascular fractions were suspended in substrate medium and inoculated in culture vessels and incubated in 37 ° C., 5% CO 2 incubator for 24 hours.
  • the solution was washed with phosphate buffer, and the substrate medium (DMEM / F12 medium containing 10% FBS), or the growth medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / ml in the substrate medium, or Epithelial growth factor (EGF) in the substrate medium was grown using a medium containing a concentration of 5 ng / ml.
  • the substrate medium DMEM / F12 medium containing 10% FBS
  • Adipose-derived mesenchymal stem cells were grown to about 80-90% of the culture vessel and obtained by separating them into single cells by trypsin treatment. The obtained cells were diluted 1: 3 to 1: 4 with proliferation medium to carry out passage culture.
  • Stem cells obtained as trypsin-treated single cells were washed three times with DMEM / F12 medium to remove FBS.
  • Cells were inoculated by adding DMEM / F12 solution to 1.3 ⁇ 10 7 stem cells / ml.
  • a DMEM / F12 solution containing 1 ng / ml basic fibroblast growth factor and 5 ng / ml epidermal growth factor was added to 500 ml per 4.0 ⁇ 10 7 cells. After 72 hours, the cell culture was recovered and refrigerated two to 11 times more by adding a new medium.
  • Stem cells obtained as trypsin-treated single cells were washed three times with DMEM / F12 medium to remove FBS.
  • the cells were suspended by adding DMEM / F12 solution containing thrombin solution to 1.3 ⁇ 10 7 stem cells / ml.
  • Cell suspensions and fibrinogen dilutions were mixed using dual syringes and then cultured in bottles or culture bags in three dimensions.
  • Cell culture medium was used DMEM / F12 solution containing 1 ng / ml basic fibroblast growth factor and 5 ng / ml epidermal growth factor. After 72 hours, the cell culture was recovered and refrigerated two to 11 times more by adding a new medium.
  • EdU cell proliferation assay was performed to confirm the immune response inhibition ability of the mesenchymal stem cell culture medium obtained in Examples 2 and 3.
  • PBMCs Peripheral Blood Mononuclear Cells
  • HLA Human Leukocyte Antigens
  • the mesenchymal stem cell culture medium obtained by the three-dimensional culture method through ⁇ Experimental Example 1> and ⁇ Experimental Example 2> does not induce an immune response and secrete an amount of inflammatory cytokines that play a role in increasing immune response. It was confirmed that it can help to heal by reducing the immune disease or inflammatory disease.
  • the mesenchymal stem cell culture obtained by the three-dimensional culture method through ⁇ Experimental Example 1> to ⁇ Experimental Example 3> does not induce an immune response and secrete an amount of inflammatory cytokines that play a role of increasing immune response. It was confirmed that it is possible to reduce and reduce the immune response or inflammatory diseases by increasing the immune response by secreting a high concentration of the immunoregulatory factor PGE2.
  • the present invention relates to a mesenchymal stem cell culture medium having significant immunosuppression and anti-inflammatory function by a three-dimensional culture method and a method for preparing the same. Or as an active ingredient of a therapeutic agent.

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Abstract

La présente invention concerne une culture de cellules souches mésenchymateuses pour la prévention ou le traitement de maladies immunitaires ou de maladies inflammatoires et un procédé de préparation associé. Sécrétant l'immunomodulateur PGE2 à une concentration élevée et présentant un effet inhibiteur remarquable sur les réponses immunitaires et la libération de cytokines inflammatoires, par rapport à des cultures de cellules souches préparées dans deux dimensions selon des procédés classiques, la culture de cellules souches préparée par la culture tridimensionnelle de cellules souches mésenchymateuses dans un milieu sans sérum à l'aide d'un échafaudage biocompatible selon le procédé de la présente invention peut être utilisée avantageusement en tant que composition pour prévenir ou traiter des maladies immunitaires ou des maladies inflammatoires.
PCT/KR2016/009112 2016-03-25 2016-08-18 Culture de cellules souches mésenchymateuses pour la prévention ou le traitement d'une maladie immunitaire ou d'une maladie inflammatoire et procédé de préparation associé Ceased WO2017164467A1 (fr)

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CN110643567A (zh) * 2018-06-27 2020-01-03 提艾勒干细胞研究所 细胞培养用培养基组合物的制备方法
IT201800020722A1 (it) * 2018-12-21 2020-06-21 Assunta Borzacchiello Biomateriale e suo utilizzo nel trattamento di patologie polmonari
US10758571B1 (en) 2019-04-09 2020-09-01 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
CN113382739A (zh) * 2018-12-13 2021-09-10 Scm生命科学有限公司 包含克隆干细胞的用于治疗胰腺炎的药学组合物
CN114561344A (zh) * 2021-11-25 2022-05-31 中国人民解放军空军军医大学 可提高干细胞免疫调控能力的无血清培养基及制备方法和应用
JP2023502236A (ja) * 2019-11-18 2023-01-23 ワイジェイ セラピューティクス インコーポレイテッド 新規化合物処理した幹細胞を含む脊髄損傷治療用組成物
US12186430B2 (en) 2020-04-07 2025-01-07 Combangio, Inc. Lyophilized mesenchymal stem cell derived secretome and uses thereof

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KR102155121B1 (ko) * 2018-08-14 2020-09-11 주식회사 티스템 세포배양용 배지 조성물 제조방법, 세포배양용 배지 조성물 제조방법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물(쉘드줄기세포) 제조방법, 이를 이용한 항관절염 치료용 조성물, 이를 이용한 항염증 치료용 조성물 및 세포재생 치료용 조성물
KR102280199B1 (ko) * 2019-12-04 2021-07-21 건국대학교 산학협력단 중간엽 줄기세포의 면역조절 활성 증진용 조성물

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Publication number Priority date Publication date Assignee Title
CN110643567A (zh) * 2018-06-27 2020-01-03 提艾勒干细胞研究所 细胞培养用培养基组合物的制备方法
CN113382739A (zh) * 2018-12-13 2021-09-10 Scm生命科学有限公司 包含克隆干细胞的用于治疗胰腺炎的药学组合物
IT201800020722A1 (it) * 2018-12-21 2020-06-21 Assunta Borzacchiello Biomateriale e suo utilizzo nel trattamento di patologie polmonari
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JP2023502236A (ja) * 2019-11-18 2023-01-23 ワイジェイ セラピューティクス インコーポレイテッド 新規化合物処理した幹細胞を含む脊髄損傷治療用組成物
JP7556584B2 (ja) 2019-11-18 2024-09-26 ワイジェイ セラピューティクス インコーポレイテッド 新規化合物処理した幹細胞を含む脊髄損傷治療用組成物
US12186430B2 (en) 2020-04-07 2025-01-07 Combangio, Inc. Lyophilized mesenchymal stem cell derived secretome and uses thereof
CN114561344A (zh) * 2021-11-25 2022-05-31 中国人民解放军空军军医大学 可提高干细胞免疫调控能力的无血清培养基及制备方法和应用

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