WO2017164467A1 - Mesenchymal stem cell culture for prevention or treatment of immune disease or inflammatory disease and preparation method thereof - Google Patents

Mesenchymal stem cell culture for prevention or treatment of immune disease or inflammatory disease and preparation method thereof 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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(주)안트로젠
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    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
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    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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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

The present invention relates to a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases and a preparation method thereof. Secreting the immunomodulator PGE2 at a high concentration and exhibiting an outstanding inhibitory effect on immune responses and the release of inflammatory cytokines, compared to stem cell cultures prepared in a two-dimensional manner according to conventional methods, the stem cell culture prepared by three-dimensionally culturing mesenchymal stem cells in a serum-free medium with the aid of a biocompatible scaffold in accordance with method of the present invention can be usefully used as a composition for preventing or treating immune diseases or inflammatory diseases.

Description

면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액 및 이의 제조방법Mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases and preparation method thereof
본 발명은 3차원 배양방법에 의해 유의적인 면역억제 및 항염기능을 보유하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액 및 이의 제조방법에 관한 것이다.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.
인간의 골수에는 몇 가지 종류의 전구세포(progenitor)가 존재하는 것이 발견되었으며, 이들 중 다분화능을 나타내는 전구세포를 중간엽줄기세포라 칭한다. 중간엽줄기세포는 골수뿐 아니라 지방, 간, 근육 등 신체 대부분의 장기에 존재하는 것으로 알려져 있다. 중간엽줄기세포는 자가증식능이 있고 골아세포(osteoblasts), 연골세포(chondrocytes), 근세포(myocytes), 골수기질세포(marrow stromal cells), 건-인대 섬유아세포(tendon-ligament fibroblasts), 지방세포(adipocytes) 등으로 분화할 수 있다.Several kinds of progenitors have been found in the human bone marrow, and progenitor cells showing pluripotency among them are called mesenchymal stem cells. 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).
또한, 중간엽줄기세포는 배양 과정에서 다양한 성장인자 및 생리활성물질을 분비하는 것으로 알려져 있다. 따라서, 줄기세포배양액은 의약품이나 화장품 조성물 등의 임상적 용도로 사용이 가능하다. In addition, mesenchymal stem cells are known to secrete various growth factors and bioactive substances in the culture process. Therefore, the stem cell culture solution can be used for clinical purposes, such as pharmaceuticals and cosmetic compositions.
그러나, 중간엽줄기세포를 배양하기 위해서는 통상적으로 소혈청을 사용하는데, 소혈청이 함유된 배양액은 임상적 적용이 부적당하다. 즉, 세포 배양액의 임상적 적용을 위해서는 소혈청과 같은 동물 유래 단백질의 사용이 제한되므로, 줄기세포의 배양에는 소혈청을 배제하는 것이 바람직하다.However, 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.
소혈청 단백질은 줄기세포의 증식 및 유지를 위해서 반드시 필요하므로, 무혈청 배지에서 중간엽줄기세포를 배양하면서도 고농도의 생리활성물질을 함유하는 배양액을 얻을 수 있는 방법이 요구된다.Since 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.
국내 특허공개 제10-2009-0001163호(2009. 01. 08)는 인간의 골수, 제대혈 또는 태반 조직으로부터 수득한 중간엽줄기세포를 혈청 배지에서 배양 후 무혈청 배지에서 계대 배양하고, 배양한 줄기세포에 저산소 배양의 물리적 자극을 가하여, 중간엽줄기세포로부터 bFGF 또는 TGF-β를 대량으로 생산하는 방법을 개시하고 있다.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-β from mesenchymal stem cells by applying physical stimulation of hypoxic culture to cells is disclosed.
유사하게, 국제공개 WO 2007/086637호(2007. 08. 02)에서는 포유동물의 지방세포에서 추출한 성체 줄기세포를 혈청 배지에서 배양 후 무혈청 배지에서 계대배양하고, 배양한 줄기세포에 저산소 배양, 자외선 조사, 영양분 결핍, 또는 기계적 마찰과 같은 물리적 자극을 가하고, 비타민 A, B, C, 또는 D를 배양액에 첨가하여 중간엽줄기세포로부터 bFGF, VEGF 또는 TGF-β를 대량으로 생산하는 방법을 개시하고 있다. Similarly, WO 2007/086637 (Aug. 02, 2007) 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. Disclosed a method for producing bFGF, VEGF or TGF-β 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.
즉, 종래의 방법에서는 줄기세포를 단순히 2차원 배양하거나, VEGF 등의 함량을 높이기 위해 저산소 조건에서 2차원 배양을 실시하고 있을 뿐으로, 무혈청 배지에서는 줄기세포가 안정적으로 유지되기 어렵다는 점을 고려할 때 줄기세포 배양액의 생리적 활성도가 저하되는 문제점이 있었다. In other words, in the conventional method, 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.
한편, 면역 질환은 포유류 면역계의 구성성분들이 포유류의 병리상태를 야기하거나, 매개하거나 또는 기타 공헌하는 질환으로서, 특히 염증성 질환은 전 세계적으로 주목을 하고 있는 질환으로 그 기저 질환을 치료할 수 있는 치료제가 시급한 질환이다. 염증은 일반적으로 외부 물질 또는 해로운 자극에 의한 숙주 침입에 대해 신체 조직의 국소화된 보호 반응이다. 염증의 원인은 박테리아, 바이러스 및 기생충과 같은 감염성 원인; 화상 또는 방사선 조사와 같은 물리적 원인; 독소, 약물 또는 산업적 제제와 같은 화학약품; 알레르기 및 자가면역 반응과 같은 면역반응, 또는 산화성 스트레스와 연관된 상태일 수 있다.On the other hand, immune diseases are diseases in which the components of the mammalian immune system cause, mediate, or contribute to the pathology of mammals. In particular, 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.
염증은 통증, 적화현상, 부기, 열 및 감염된 영역의 궁극적인 기능 손실을 그 특징으로 한다. 이들 증상은 면역계의 세포 사이에서 일어나는 일련의 복잡한 상호작용의 결과이다. 세포의 반응으로 인해 결과적으로 여러 그룹의 염증 매개자의 상호작용 네트워크가 생성된다: 단백질(예를 들면, 사이토카인, 효소(예를 들면 프로테아제, 퍼옥시다제), 주요 염기성 단백질, 점착 분자(ICAM, VCAM), 지질 매개자(예를 들면, 에이코사노이드, 프로스타글란딘, 류코트라이엔, 혈소판 활성화 인자(PAF)), 반응성 산소 종(예를 들면, 하이드로퍼옥사이드, 슈퍼옥사이드 음이온 O2-, 산화질소(NO) 등). 그러나 염증의 이들 매개자 중 대부분은 또한 정상적인 세포 활성의 조절자이다. 따라서 염증 반응의 결핍으로 인해 숙주가 제어되지 않으면서 손상(즉, 감염)되고, 또는 만성 염증으로 인해 부분적으로는 상기 언급된 매개자 중 여럿이 과다 생성됨으로써 매개되는 염증성 질환이 야기된다.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. hydroperoxide, superoxide anion O2-, nitric oxide (NO) However, most of these mediators of inflammation are also regulators of normal cellular activity, thus the host is compromised (ie, infected) uncontrolled due to lack of an inflammatory response, or in part due to chronic inflammation. Overproduction of several of the aforementioned mediators results in inflammatory diseases mediated.
또한, 면역 질환 중 하나인 자가면역 질환은 면역 체계가 그 자신의 기관을 공격하여 자발적인 반응을 일으키는 것을 특징으로 한다. 이러한 반응들은 T 림프구에 의한 자가항원(auto-antigen)의 인식에 기인하며, 이로 인하여 체액상(자가항체 생성) 및 세포상(림프구 및 대식세포 세포독성 활성 증가) 면역 반응이 유발된다. 자가면역 질환으로는 류마티스성 질환, 건선, 전신성 피부근염, 다발성 경화증, 홍반성 낭창, 또는 항원에 의한 면역반응 악화, 즉, 천식, 약물 또는 음식에 대한 알레르기 등이 있다. 이러한 질환들은 모두 제한성이고 만성인 질환들이며, 경우에 따라서는 치명적이고, 현재까지 상기 질환들을 치료할 수 있는 효과적인 치료 방법이 존재하지 않는 실정이다. 그러므로 당해 질환의 진행 중에 질환을 경감시키거나 완화시킬 수 있는 약물, 의약 또는 매체라면 환자의 건강을 위해서 중요한 해결 수단이 된다고 할 것이다.In addition, 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.
이에, 본 발명자들은 면역 질환 또는 염증성 질환 치료제 적용을 위한 임상 용도로 사용 가능한 줄기세포 배양액을 개발하기 위해 노력한 결과, 기존 2차원 배양에 따라 수득한 배양액을 비교하여 무혈청 배지에서 생체적합성 스캐폴드를 이용하여 3차원 배양에 따라 수득한 중간엽줄기세포 배양액이 면역반응을 유발하지 않고, 면역반응성을 증가시키는 역할을 하는 염증성 사이토카인인 TNF-α 및 IFN-γ의 분비량을 감소시키며, 면역조절인자인 PGE2를 고농도로 분비하는 것을 확인함으로써, 상기 방법에 따라 수득한 배양액을 면역 질환 또는 염증성 질환의 예방 또는 치료에 이용할 수 있음을 밝힘으로써, 본 발명을 완성하였다.Accordingly, 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. As a result, 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-α and IFN-γ 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.
본 발명의 목적은 면역 질환 또는 염증성 질환 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법을 제공하기 위한 것이다.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.
본 발명의 다른 목적은 상기 제조방법으로 제조된 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액을 제공하기 위한 것이다.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.
본 발명의 다른 목적은 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지를 유효성분으로 함유하는 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물을 제공하기 위한 것이다.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. .
본 발명의 다른 목적은 상기 제조방법으로 제조된 중간엽줄기세포 배양액을 유효성분으로 함유하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하기 위한 것이다.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.
본 발명의 다른 목적은 상기 제조방법으로 제조된 중간엽줄기세포 배양액을 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 예방 또는 개선방법을 제공하기 위한 것이다.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.
본 발명의 다른 목적은 상기 제조방법으로 제조된 중간엽줄기세포 배양액을 면역 질환 또는 염증성 질환에 걸린 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 치료방법을 제공하기 위한 것이다.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.
본 발명의 다른 목적은 면역 질환 또는 염증성 질환 예방 또는 치료용 조성물로 사용하기 위한, 상기 제조방법으로 제조된 중간엽줄기세포 배양액의 용도를 제공하기 위한 것이다.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.
본 발명의 다른 목적은 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물로 사용하기 위한 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지의 용도를 제공하기 위한 것이다.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. .
본 발명의 목적을 달성하기 위하여 본 발명은In order to achieve the object of the present invention
(a) 중간엽줄기세포를 수집하여 무혈청 배지에서 생체적합성 스캐폴드와 3차원 배양하는 단계; 및(a) collecting mesenchymal stem cells and culturing them three-dimensionally with a biocompatible scaffold in a serum-free medium; And
(b) 배양 배지를 수집하는 단계를 포함하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법을 제공한다.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.
또한, 본 발명은 상기 제조방법으로 제조된 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액을 제공한다.In addition, 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.
또한, 본 발명은 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지를 유효성분으로 함유하는 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물을 제공한다.In addition, 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.
또한, 본 발명은 상기 제조방법으로 제조된 중간엽줄기세포 배양액을 유효성분으로 함유하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.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.
또한, 본 발명은 상기 제조방법으로 제조된 중간엽줄기세포 배양액을 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 예방 또는 개선방법을 제공한다.In addition, 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.
또한, 본 발명은 상기 제조방법으로 제조된 중간엽줄기세포 배양액을 면역 질환 또는 염증성 질환에 걸린 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 치료방법을 제공한다.In another aspect, 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.
또한, 본 발명은 면역 질환 또는 염증성 질환 예방 또는 치료용 조성물로 사용하기 위한, 상기 제조방법으로 제조된 중간엽줄기세포 배양액의 용도를 제공한다.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.
또한, 본 발명은 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물로 사용하기 위한 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지의 용도를 제공한다.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.
본 발명의 방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻어지는 줄기세포 배양액은, 종래의 방법에 따라 2차원 배양하여 얻어지는 줄기세포 배양액보다 면역조절인자인 PGE2가 고농도로 분비되고, 면역반응 억제 및 염증성 사이토카인 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환을 예방 또는 치료하는 조성물로 유용하게 사용될 수 있다.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.
도 1은 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)을 농도별로 처리하고 말초혈액단핵세포 증식 억제율을 나타낸 도이다.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.
도 2는 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)을 농도별로 처리하고 말초혈액단핵세포에서 분비되는 TNF-α 분비 억제율을 나타낸 도이다.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-α secretion.
도 3은 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)을 농도별로 처리하고 말초혈액단핵세포에서 분비되는 IFN-γ 분비 억제율을 나타낸 도이다.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-γ secretion inhibition rate.
도 4는 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)에서의 PGE2(prostaglandin E2)의 분비량을 나타낸 도이다.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.
이하, 본 발명의 용어를 하기와 같이 정의한다.Hereinafter, the terms of the present invention are defined as follows.
본 명세서에서 "중간엽줄기세포(mesenchymal stem cells)"는 자기 증식이 가능하고 다분화능을 가지고 있으며, CD73+, CD90+, CD105+, CD14-, CD20-, CD34-, CD45-인 세포 표현형을 나타내는 세포를 의미하고, 골수, 지방조직, 제대혈 등에서 분리될 수 있으며, 이에 한정되지는 않는다."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.
본 명세서에서 "생리활성물질"은 세포나 신체의 기능에 영향을 미칠 수 있는 사이토카인, 세포성장인자, 면역조절인자 등을 통칭하여 나타낸다. 생리활성물질의 예로는 VEGF(vascular endothelial growth factor), EGF(epidermal growth factor), HGF(hepatocyte growth factor), TGF-beta(Tumor growth factor-beta), IGF(Insulin growth factor)등을 포함하며, 이에 한정되지는 않는다.As used herein, the term "physiologically active substance" refers collectively to cytokines, cell growth factors, and immunomodulators that may affect the function of cells or the body. Examples of 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.
본 명세서에서 "줄기세포 배양액"은 줄기세포를 배양한 세포배양 상등액을 지칭한다. 줄기세포 배양액은 줄기세포 배양 과정에서 세포로부터 분비되는 여러 가지 생리활성 물질을 함유하고 있다.As used herein, "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.
본 명세서에서 생체적합성 스캐폴드는 세포와 친화성을 가지며 이른바 세포 접착성인 표면을 지닌 재료로 만들어지며 세포를 3차원적으로 부착하고 배양시킬 수 있는 지지체를 의미한다. 천연 유래 지지체의 예로는 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스 등이 있고 이에 한정되지는 않는다. 합성 고분자의 예를 들면 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 폴리안하이드라이드 등이 있으며 이에 한정되지는 않는다.As used herein, 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. Examples of naturally occurring supports include, but are not limited to, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, and the like. Examples of the synthetic polymer include, but are not limited to, poly (alpha-hydroxy acid) series, poly (vinyl alcohol), polyanhydride, and the like.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은The present invention
(a) 중간엽줄기세포를 수집하여 무혈청 배지에서 생체적합성 스캐폴드와 3차원 배양하는 단계; 및(a) collecting mesenchymal stem cells and culturing them three-dimensionally with a biocompatible scaffold in a serum-free medium; And
(b) 배양 배지를 수집하는 단계를 포함하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법을 제공한다.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.
상기 단계 (a)에서 중간엽줄기세포는 지방, 골수, 간 및 근육으로 이루어진 군으로부터 선택되는 어느 하나로부터 분리된 것이 바람직하다.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.
상기 단계 (a)에서 중간엽줄기세포는 기질배지 및 증식배지에서 배양한 후 계대 배양한 것이 바람직하다.The mesenchymal stem cells in step (a) is preferably cultured in a substrate medium and proliferation medium and then subcultured.
상기 단계 (a)에서 3차원 배양은 3일 간격으로 배지를 교환하면서 3회 이상 줄기세포 배양액을 얻는 것이 바람직하다.In the step (a), the three-dimensional culture is preferably obtained at least three times the stem cell culture medium exchange medium at intervals of three days.
상기 단계 (a)에서 무혈청 배지에 bFGF(basic fibroblast growth factor) 또는 EGF(epidermal growth factor) 중 어느 하나 이상을 첨가하여 배양하는 것이 바람직하다.In 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.
상기 단계 (b)에서 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포 지지체로서 천연 또는 합성 고분자인 것이 바람직하고, 예를 들어 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스, 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 폴리안하이드라이드 등 일 수 있으나, 이에 한정되지 않는다.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.
상기 면역 질환은 베체트병, 다발성 근육염, 피부 근육염, 자가면역 혈구감소증, 자가면역 심근염, 아토피피부염, 알레르기, 천식, 일차성간경변, 피부근염, 굿파이처 증후군, 자가면역 뇌수막염, 쇼그렌 증후군, 전신 홍반성 루프스, 애디슨병, 원형 탈모증, 강직성 척수염, 자가면역성 간염, 자가면역성 이하선염, 크론병, 인슐린 의존성 당뇨병, 이영양성 수포성 표피박리증, 부고환염, 사구체 신염, 그레이브스병, 길랑바레 증후군, 하시모토병, 용혈성 빈혈, 다발성 경화증, 중증 근무력증, 심상천포창, 건선, 류마티스열, 류마티스 관절염, 유육종증, 피부 경화증, 척추관절증, 갑상선염, 혈관염, 백반증, 점액수종, 악성빈혈, 궤양성 대장염, 이식편대숙주질환 또는 비만일 수 있으나 이에 한정되지 않는다.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, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, graft versus host disease or obesity But it is not limited thereto.
상기 염증성 질환은 상기 염증성 질환은 위염, 장염, 신장염, 간염, 만성 폐쇄성 폐질환(COPD), 폐섬유증, 과민성 대장 증후군, 염증성 통증, 편두통, 두통, 허리 통증, 섬유근육통, 근막 질환, 바이러스 감염, 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, PGE 과다 증후군, 아테롬성 동맥 경화증, 통풍, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염 또는 급성 및 만성 염증성 질환일 수 있으나 이에 한정되지 않는다.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.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by an immune response and immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.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.
본 발명의 구체적인 실시예에서, 본 발명자들은 면역반응을 유발한 말초혈액단핵세포에 상기 3차원 배양방법에 따라 수득한 중간엽줄기세포 배양액을 처리한 결과, 상기 말초혈액단핵세포의 세포 증식이 억제되는 것을 확인하였다(도 1 참조).In a specific embodiment of the present invention, 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).
또한, 본 발명자들은 면역반응을 유발한 말초혈액단핵세포에 상기 3차원 배양방법에 따라 수득한 중간엽줄기세포 배양액을 처리한 결과, 상기 말초혈액단핵세포에서 TNF-α 및 IFN-γ 분비를 억제하는 것을 확인하였다(도 2 및 도 3 참조).In addition, 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-α and IFN-γ secretion from the peripheral blood mononuclear cells. It was confirmed that (see Fig. 2 and 3).
또한, 본 발명자들은 상기 3차원 배양방법에 따라 수득한 중간엽줄기세포 배양액에서 면역조절인자인 PGE2가 2차원 배양방법에 따라 수득한 중간엽줄기세포 배양액보다 현저하게 증가하는 것을 확인하였다(도 4 참조).In addition, 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).
따라서, 본 발명의 방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 줄기세포 배양액이 PGE2를 고농도 포함하고, 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수함을 확인함으로써, 본 발명은 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법에 유용하게 사용될 수 있다.Therefore, 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 By identifying the present invention, 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.
본 발명에 따른 중간엽줄기세포의 배양은 다음 과정에 따라 실시될 수 있으며, 세포배양에 사용되는 배지는 이에 한정되지 않는다.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.
(1) 중간엽줄기세포의 배양(1) Cultivation of mesenchymal stem cells
해당 조직에서 얻은 중간엽줄기세포를 기질배지에 현탁시켜 10,000~40,000 cells/㎠의 농도로 배양용기에 접종한 후 배양한다. 기질배지는 10% 우혈청이 포함되어 있는 DMEM 또는 DMEM/F12(Dulbecco's Modified Eagle Medium/Ham's F-12 Nutrient Broth) 배지로, 약 24시간 배양한다.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 / ㎠ 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.
(2) 증식배지(expansion media)에서의 배양(2) Cultivation in expansion media
기질배지를 제거한 후, 증식배지에서 배양하여 부착성 세포를 증식시킨다. 증식배지는 10% 우혈청, 0.1~100 ng/㎖ 농도의 EGF(epidermal growth factor) 및/또는 0.1~100 ng/㎖ 농도의 bFGF(basic fibroblast growth factor)를 포함하는 DMEM 또는 DMEM/F12로, 부착성인 중간엽줄기세포를 신속하게 증식시켜 세포 양을 단기간에 대량으로 증가시키는 작용을 한다.After removing the substrate medium, the cells are grown in the growth medium to proliferate adherent cells. 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.
(3) 계대 배양(3) subculture
세포가 배양용기 바닥의 80 내지 90%를 채우면 증식배지를 제거하고 트립신 처리를 통해 세포를 배양용기에서 떼어낸다. 계대배양을 위해서는 세포를 1:3~1:4로 희석하여 새로운 배양용기에서 증식배지와 함께 배양한다. 이와 같은 방법으로 추가적인 계대배양이 가능하다.When cells fill 80 to 90% of the bottom of the culture vessel, the growth medium is removed and cells are removed from the culture vessel by trypsin treatment. For subculture, 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.
(4) 생체적합성 스캐폴드와 함께 배양(4) incubation with biocompatible scaffolds
배양한 세포는 PBS로 3회 이상 세척하여 FBS를 제거해주고, 무혈청 배지에서 생체적합성 스캐폴드에 부착된 형태로 배양한다. 스캐폴드 내에서의 세포배양은 통상적인 세포배양 용기를 필요로 하지 않으므로, 무균 바틀 또는 culture bag에서 다량으로 배양이 가능하여 보다 낮은 비용으로 편리하게 배양할 수 있는 장점이 있다. 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.
또한, 본 발명은 본 발명의 제조방법에 따라 제조된 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액을 함유하는 조성물을 제공한다.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.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by an immune response and immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.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.
본 발명의 제조방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻은 중간엽줄기세포 배양액은 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환의 예방 또는 치료용 조성물로 유용하게 사용될 수 있다.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.
또한, 본 발명은 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지를 유효성분으로 함유하는 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물을 제공한다.In addition, 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.
상기 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포 지지체로서 천연 또는 합성 고분자인 것이 바람직하고, 예를 들어 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스, 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 폴리안하이드라이드 등 일 수 있으나, 이에 한정되지 않는다.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.
상기 무혈청 배지는 당업계에 알려진 기본 배지를 제한 없이 사용할 수 있으며, 다만 혈청이 첨가되지 않은 것을 사용한다. 기본 배지는 인위적으로 합성하여 제조할 수 있으며, 상업적으로 제조된 배지를 사용할 수도 있다. 상업적으로 제조되는 배지의 예를 들면, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium) 및 Isocove's Modified Dulbecco's Medium 등이 있으나, 이에 한정되는 것은 아니며, 바람직하게는 DMEM 또는 DMEM/F12 를 사용할 수 있다.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. Examples of 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, α-MEM (α-Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium, but are not limited thereto. Preferably, DMEM or DMEM / F12 may be used.
상기 무혈청 배지에 bFGF는 0.5 ~ 2 ng/㎖ 첨가된 것이 바람직하고, 0.7 ~ 1.5 ng/㎖ 첨가된 것이 보다 바람직하고, 1 ng/㎖ 첨가하는 것이 보다 더 바람직하다.In the serum-free medium, 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.
상기 무혈청 배지에 EGF는 3 ~ 7 ng/㎖ 첨가된 것이 바람직하고, 4 ~ 6 ng/㎖ 첨가된 것이 보다 바람직하고, 5 ng/㎖ 첨가된 것이 보다 더 바람직하다.In the serum-free medium, EGF is preferably added 3-7 ng / ml, more preferably 4-6 ng / ml, and even more preferably 5 ng / ml.
또한, 본 발명은 본 발명의 제조방법에 따라 제조된 중간엽줄기세포 배양액을 유효성분으로 함유하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.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.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by an immune response and immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.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.
본 발명의 제조방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻은 중간엽줄기세포 배양액은 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다.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.
또한, 본 발명은 본 발명의 제조방법에 따라 제조된 중간엽줄기세포 배양액을 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 예방 또는 개선방법을 제공한다.In addition, 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.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by an immune response and immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.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.
본 발명의 제조방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻은 중간엽줄기세포 배양액은 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수하므로, 개체에 투여하여 면역 질환 또는 염증성 질환을 예방 또는 개선하는 방법으로 유용하게 사용될 수 있다.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.
또한, 본 발명은 본 발명의 제조방법에 따라 제조된 중간엽줄기세포 배양액을 면역 질환 또는 염증성 질환에 걸린 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 치료방법을 제공한다.In addition, 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.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by an immune response and immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.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.
본 발명의 제조방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻은 중간엽줄기세포 배양액은 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환에 걸린 개체에 투여하여 면역 질환 또는 염증성 질환을 치료하는 방법으로 유용하게 사용될 수 있다.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.
또한, 본 발명은 면역 질환 또는 염증성 질환 예방 또는 치료용 조성물로 사용하기 위한, 본 발명의 제조방법에 따라 제조된 중간엽줄기세포 배양액의 용도를 제공한다.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.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by an immune response and immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture is preferably, but not limited to, reducing immune rejection and inflammatory responses during xenotransplantation.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.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.
본 발명의 제조방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻은 중간엽줄기세포 배양액은 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환 예방 또는 치료용 조성물로 유용하게 사용될 수 있다.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.
또한, 본 발명은 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물로 사용하기 위한 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지의 용도를 제공한다.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.
상기 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포 지지체로서 천연 또는 합성 고분자인 것이 바람직하고, 예를 들어 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스, 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 폴리안하이드라이드 등 일 수 있으나, 이에 한정되지 않는다.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.
상기 무혈청 배지는 당업계에 알려진 기본 배지를 제한 없이 사용할 수 있으며, 다만 혈청이 첨가되지 않은 것을 사용한다. 기본 배지는 인위적으로 합성하여 제조할 수 있으며, 상업적으로 제조된 배지를 사용할 수도 있다. 상업적으로 제조되는 배지의 예를 들면, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium) 및 Isocove's Modified Dulbecco's Medium 등이 있으나, 이에 한정되는 것은 아니며, 바람직하게는 DMEM 또는 DMEM/F12 를 사용할 수 있다.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. Examples of 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, α-MEM (α-Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium, but are not limited thereto. Preferably, DMEM or DMEM / F12 may be used.
상기 무혈청 배지에 bFGF는 0.5 ~ 2 ng/㎖ 첨가된 것이 바람직하고, 0.7 ~ 1.5 ng/㎖ 첨가된 것이 보다 바람직하고, 1 ng/㎖ 첨가하는 것이 보다 더 바람직하다.In the serum-free medium, 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.
상기 무혈청 배지에 EGF는 3 ~ 7 ng/㎖ 첨가된 것이 바람직하고, 4 ~ 6 ng/㎖ 첨가된 것이 보다 바람직하고, 5 ng/㎖ 첨가된 것이 보다 더 바람직하다.In the serum-free medium, EGF is preferably added 3-7 ng / ml, more preferably 4-6 ng / ml, and even more preferably 5 ng / ml.
이하 본 발명을 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다.However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.
<실시예 1> 인간 지방유래 중간엽줄기세포의 배양 방법Example 1 Culture Method of Human Adipose-derived Mesenchymal Stem Cells
지방 조직은 보통 지방 흡입술로 얻을 수 있지만, 이에 한정되지는 않는다. 지방 흡입에 의해 얻어진 지방 조직으로부터 다음과 같이 지방유래 중간엽줄기세포를 분리하였다: 혈액을 제거하기 위해 지방 조직을 같은 부피의 KRB 용액으로 3~4회 세척하였다. 그 다음, 지방 조직과 같은 부피의 콜라게나제 용액을 넣어 37℃ 수욕에서 반응시켰다. 이를 원심분리용 튜브에 옮겨 넣고 20, 1500 rpm에서 10분 동안 원심분리하였다. 원심분리 후, 상층액인 지방층을 제거하고 아래층인 콜라게나제 용액을 흔들리지 않도록 조심해서 분리하였다. 기질배지를 넣어 현탁시킨 후, 20℃, 1500 rpm에서 5분 동안 원심분리하였다. 이때, 아래에 가라앉는 것이 스트로마-혈관 분획으로, 상층액을 제거하였다. 스트로마-혈관 분획을 기질배지에 현탁시켜 배양용기에 접종하고, 37℃, 5% CO2 인큐베이터에서 24시간 동안 배양하였다. 배양액 제거 후 인산염 완충용액으로 세척하고, 기질배지(10% FBS가 함유된 DMEM/F12배지), 또는 기질배지에 염기성 섬유아세포 성장인자(bFGF)가 1 ng/㎖ 농도로 포함된 증식배지, 또는 기질배지에 표피세포 성장인자(EGF)가 5 ng/㎖ 농도로 포함된 배지를 이용하여 증식시켰다. 지방유래 중간엽줄기세포가 배양용기의 80~90% 정도로 자라면 트립신 처리하여 단일 세포로 분리하여 수득하였다. 얻어진 세포를 증식배지로 1:3~1:4로 희석하여 계대 배양을 실시하였다.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. After the substrate medium was suspended and centrifuged for 5 minutes at 20 ℃, 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. After removal of the culture solution, 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 / ㎖. 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.
<실시예 2> 인간 지방유래 중간엽줄기세포를 2차원 배양하는 방법Example 2 A Two-Dimensional Culture of Human Adipose Mesenchymal Stem Cells
트립신 처리하여 단일세포로 얻어진 줄기세포를 DMEM/F12 배지로 3회 세척하여 FBS를 제거하였다. 1.3 × 107개/㎖ 줄기세포에 DMEM/F12 용액을 첨가하여 세포를 현탁하여 접종하였다. 그 다음, 1 ng/㎖ 염기성 섬유아세포 성장인자 및 5 ng/㎖ 표피세포 성장인자가 포함된 DMEM/F12 용액을 세포 4.0 × 107개 당 500 ㎖가 되도록 첨가하였다. 72시간 후 세포 배양액을 회수하고 새 배지를 첨가하는 방법으로 2~11회 더 세포 배양액을 회수하여 냉장 보관하였다.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. Then, 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.
<실시예 3> 인간 지방유래 중간엽줄기세포를 피브린 글루와 3차원 배양하는 방법Example 3 Method of 3-D Culture of Human Adipose-derived Mesenchymal Stem Cells with Fibrin Glue
트립신 처리하여 단일세포로 얻어진 줄기세포를 DMEM/F12 배지로 3회 세척하여 FBS를 제거하였다. 1.3 × 107개/㎖ 줄기세포에 트롬빈 용액이 포함된 DMEM/F12 용액을 첨가하여 세포를 현탁하였다. 듀얼시린지를 이용하여 세포현탁액과 피브리노겐 희석액이 혼합되도록 한 후 바틀 또는 culture bag에서 3차원배양하였다. 세포배양배지는 1 ng/㎖ 염기성 섬유아세포 성장인자 및 5ng/㎖ 표피세포 성장인자가 포함된 DMEM/F12 용액을 사용하였다. 72시간 후 세포 배양액을 회수하고 새 배지를 첨가하는 방법으로 2~11회 더 세포 배양액을 회수하여 냉장 보관하였다.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.
<실험예 1> 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액의 면역반응Experimental Example 1 Immune Response of Mesenchymal Stem Cell Cultures Obtained by Three-Dimensional Culture Method 억제 확인Suppress Confirmation
상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포 배양액의 면역반응 억제능을 확인하기 위하여 EdU 세포 증식 분석법을 수행하였다.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.
구체적으로, 조직 적합성 항원 (Human Leukocyte Antigen; HLA)이 다른 공여자에서 얻은 말초혈액단핵세포 (peripheral Blood Mononuclear Cell; PBMC) 5 × 105개를 24-웰 플레이트에 첨가하였다. 양성대조군으로는 말초혈액단핵세포에 마이토겐 (mitogen)인 PHA (phyto-hemagglutinin) 1.0 ㎍/㎖를 첨가하여 말초혈액단핵세포의 면역반응을 유발하였다. 그 다음, 상기 <실시예 2>의 2차원 배양방법으로 수득한 중간엽줄기세포 배양액 또는 <실시예 3>의 3차원 배양방법으로 수득한 중간엽줄기세포 배양액 두 세트(Lot#1 및 Lot#2)를 10% 또는 20%의 농도로 처리하여 반응 시작 후 3일째 상등액을 수집하여 말초혈액단핵세포의 증식능력을 FACS 기법으로 측정하였다.Specifically, 5 x 10 5 Peripheral Blood Mononuclear Cells (PBMCs) from which tissue compatible antigens (Human Leukocyte Antigens (HLA)) were obtained from other donors were added to 24-well plates. As a positive control group, 1.0 μg / ml of mitogen PHA (phyto-hemagglutinin) was added to peripheral blood mononuclear cells to induce an immune response of peripheral blood mononuclear cells. Next, two sets of mesenchymal stem cell culture medium obtained by the two-dimensional culture method of <Example 2> or mesenchymal stem cell culture solution obtained by the three-dimensional culture method of <Example 3> (Lot # 1 and Lot # 2) was treated at a concentration of 10% or 20% to collect the supernatant 3 days after the start of the reaction to determine the proliferation capacity of peripheral blood mononuclear cells by FACS technique.
그 결과, 도 1에 나타낸 바와 같이, 상기 <실시예 2>의 2차원 배양방법에 의해 수득한 중간엽줄기세포 배양액을 처리한 군에서는 세포 증식이 억제되지 않는 반면, 상기 <실시예 3>의 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액을 처리한 군에서는 세포 증식이 억제되므로, 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액이 면역반응을 유의적으로 억제함을 확인하였다(도 1).As a result, as shown in FIG. 1, the cell proliferation was not inhibited in the group treated with the mesenchymal stem cell culture solution obtained by the two-dimensional culture method of <Example 2>, whereas in <Example 3> Cell proliferation was inhibited in the group treated with the mesenchymal stem cell culture obtained by the three-dimensional culture method, it was confirmed that the mesenchymal stem cell culture obtained by the three-dimensional culture method significantly inhibited the immune response ( 1).
<< 실험예Experimental Example 2> 3차원 배양방법에 의해  2> By 3D culture method 수득된Obtained 중간엽줄기세포Mesenchymal stem cells 배양액의 염증성 사이토카인 분비 억제 확인 Inhibition of inflammatory cytokine secretion in culture
상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포 배양액의 염증성 사이토카인 분비 억제 여부를 확인하기 위하여, ELISA 분석법을 수행하였다.In order to confirm whether the mesenchymal stem cell culture medium obtained in <Example 2> and <Example 3> inhibits the inflammatory cytokine secretion, ELISA analysis was performed.
구체적으로, 상기 <실험예 1>과 같이 말초혈액단핵세포 5 × 105개를 24-웰 플레이트에 넣고 PHA로 활성화시켜 면역반응을 유발하였다. 그 다음, 상기 <실시예 2>의 2차원 배양방법으로 수득한 중간엽줄기세포 배양액 또는 <실시예 3>의 3차원 배양방법으로 수득한 중간엽줄기세포 배양액 두 세트(Lot#1 및 Lot#2)를 10% 또는 20%의 농도로 첨가하였다. 반응 3일째 상등액을 수집하여 TNF-α와 IFN-γ의 분비량을 ELISA 분석으로 측정하였다. Specifically, 5 × 10 5 peripheral blood mononuclear cells as in <Experimental Example 1> were put into a 24-well plate and activated with PHA to induce an immune response. Next, two sets of mesenchymal stem cell culture medium obtained by the two-dimensional culture method of <Example 2> or mesenchymal stem cell culture solution obtained by the three-dimensional culture method of <Example 3> (Lot # 1 and Lot # 2) was added at a concentration of 10% or 20%. On day 3 of the reaction, the supernatant was collected and the secretion amount of TNF-α and IFN-γ was measured by ELISA analysis.
그 결과, 도 2 및 도 3에 나타낸 바와 같이, 상기 <실시예 3>의 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액을 처리한 군에서는 TNF-α와 IFN-γ의 분비량이 현저히 감소함을 확인하였다(도 2 및 도 3).As a result, as shown in Figures 2 and 3, in the group treated with the mesenchymal stem cell culture obtained by the three-dimensional culture method of <Example 3> the secretion amount of TNF-α and IFN-γ is significantly reduced It was confirmed that (Fig. 2 and 3).
따라서, 상기 <실험예 1> 및 <실험예 2>를 통해 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액은 면역반응을 유발하지 않으며, 면역반응성을 증가시키는 역할을 하는 염증성 사이토카인의 분비량을 감소시켜 면역 질환 또는 염증성 질환을 완화시켜 치유되도록 도울 수 있음을 확인하였다.Therefore, 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.
<실험예 3> 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액의 면역조절인자 확인Experimental Example 3 Identification of Immunomodulators in Mesenchymal Stem Cell Cultures Obtained by Three-Dimensional Culture Method
상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포 배양액에서 면역조절인자인 prostaglandin E2(PGE2)의 분비 정도를 확인하기 위하여, 상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포액을 ELISA 분석을 수행하고, 450 nm에서 흡광도를 측정하였다.In order to check the secretion level of the immunoregulatory prostaglandin E2 (PGE2) in the mesenchymal stem cell culture medium obtained in the above <Example 2> and <Example 3>, the <Example 2> and <Example 3> The mesenchymal stem cell fluid obtained at was subjected to ELISA analysis and absorbance at 450 nm was measured.
그 결과, 도 4에 나타낸 바와 같이, 상기 <실시예 3>의 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액(Lot #1 및 Lot #2)에서는 PGE2의 분비량이 현저히 증가함을 확인하였다(도 4).As a result, as shown in Figure 4, it was confirmed that the secretion amount of PGE2 significantly increased in the mesenchymal stem cell culture solution (Lot # 1 and Lot # 2) obtained by the three-dimensional culture method of <Example 3>. (FIG. 4).
따라서, 상기 <실험예 1> 내지 <실험예 3>을 통해 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액은 면역반응을 유발하지 않으며, 면역반응성을 증가시키는 역할을 하는 염증성 사이토카인의 분비량을 감소시키고, 면역조절인자인 PGE2를 고농도로 분비하여 면역 반응성을 증가시켜 면역 질환 또는 염증성 질환을 완화시켜 치유되도록 도울 수 있음을 확인하였다.Therefore, 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.
본 발명은 3차원 배양방법에 의해 유의적인 면역억제 및 항염기능을 보유하는 중간엽줄기세포 배양액 및 이의 제조방법에 관한 것으로, 본 발명에 따라 제조된 중간엽줄기세포 배양액을 면역 질환 또는 염증성 질환 예방 또는 치료제의 유효성분으로 유용하게 이용 가능하다.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.

Claims (24)

  1. (a) 중간엽줄기세포를 수집하여 무혈청 배지에서 생체적합성 스캐폴드와 3차원 배양하는 단계; 및(a) collecting mesenchymal stem cells and culturing them three-dimensionally with a biocompatible scaffold in a serum-free medium; And
    (b) 배양 배지를 수집하는 단계를 포함하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법.(B) a method for producing a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases comprising the step of collecting the culture medium.
  2. 제 1항에 있어서, 상기 (a)에서 중간엽줄기세포는 지방, 골수, 간 및 근육으로 이루어진 군으로부터 선택되는 어느 하나로부터 분리된 것을 특징으로 하는 방법.[Claim 2] The method of claim 1, wherein the mesenchymal stem cells in (a) are isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.
  3. 제 1항에 있어서, 상기 (a)에서 중간엽줄기세포는 기질배지 및 증식배지에서 배양한 후 계대 배양한 것을 특징으로 하는 방법.[Claim 2] The method of claim 1, wherein the mesenchymal stem cells in (a) are cultured in a matrix medium and a growth medium and then passaged.
  4. 제 1항에 있어서, 상기 (a)에서 3차원 배양은 3일 간격으로 배지를 교환하면서 3회 이상 줄기세포 배양액을 얻는 것을 특징으로 하는 방법.The method of claim 1, wherein the three-dimensional culture in (a) is characterized in that the stem cell culture solution is obtained at least three times while the medium is exchanged every three days.
  5. 제 1항에 있어서, 상기 (a)에서 무혈청 배지에 bFGF(basic fibroblast growth factor) 또는 EGF(epidermal growth factor) 중 어느 하나 이상을 첨가하여 배양하는 것을 특징으로 하는 방법.The method of claim 1, wherein (a) in the serum-free medium is cultured by adding any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor).
  6. 제 1항에 있어서, 상기 (b)에서 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포지지체로서 천연 또는 합성 고분자인 것을 특징으로 하는 방법.2. The method of claim 1 wherein the biocompatible scaffold in (b) is a cell support having a cell adhesive surface, which is a natural or synthetic polymer.
  7. 제 6항에 있어서, 상기 생체적합성 스캐폴드는 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스, 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 및 폴리안하이드라이드로 구성된 군으로부터 선택된 어느 하나인 것을 특징으로 하는 방법.7. The biocompatible scaffold of claim 6, wherein the biocompatible scaffold is from the group consisting of alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) series, poly (vinyl alcohol), and polyanhydride Any one selected.
  8. 제 1항에 있어서, 상기 면역 질환은 베체트병, 다발성 근육염, 피부 근육염, 자가면역 혈구감소증, 자가면역 심근염, 아토피피부염, 알레르기, 천식, 일차성간경변, 피부근염, 굿파이처 증후군, 자가면역 뇌수막염, 쇼그렌 증후군, 전신 홍반성 루프스, 애디슨병, 원형 탈모증, 강직성 척수염, 자가면역성 간염, 자가면역성 이하선염, 크론병, 인슐린 의존성 당뇨병, 이영양성 수포성 표피박리증, 부고환염, 사구체 신염, 그레이브스병, 길랑바레 증후군, 하시모토병, 용혈성 빈혈, 다발성 경화증, 중증 근무력증, 심상천포창, 건선, 류마티스열, 류마티스 관절염, 유육종증, 피부 경화증, 척추관절증, 갑상선염, 혈관염, 백반증, 점액수종, 악성빈혈, 궤양성 대장염, 이식편대숙주질환 및 비만으로 이루어진 군으로부터 선택되는 어느 하나인 것을 특징으로 하는 방법.The method of claim 1, wherein the immune disease is 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 lupus erythematosus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, dystrophic bullous 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, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, graft Characterized in that it is any one selected from the group consisting of host disease and obesity Way.
  9. 제 1항에 있어서, 상기 염증성 질환은 위염, 장염, 신장염, 간염, 만성 폐쇄성 폐질환(COPD), 폐섬유증, 과민성 대장 증후군, 염증성 통증, 편두통, 두통, 허리 통증, 섬유근육통, 근막 질환, 바이러스 감염, 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, PGE 과다 증후군, 아테롬성 동맥 경화증, 통풍, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염 및 급성 및 만성 염증성 질환으로 이루어진 군으로부터 선택되는 어느 하나인 것을 특징으로 하는 방법.The method of claim 1, wherein the inflammatory disease is gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascia disease, virus Infections, bacterial infections, fungal infections, burns, surgical or dental surgery wounds, PGE hyperplasia, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iris, scleritis, uveitis and acute and chronic inflammatory diseases It is any one selected from the group consisting of.
  10. 제 1항에 있어서, 상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것을 특징으로 하는 방법.The method of claim 1, wherein the mesenchymal stem cell culture is characterized in that the suppression of the immune response and secretion of TNF-α or IFN-γ by the immune response.
  11. 제 1항에 있어서, 상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것을 특징으로 하는 방법.The method of claim 1, wherein the mesenchymal stem cell culture is characterized in that for reducing xenograft immune rejection and inflammatory response.
  12. 제 1항에 있어서, 상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것을 특징으로 하는 방법.The method of claim 1, wherein the mesenchymal stem cell culture is characterized in that it comprises PGE2 (prostaglandin E2).
  13. 제 1항의 방법으로 제조된 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액을 함유하는 조성물.A composition containing mesenchymal stem cell culture for the prophylaxis or treatment of immune diseases or inflammatory diseases prepared by the method of claim 1.
  14. 제 13항에 있어서, 상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ의 분비를 억제하는 것을 특징으로 하는 중간엽줄기세포 배양액을 함유하는 조성물.The composition of claim 13, wherein the mesenchymal stem cell culture solution contains a mesenchymal stem cell culture solution, wherein the mesenchymal stem cell culture solution suppresses the secretion of TNF-α or IFN-γ by an immune response.
  15. 제 13항에 있어서, 상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것을 특징으로 하는 중간엽줄기세포 배양액을 함유하는 조성물.The composition of claim 13, wherein the mesenchymal stem cell culture medium contains a mesenchymal stem cell culture medium, wherein the mesenchymal stem cell culture medium reduces immune rejection and inflammatory responses.
  16. 제 13항에 있어서, 상기 중간엽줄기세포 배양액은 PGE2를 포함하는 것을 특징으로 중간엽줄기세포 배양액을 함유하는 조성물.The composition containing mesenchymal stem cell culture according to claim 13, wherein the mesenchymal stem cell culture comprises PGE2.
  17. 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지를 유효성분으로 함유하는 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물.A biocompatible scaffold and a composition for producing mesenchymal stem cell culture for the treatment of immune diseases or inflammatory diseases containing a serum-free medium comprising any one or more of bEGF or EGF as an active ingredient.
  18. 제 17항에 있어서, 상기 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포지지체로서 천연 또는 합성 고분자인 것을 특징으로 하는 중간엽줄기세포 배양액 제조용 조성물.18. The composition of claim 17, wherein the biocompatible scaffold is a cell support having a cell adhesive surface, which is a natural or synthetic polymer.
  19. 제 17항에 있어서, 상기 bFGF는 0.5 ~ 2 ng/㎖, 상기 EGF는 3 ~ 7 ng/㎖ 첨가된 것을 특징으로 하는 중간엽줄기세포 배양액 제조용 조성물.18. The composition of claim 17, wherein the bFGF is 0.5-2 ng / ml and the EGF is 3-7 ng / ml.
  20. 제 1항의 방법으로 제조된 중간엽줄기세포 배양액을 유효성분으로 함유하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases comprising the mesenchymal stem cell culture prepared by the method of claim 1 as an active ingredient.
  21. 제 1항의 방법으로 제조된 중간엽줄기세포 배양액을 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 예방 또는 개선방법.A method for preventing or ameliorating an autoimmune disease or inflammatory disease comprising administering to the subject a mesenchymal stem cell culture prepared by the method of claim 1.
  22. 제 1항의 방법으로 제조된 중간엽줄기세포 배양액을 면역 질환 또는 염증성 질환에 걸린 개체에 투여하는 단계를 포함하는 면역 질환 또는 염증성 질환 치료방법.A method for treating an autoimmune disease or inflammatory disease comprising administering a mesenchymal stem cell culture prepared by the method of claim 1 to an individual suffering from an autoimmune disease or an inflammatory disease.
  23. 면역 질환 또는 염증성 질환 예방 또는 치료용 약학적 조성물로 사용하기 위한, 제 1항의 방법으로 제조된 중간엽줄기세포 배양액의 용도.Pharmaceutical for preventing or treating autoimmune or inflammatory diseases Use of mesenchymal stem cell culture prepared by the method of claim 1 for use as a composition.
  24. 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물로 사용하기 위한 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지의 용도.Use of a biocompatible scaffold and a serum-free medium comprising any one or more of bEGF or EGF for use as a composition for preparing mesenchymal stem cell culture for the treatment of immune diseases or inflammatory diseases.
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