KR20200000415A - Mesenchymal stem cell culture media for preventing or treating immune disease or inflammatory disease and a method for preparing the same - Google Patents

Abstract

The present invention relates to mesenchymal stem cell culture media for preventing or treating immune diseases or inflammatory diseases, and a method for preparing the same. The stem cell culture media, obtained by culturing mesenchymal stem cells in a three-dimensional culture by using a biocompatible scaffold in a serum-free medium, secretes a higher concentration of PGE2 as an immunoregulatory factor than the same of stem cell culture media obtained by a two-dimensional culture according to a conventional method, and has an excellent inhibition effect against an immune response and an inflammatory cytokine secretion. Thus, the stem cell culture media can be usefully used as a composition for preventing or treating immune diseases or inflammatory diseases.

Description

Mesenchymal stem cell culture media for preventing or treating immune disease or inflammatory disease and a method for preparing the same

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.

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 self-proliferative capacity, osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon-ligament fibroblasts, adipocytes, etc. Can differentiate into

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Still another object of the present invention is to provide a composition for preparing 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. will be.

In order to achieve the object of the present invention

(a) collecting mesenchymal stem cells and culturing them three-dimensionally with a biocompatible scaffold in a serum-free medium; And

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 also provides a mesenchymal stem cell culture for the prevention or treatment of immune diseases or inflammatory diseases prepared by the above method.

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.

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 by 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 peripheral blood mononuclear cell proliferation inhibition rate The figure shown.
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.
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 IFN-γ secretion inhibition rate.
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.

Hereinafter, the terms of the present invention are defined as follows.

"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.

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.

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.

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.

Hereinafter, the present invention will be described in more detail.

The present invention

(a) collecting mesenchymal stem cells and culturing them three-dimensionally with a biocompatible scaffold in a serum-free medium; And

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.

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.

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.

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, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, graft versus host disease or obesity But it is not limited thereto.

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, It can be a bacterial infection, fungal infection, burns, wounds from surgical or dental surgery, excessive PGE syndrome, 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-α or IFN-γ 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.

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 was confirmed that (see FIG. 1).

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).

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).

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.

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) Cultivation of mesenchymal stem cells

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) Cultivation in expansion media

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) subculture

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) incubation with biocompatible scaffolds

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.

In addition, the present invention 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-α or IFN-γ 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.

According to the preparation method of the present invention, the mesenchymal stem cells are cultured three-dimensionally using a biocompatible scaffold in a serum-free medium, and the stem cell culture medium has excellent immune response and secretion effect of inflammatory cytokines. Or it may be usefully used as a composition for the prevention or treatment of inflammatory diseases.

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.

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.

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.

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.

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.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

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.

Example 1 Culture Method of Human Adipose-derived Mesenchymal Stem Cells

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 cells were washed with phosphate buffer, substrate medium (DMEM / F12 medium containing 10% FBS), or 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 into single cells by trypsin treatment. The obtained cells were diluted 1: 3 to 1: 4 with proliferation medium to carry out passage culture.

Example 2 A Two-Dimensional Culture of Human Adipose Mesenchymal Stem Cells

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.

Example 3 Method of 3-D Culture of Human Adipose-derived Mesenchymal Stem Cells with Fibrin Glue

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 a dual syringe, and then three-dimensionally cultured in a bottle or culture bag. 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.

Experimental Example 1 Immune Response of Mesenchymal Stem Cell Cultures Obtained by Three-Dimensional Culture Method Suppress Confirmation

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.

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.

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).

Experimental Example 2 Inhibition of Inflammatory Cytokine Secretion in Mesenchymal Stem Cell Cultures Obtained by Three-Dimensional Culture Method

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.

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.

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).

Therefore, the mesenchymal stem cell culture 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.

Experimental Example 3 Identification of Immunomodulators in Mesenchymal Stem Cell Cultures Obtained by Three-Dimensional Culture Method

In order to confirm the secretion level of the immunomodulatory prostaglandin E2 (PGE2) in the mesenchymal stem cell culture medium obtained in the <Example 2> and <Example 3>, the <Example 2> and <Example 3> The mesenchymal stem cell fluid obtained at was subjected to ELISA analysis, and the absorbance was measured at 450 nm.

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).

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.

Claims (6)

(a) collecting mesenchymal stem cells and incubating three-dimensionally with a biocompatible scaffold in a serum-free medium to which at least one of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) is added; And
(b) collecting the culture medium to obtain mesenchymal stem cell culture comprising PGE ₂ (prostaglandin E2),
Wherein the mesenchymal stem cell culture is for the prevention or treatment of immune diseases that suppress the secretion of TNF-α or IFN-γ by immune response and immune response,
Immune diseases include Behcet's disease, multiple myositis, cutaneous myositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergies, asthma, primary cirrhosis, dermatitis, Goodfiction syndrome, autoimmune meningitis, Sjogren's syndrome, systemic redness 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 ulcer, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and graft-versus-host disease Any one selected from, Method for producing mesenchymal stem cell culture.
[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.
[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.
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.
The method of claim 1, wherein the biocompatible scaffold in (a) is a cell support having a cell adhesive surface, characterized in that the natural or synthetic polymer.
6. The biocompatible scaffold of claim 5, 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.

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