KR20220139844A - Mesenchymal stem cell culture media for preventing or treating immune 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 manufacturing 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 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 or inflammatory diseases having significant immunosuppressive and anti-inflammatory functions by a three-dimensional culture method and a method for preparing the same.

It has been found that several types of progenitors exist in the human bone marrow, and among them, progenitor cells showing pluripotency are called mesenchymal stem cells. Mesenchymal stem cells are known to exist in most organs of the body, such as fat, liver, and muscle, as well as bone marrow. Mesenchymal stem cells have self-renewal ability and include osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon ligament fibroblasts, adipocytes, etc. can be differentiated into

In addition, mesenchymal stem cells are known to secrete various growth factors and physiologically active substances during the culture process. Therefore, the stem cell culture medium can be used for clinical purposes such as pharmaceuticals or cosmetic compositions.

However, bovine serum is usually used for culturing mesenchymal stem cells, and a culture solution containing bovine serum is inappropriate for clinical application. That is, since the use of animal-derived proteins such as bovine serum is limited for clinical application of cell culture, it is preferable to exclude bovine serum from culturing stem cells.

Since bovine serum protein is absolutely necessary for the proliferation and maintenance of stem cells, a method for culturing mesenchymal stem cells in a serum-free medium while obtaining a culture medium containing a high concentration of physiologically active substances is required.

Korean Patent Publication No. 10-2009-0001163 (Jan. 08, 2009) discloses that mesenchymal stem cells obtained from human bone marrow, umbilical cord blood, or placental tissue are cultured in a serum medium, then subcultured in a serum-free medium, and the cultured stem Disclosed is a method for mass production of bFGF or TGF-β from mesenchymal stem cells by applying physical stimulation of hypoxic culture to cells.

Similarly, in International Publication No. WO 2007/086637 (2007. 08. 02), adult stem cells extracted from adipocytes of mammals are cultured in a serum medium, subcultured in a serum-free medium, and the cultured stem cells are hypoxic cultured, Disclosed is a method for mass production of bFGF, VEGF or TGF-β from mesenchymal stem cells by applying a physical stimulus such as UV irradiation, nutrient deficiency, or mechanical friction, and adding vitamins A, B, C, or D to the culture medium are doing

That is, in the conventional method, stem cells are simply two-dimensionally cultured, or two-dimensional culture is performed under hypoxic conditions to increase the content of VEGF, etc. Considering that it is difficult to stably maintain stem cells in a serum-free medium. There was a problem in that the physiological activity of the stem cell culture medium was lowered.

On the other hand, immune diseases are diseases in which components of the mammalian immune system cause, mediate, or otherwise contribute to the pathology of mammals. In particular, inflammatory diseases are attracting worldwide attention. It is an urgent disease. Inflammation is generally a localized protective response of body tissues against host invasion by foreign substances or noxious stimuli. Inflammation can be caused by infectious causes such as bacteria, viruses, and parasites; physical causes such as burns or radiation; chemicals such as toxins, drugs or industrial agents; immune responses, such as allergies and autoimmune responses, or conditions associated with oxidative stress.

Inflammation is characterized by pain, redness, swelling, fever, and eventual loss of function of the affected area. These symptoms are the result of a complex series of interactions between the cells of the immune system. The cellular response results in an interactive network of several groups of inflammatory mediators: proteins (eg cytokines, enzymes (eg proteases, peroxidases), major basic proteins, adhesion molecules (ICAM, VCAM), lipid mediators (e.g., eicosanoids, prostaglandins, leukotrienes, platelet activating factor (PAF)), reactive oxygen species (e.g., hydroperoxide, superoxide anion O2-, nitric oxide (NO) ) etc.) However, most of these mediators of inflammation are also modulators of normal cellular activity, so the host is uncontrolled and damaged (i.e. infection) due to a lack of inflammatory response, or partially due to chronic inflammation Overproduction of several of the above-mentioned mediators results in mediated inflammatory diseases.

In addition, autoimmune disease, which is one of the immune diseases, is characterized in that the immune system attacks its own organs to cause a spontaneous reaction. These responses are due to the recognition of auto-antigens by T lymphocytes, which elicits both humoral (autoantibody production) and cellular (lymphocyte and macrophage cytotoxic activity) immune responses. Autoimmune diseases include rheumatic diseases, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or exacerbation of immune responses by antigens, ie, asthma, allergy to drugs or food. These diseases are all limited and chronic diseases, in some cases fatal, and there is no effective treatment method for treating the diseases to date. Therefore, any drug, drug or medium capable of alleviating or alleviating 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 purposes for the application of therapeutic agents for immune diseases or inflammatory diseases. As a result, biocompatible scaffolds were obtained in serum-free medium by comparing the culture medium obtained according to the existing two-dimensional culture. The mesenchymal stem cell culture medium obtained according to the three-dimensional culture using By confirming that phosphorus PGE2 is secreted at a high concentration, 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.

It is an object of the present invention to provide a method for preparing a culture medium for mesenchymal stem cells for preventing or treating immune diseases or inflammatory diseases.

Another object of the present invention is to provide a mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases prepared by the above production method.

Another object of the present invention is to provide a composition for preparing a mesenchymal stem cell culture medium for the treatment of immune or inflammatory diseases containing a biocompatible scaffold and a serum-free medium comprising at least one of bEGF or EGF as an active ingredient. will be.

In order to achieve the object of the present invention, the present invention

(a) collecting the mesenchymal stem cells and three-dimensional culture with a biocompatible scaffold in a serum-free medium; and

(b) provides a method for producing a mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases comprising the step of collecting the culture medium.

In addition, the present invention provides a mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases prepared by the above method.

In addition, the present invention provides a biocompatible scaffold and a composition for preparing a mesenchymal stem cell culture medium for treatment of immune or inflammatory diseases containing a serum-free medium containing at least one of bEGF and EGF as an active ingredient.

Stem cell culture medium obtained by three-dimensionally culturing mesenchymal stem cells in serum-free medium using biocompatible scaffolds according to the method of the present invention is an immunomodulatory factor rather than stem cell culture medium obtained by two-dimensional culture according to the conventional method. Since PGE2 is secreted at a high concentration and has excellent effects of suppressing immune response and inhibiting secretion of inflammatory cytokines, it can be usefully used as a composition for preventing or treating immune diseases or inflammatory diseases.

1 is a mesenchymal stem cell culture medium (2D CM) obtained by a two-dimensional culture method or a mesenchymal stem cell culture medium (3D CM) obtained by a three-dimensional culture method by concentration and the inhibition rate of peripheral blood mononuclear cell proliferation is the diagram shown.
Figure 2 shows the mesenchymal stem cell culture medium (2D CM) obtained by the two-dimensional culture method or the mesenchymal stem cell culture medium (3D CM) obtained by the three-dimensional culture method is treated by concentration and secreted from peripheral blood mononuclear cells. It is a diagram showing the inhibition rate of TNF-α secretion.
Figure 3 shows the mesenchymal stem cell culture medium (2D CM) obtained by the two-dimensional culture method or the mesenchymal stem cell culture medium (3D CM) obtained by the three-dimensional culture method is treated by concentration and secreted from peripheral blood mononuclear cells. A diagram showing the inhibition rate of IFN-γ secretion.
4 is a diagram showing the secretion amount of PGE2 (prostaglandin E2) in the mesenchymal stem cell culture medium (2D CM) obtained by the two-dimensional culture method or the mesenchymal stem cell culture medium (3D CM) obtained by the three-dimensional culture method to be.

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

As used herein, "mesenchymal stem cells" are capable of self-proliferation and pluripotency, and exhibit a cell phenotype that is CD73 ⁺ , CD90 ⁺ , CD105 ⁺ , CD14 ^- , CD20 ^- , CD34 ^- , CD45 ^- It means a cell represented, and may be isolated from bone marrow, adipose tissue, umbilical cord blood, etc., but is not limited thereto.

As used herein, the term "biologically active substance" refers to cytokines, cell growth factors, immunomodulatory factors, and the like that can affect the functions of cells or the body. Examples of physiologically active substances include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), Tumor growth factor-beta (TGF-beta), Insulin growth factor (IGF), and the like, However, the present invention is not limited thereto.

As used herein, "stem cell culture medium" refers to a cell culture supernatant in which stem cells are cultured. Stem cell culture medium contains various physiologically active substances secreted from cells during the stem cell culture process.

In the present specification, the biocompatible scaffold refers to a support that is made of a material having a cell-adhesive surface and has affinity for cells, and can attach and culture cells in three dimensions. Examples of naturally-derived 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 the mesenchymal stem cells and three-dimensional culture with a biocompatible scaffold in a serum-free medium; and

(b) provides a method for producing a mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases comprising the step of collecting the culture medium.

In step (a), the mesenchymal stem cells are preferably isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.

In step (a), the mesenchymal stem cells are preferably subcultured after culturing in a stromal medium and a proliferation medium.

In the three-dimensional culture in step (a), it is preferable to obtain a stem cell culture medium three or more times while exchanging the medium at three-day intervals.

In step (a), it is preferable to add any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) to the serum-free medium for culturing.

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-hyd) hydroxy acid) series, poly(vinyl alcohol), polyanhydride, and the like, but is not limited thereto.

The immune disease is Behcet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary liver cirrhosis, dermatomyositis, Goodpeitzer syndrome, autoimmune meningitis, Sjogren's syndrome, systemic redness Lupus reflex, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic bullous epidermolysis, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic Anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, graft-versus-host disease or obesity. can, but is not limited thereto.

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, myofascial disease, viral infection, Bacterial infection, fungal infection, burns, surgical or dental wounds, PGE excess syndrome, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis or acute and chronic inflammatory diseases However, the present invention is not limited thereto.

The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by the immune response and the immune response, but is not limited thereto.

The mesenchymal stem cell culture medium preferably reduces immune rejection and inflammatory responses during xenotransplantation, but is not limited thereto.

The mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 pg/ml to 10000 pg/ml PGE2, and 7900 pg/ml to 9800 pg/ml PGE2 it is more preferable

In a specific embodiment of the present invention, the present inventors treated peripheral blood mononuclear cells inducing an immune response with the mesenchymal stem cell culture medium obtained according to the three-dimensional culture method, as a result, the cell proliferation of the peripheral blood mononuclear cells was inhibited. It was confirmed that (see FIG. 1).

In addition, the present inventors treated peripheral blood mononuclear cells inducing an immune response with the mesenchymal stem cell culture medium obtained according to the three-dimensional culture method, and inhibited TNF-α and IFN-γ secretion in the peripheral blood mononuclear cells. It was confirmed that (see FIGS. 2 and 3).

In addition, the present inventors confirmed that the immunomodulatory factor, PGE2, in the mesenchymal stem cell culture medium obtained according to the three-dimensional culture method significantly increased than in the mesenchymal stem cell culture medium obtained according to the two-dimensional culture method (Fig. 4). Reference).

Therefore, according to the method of the present invention, the mesenchymal stem cells are three-dimensionally cultured using a biocompatible scaffold in a serum-free medium. By confirming, the present invention can be usefully used in a method for producing a mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases.

The culture of mesenchymal stem cells according to the present invention may be carried out according to the following procedure, and the medium used for cell culture is not limited thereto.

(1) Culturing of mesenchymal stem cells

The mesenchymal stem cells obtained from the tissue are suspended in a stromal medium, inoculated in a culture vessel at a concentration of 10,000~40,000 cells/cm2, and then cultured. The substrate medium is DMEM or DMEM/F12 (Dulbecco's Modified Eagle Medium/Ham's F-12 Nutrient Broth) containing 10% bovine serum, and cultured for about 24 hours.

(2) Cultivation in expansion media

After removing the matrix medium, the adherent cells are proliferated by culturing in the growth medium. The proliferation medium is DMEM or DMEM/F12 containing 10% bovine serum, 0.1-100 ng/ml of EGF (epidermal growth factor) and/or 0.1-100 ng/ml of bFGF (basic fibroblast growth factor), It rapidly proliferates adherent mesenchymal stem cells and increases the amount of cells in a short period of time.

(3) subculture

When the cells fill 80 to 90% of the bottom of the culture vessel, the growth medium is removed and the cells are removed from the culture vessel through trypsinization. For subculture, the cells are diluted 1:3 to 1:4 and incubated with the growth 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 in a form attached to the biocompatible scaffold. Since cell culture in the scaffold does not require a conventional cell culture vessel, it can be cultured in a large amount in a sterile bottle or culture bag, which has the advantage of being able to culture conveniently at a lower cost.

In addition, there is provided a composition containing the mesenchymal stem cell culture medium for the prevention or treatment of immune diseases or inflammatory diseases prepared according to the production method of the present invention.

The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-α or IFN-γ by the immune response and the immune response, but is not limited thereto.

The mesenchymal stem cell culture medium preferably reduces immune rejection and inflammatory responses during xenotransplantation, but is not limited thereto.

The mesenchymal stem cell culture medium preferably contains PGE2 (prostaglandin E2), more preferably 5000 pg/ml to 10000 pg/ml PGE2, and 7900 pg/ml to 9800 pg/ml PGE2 it is more preferable

According to the production method of the present invention, mesenchymal stem cells are three-dimensionally cultured using a biocompatible scaffold in a serum-free medium. Or it may be usefully used as a composition for preventing or treating inflammatory diseases.

In addition, the present invention provides a biocompatible scaffold and a composition for preparing a mesenchymal stem cell culture medium for treatment of immune or inflammatory diseases containing a serum-free medium containing at least one of bEGF and EGF as an active ingredient.

The biocompatible scaffold 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-hydroxy acid) series, poly(vinyl alcohol), polyanhydride, etc., but is not limited thereto.

As the serum-free medium, any basic medium known in the art may be used without limitation, but serum-free medium is used. The basal medium may be artificially synthesized and manufactured, or a commercially prepared medium may be used. Examples of commercially prepared media include 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) and Isocove's Modified Dulbecco's Medium, but are not limited thereto, and DMEM or DMEM/F12 may be preferably used.

0.5 to 2 ng/ml of bFGF is preferably added to the serum-free medium, more preferably 0.7 to 1.5 ng/ml, and even more preferably 1 ng/ml.

It is preferable that 3-7 ng/ml of EGF is added to the serum-free medium, more preferably 4-6 ng/ml is added, and even more preferably 5 ng/ml is added.

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

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Example 1> Culturing method of human adipose-derived mesenchymal stem cells

Adipose tissue can usually be obtained by liposuction, but is not limited thereto. Adipose-derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction as follows: To remove blood, adipose tissue was washed 3 to 4 times with an equal volume of KRB solution. Then, a collagenase solution of the same volume as the adipose tissue was added and reacted in a water bath at 37°C. This was transferred to a centrifuge tube and centrifuged at 20 and 1500 rpm for 10 minutes . After centrifugation, the supernatant, the fat layer, was removed, and the collagenase solution, which was the lower layer, was carefully separated so as not to be shaken. After the suspension was put in a substrate medium, centrifuged at 20 ℃, 1500 rpm for 5 minutes. At this time, what sinks to the bottom is the stroma-vascular fraction, and the supernatant is removed. The stroma-vascular fraction was suspended in a substrate medium and inoculated into a culture vessel, and cultured at 37° C., 5% CO ₂ in an incubator for 24 hours. After removing the culture medium, washing with a phosphate buffer solution, a substrate medium (DMEM/F12 medium containing 10% FBS), or a growth medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng/ml in the substrate medium, or It was grown using a medium containing epidermal growth factor (EGF) at a concentration of 5 ng/ml in the stromal medium. When the adipose-derived mesenchymal stem cells grew to about 80-90% of the culture vessel, trypsin-treated them to obtain single cells. The obtained cells were diluted 1:3 to 1:4 with a growth medium and subcultured.

<Example 2> Method of culturing human adipose-derived mesenchymal stem cells in two dimensions

Stem cells obtained as single cells by trypsinization were washed three times with DMEM/F12 medium to remove FBS. DMEM/F12 solution was added to 1.3 × 10 ⁷ cells/ml stem cells, and the cells were suspended and inoculated. Then, a DMEM/F12 solution containing 1 ng/ml basic fibroblast growth factor and 5 ng/ml epidermal growth factor was added to make 500 ml per 4.0 × 10 ⁷ cells. After 72 hours, the cell culture medium was recovered and a new medium was added. The cell culture medium was recovered 2 to 11 more times and stored in a refrigerator.

<Example 3> Method of three-dimensional culture of human adipose-derived mesenchymal stem cells with fibrin glue

Stem cells obtained as single cells by trypsinization were washed three times with DMEM/F12 medium to remove FBS. DMEM/F12 solution containing thrombin solution was added to 1.3 × 10 ⁷ cells/ml stem cells to suspend the cells. Using a dual syringe, the cell suspension and the fibrinogen dilution were mixed, followed by three-dimensional culture in a bottle or culture bag. As the cell culture medium, DMEM/F12 solution containing 1 ng/ml basic fibroblast growth factor and 5 ng/ml epidermal growth factor was used. After 72 hours, the cell culture medium was recovered and a new medium was added. The cell culture medium was recovered 2 to 11 more times and stored in a refrigerator.

<Experimental Example 1> Immune response of mesenchymal stem cell culture medium obtained by three-dimensional culture method Check suppression

EdU cell proliferation assay was performed to confirm the immune response inhibitory ability of the mesenchymal stem cell culture medium obtained in <Example 2> and <Example 3>.

Specifically, 5 × 10 ⁵ peripheral blood mononuclear cells (PBMC) obtained from donors having different histocompatibility antigens (Human Leukocyte Antigen; HLA) were added to a 24-well plate. As a positive control, an immune response of peripheral blood mononuclear cells was induced by adding 1.0 μg/ml of phyto-hemagglutinin (PHA), a mitogen, to peripheral blood mononuclear cells. Then, two sets of mesenchymal stem cell culture medium obtained by the two-dimensional culture method of <Example 2> or the three-dimensional culture method of <Example 3>(Lot#1 and Lot#) 2) was treated at a concentration of 10% or 20%, and the supernatant was collected on the 3rd day after the start of the reaction, and the proliferation ability of peripheral blood mononuclear cells was measured by FACS technique.

As a result, as shown in FIG. 1, cell proliferation was not inhibited in the group treated with the mesenchymal stem cell culture medium obtained by the two-dimensional culture method of <Example 2>, whereas the <Example 3> Since cell proliferation was inhibited in the group treated with the mesenchymal stem cell culture medium obtained by the three-dimensional culture method, it was confirmed that the mesenchymal stem cell culture medium obtained by the three-dimensional culture method significantly suppressed the immune response ( Fig. 1).

<Experimental Example 2> Confirmation of inhibition of inflammatory cytokine secretion in mesenchymal stem cell culture medium obtained by three-dimensional culture method

In order to confirm whether the mesenchymal stem cell culture medium obtained in <Example 2> and <Example 3> inhibited the secretion of inflammatory cytokines, ELISA analysis was performed.

Specifically, as in <Experimental Example 1>, 5 × 10 ⁵ peripheral blood mononuclear cells were placed in a 24-well plate and activated with PHA to induce an immune response. Then, two sets of mesenchymal stem cell culture medium obtained by the two-dimensional culture method of <Example 2> or the three-dimensional culture method of <Example 3>(Lot#1 and Lot#) 2) was added at a concentration of 10% or 20%. On the third day 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 FIGS. 2 and 3, the secretion amount of TNF-α and IFN-γ was significantly reduced in the group treated with the mesenchymal stem cell culture medium obtained by the three-dimensional culture method of <Example 3>. It was confirmed that (Figs. 2 and 3).

Therefore, the mesenchymal stem cell culture medium obtained by the three-dimensional culture method through the <Experimental Example 1> and <Experimental Example 2> does not induce an immune response, and the secretion amount of inflammatory cytokines that play a role in increasing the immune reactivity It was confirmed that it can help heal by alleviating immune diseases or inflammatory diseases by reducing .

<Experimental Example 3> Identification of immunomodulatory factors of mesenchymal stem cell culture medium obtained by three-dimensional culture method

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

As a result, as shown in FIG. 4 , it was confirmed that the secretion amount of PGE2 was significantly increased in the mesenchymal stem cell culture medium (Lot #1 and Lot #2) obtained by the three-dimensional culture method of <Example 3>. (Fig. 4).

Therefore, the mesenchymal stem cell culture medium obtained by the three-dimensional culture method through <Experimental Example 1> to <Experimental Example 3> does not induce an immune response, and the secretion amount of inflammatory cytokines that play a role in increasing immune reactivity It was confirmed that it can help heal by alleviating immune diseases or inflammatory diseases by increasing immune reactivity by secreting PGE2, an immunomodulatory factor, at a high concentration.

Claims (7)

(a) collecting mesenchymal stem cells and three-dimensionally culturing with a biocompatible scaffold in a serum-free medium to which one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added; and
(B) through the step of collecting the culture medium, 5,000 pg / mL to 10,000 pg / mL of PGE ₂ (prostaglandin E2) is prepared including the step of obtaining a mesenchymal stem cell culture solution,
Wherein the mesenchymal stem cell culture medium is to suppress the secretion of TNF-α or IFN-γ by the immune response and immune response,
A pharmaceutical composition for the prevention or treatment of immune diseases, comprising a mesenchymal stem cell culture medium as an active ingredient.
The method of claim 1,
In the above (a), the mesenchymal stem cells are a pharmaceutical composition, characterized in that isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.
The method of claim 1,
In (a), the mesenchymal stem cells are cultured in a stromal medium and a proliferation medium, and then subcultured in a pharmaceutical composition.
The method of claim 1,
The three-dimensional culture in (a) is a pharmaceutical composition, characterized in that obtaining a stem cell culture medium three or more times while exchanging the medium at three-day intervals.
The method of claim 1,
In (a), the biocompatible scaffold is a cell support having a cell-adhesive surface, and a pharmaceutical composition, characterized in that it is a natural or synthetic polymer.
6. The method of claim 5,
The biocompatible scaffold is characterized in that any one selected from the group consisting of alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) series, poly (vinyl alcohol), and polyanhydride A pharmaceutical composition comprising.
The method of claim 1,
Immune diseases include Behcet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary liver cirrhosis, dermatomyositis, Goodpeitzer syndrome, autoimmune meningitis, Sjogren's syndrome, systemic erythematosus Lupus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia , multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthrosis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis and graft-versus-host disease. Which one is selected, the pharmaceutical composition.

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