KR20180072644A - 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 a mesenchymal stem cell culture for prevention or treatment of immune diseases or inflammatory diseases and to 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 prevention or treatment of the immune diseases or inflammatory diseases.

Description

[0001] The present invention relates to a mesenchymal stem cell culture medium for preventing or treating an immunological disease or an inflammatory disease, and a method for preparing the same,

The present invention relates to a mesenchymal stem cell culture medium for preventing or treating an immunological disease or inflammatory disease which has significant immunosuppressive and anti-inflammatory functions by a three-dimensional culture method, and a method for producing the same.

Several types of progenitors have been found in human bone marrow. Among them, precursor cells exhibiting multipotentiality are called mesenchymal stem cells. Mesenchymal stem cells are known to exist not only in bone marrow, but also in most organs of the body, including fat, liver, and muscle. The mesenchymal stem cells are self-renewing and have an ability to function as osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon ligament fibroblasts, adipocytes, etc. . ≪ / RTI >

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

However, in order to cultivate mesenchymal stem cells, bovine serum is usually used, but the culture medium containing bovine serum is inadequate for clinical application. That is, since the use of animal-derived proteins such as bovine serum is limited for clinical application of cell culture media, it is preferable to exclude bovine serum for culture of stem cells.

Bovine serum proteins are necessary for the proliferation and maintenance of stem cells. Therefore, a method of obtaining a culture solution containing a high concentration of physiologically active substance while culturing mesenchymal stem cells in a serum-free medium is required.

Korean Patent Laid-Open No. 10-2009-0001163 (2009. 01. 08) discloses that mesenchymal stem cells obtained from human bone marrow, umbilical cord blood or placenta tissue are cultured in serum medium, subcultured in serum-free medium, Discloses a method of mass-producing bFGF or TGF-? From mesenchymal stem cells by applying physical stimulation of hypoxic culture to the cells.

Similarly, in International Publication WO 2007/086637 (Aug. 02, 2007), adult stem cells extracted from adipocytes of mammals are cultured in serum medium, subcultured in serum-free medium, cultured in hypoxic culture, A method of mass-producing bFGF, VEGF or TGF-? From mesenchymal stem cells by adding physical stimulation such as ultraviolet irradiation, nutrient deficiency, or mechanical friction and adding vitamin A, B, C, .

That is, in the conventional method, only two-dimensional culture is carried out under hypoxic condition in order to increase the content of VEGF or the like in a simple two-dimensional culture of stem cells. Considering that the stem cells are not stably maintained in serum-free medium The physiological activity of the stem cell culture solution is lowered.

On the other hand, immunological diseases are diseases in which components of the mammalian immune system cause, mediate, or contribute to mammalian pathological conditions, in particular, inflammatory diseases are globally noted diseases, and therapeutic agents capable of treating the underlying diseases It is an urgent disease. Inflammation is generally a localized protective response of the body tissue to host infiltration by foreign substances or by 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 formulations; An immune response such as an allergic and autoimmune response, or a condition associated with oxidative stress.

Inflammation is characterized by pain, numbness, swelling, heat and ultimate loss of function in the affected area. These symptoms are the result of a series of complex interactions that take place between cells of the immune system. The reaction of cells results in a network of interaction groups of inflammatory mediators as a result: proteins (eg, cytokines, enzymes (eg proteases, peroxidases), major basic proteins, (E.g., VCAM), lipid mediators (e.g., eicosanoids, prostaglandins, leukotrienes, platelet activating factors (PAF)), reactive oxygen species such as hydroperoxides, superoxide anions O2-, ), Etc. However, many of these mediators of inflammation are also regulators of normal cellular activity, and thus are either damaged (i.e., infected) without control of the host due to the deficiency of the inflammatory response, or partly due to chronic inflammation Inflammatory diseases mediated by the overproduction of several of the above-mentioned mediators.

Also, an autoimmune disease, one of the immune diseases, is characterized by the immune system attacking its own organ and causing a spontaneous reaction. These reactions are caused by the recognition of auto-antigen by T lymphocytes, which leads to immune responses in the body fluid (autoantibody production) and in the cellular phase (increased lymphocyte and macrophage cytotoxic activity). Examples of autoimmune diseases include rheumatic diseases, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or aggravation of immune responses by antigens such as asthma, drugs or food allergies. These diseases are all limited and chronic diseases, and in some cases, they are fatal, and thus there is no effective treatment method for treating the diseases. Therefore, drugs, medicines or medicines that can alleviate or alleviate the disease during the course of the disease will be said to be an important solution for the health of the patient.

Accordingly, the present inventors have made efforts to develop a stem cell culture solution which can be used for clinical use for the treatment of immunological diseases or inflammatory diseases. As a result, the inventors of the present invention found that biocompatible scaffolds The mesenchymal stem cell culture obtained by the three-dimensional culture does not induce an immune response and reduces the secretion amount of TNF-a and IFN-y, which are inflammatory cytokines that increase the immunoreactivity, And that the culture solution obtained according to the above method can be used for the prophylaxis or treatment of an immunological disease or an inflammatory disease by confirming that PGE2 is secreted at a high concentration.

It is an object of the present invention to provide a method for producing a mesenchymal stem cell culture solution for the prevention or treatment of an immune disease or an inflammatory disease.

Another object of the present invention is to provide a mesenchymal stem cell culture solution for the prevention or treatment of an immunological disease or inflammatory disease produced by the above production method.

Yet another object of the present invention is to provide a composition for preparing a mesenchymal stem cell culture liquid for the treatment of an immunological disease or inflammatory disease, which comprises a biocompatible scaffold and a serum-free medium containing 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 in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And

(b) collecting the culture medium. The present invention also provides a method for producing a mesenchymal stem cell culture solution for preventing or treating an immunological disease or an inflammatory disease.

In addition, the present invention provides a mesenchymal stem cell culture solution for preventing or treating an immunological disease or inflammatory disease produced by the above method.

The present invention also provides a composition for the manufacture of a mesenchymal stem cell culture for the treatment of an immunological disease or inflammatory disease, which comprises a biocompatible scaffold and a serum-free medium containing any one or more of bEGF or EGF as an active ingredient.

According to the method of the present invention, a stem cell culture obtained by three-dimensionally culturing mesenchymal stem cells in a serum-free medium using a biocompatible scaffold can be used as an immunomodulator PGE2 is secreted at a high concentration and has excellent immunosuppressive and inflammatory cytokine secretion inhibiting effects, and thus can be effectively used as a composition for preventing or treating immunological diseases or inflammatory diseases.

FIG. 1 is a graph showing the results of treatment of mesenchymal stem cell culture (2D CM) obtained by a two-dimensional culture method or mesenchymal stem cell culture (3D CM) obtained by a three-dimensional culture method by concentration and inhibition of peripheral blood mononuclear cell proliferation Fig.
FIG. 2 is a graph showing the results obtained by treating 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- TNF-a < / RTI >
FIG. 3 is a graph showing the results obtained by treating 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- 0.0 > IFN-y < / RTI > secretion inhibition rate.
4 is a graph showing the secretion amount of PGE2 (prostaglandin E2) in the mesenchymal stem cell culture solution (2D CM) obtained by the two-dimensional culture method or the mesenchymal stem cell culture solution (3D CM) obtained by the three- to be.

Hereinafter, terms of the present invention will be 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 And may be isolated from bone marrow, adipose tissue, umbilical cord blood, etc., but is not limited thereto.

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

As used herein, the term " stem cell culture fluid " refers to a cell culture supernatant in which stem cells have been cultured. Stem cell cultures contain various physiologically active substances secreted from cells during stem cell culture.

In this specification, the biocompatible scaffold refers to a scaffold that has affinity with a cell and is made of a material having a so-called cell adhesion surface, and capable of adhering and culturing cells three-dimensionally. Examples of natural-derived supports include, but are not limited to, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, and the like. Examples of the synthetic polymer include 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 in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And

(b) collecting the culture medium. The present invention also provides a method for producing a mesenchymal stem cell culture solution for preventing or treating an immunological disease or an inflammatory disease.

In the 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 cultured in a medium and a proliferation medium, followed by subculture.

In the step (a), it is preferable to obtain a stem cell culture solution three times or more while exchanging the medium at 3-day intervals.

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

In the step (b), the biocompatible scaffold is preferably a natural or synthetic polymer as a cell support having a cell adhesion surface, and examples thereof include alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, Poly (vinyl alcohol), polyanhydride, and the like, but are not limited thereto.

The immunological diseases are selected from the group consisting of Behcet's disease, multiple myositis, skin myositis, autoimmune hemodilution, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatomyositis, Goodpie syndrome, autoimmune meningitis, Sjogren's syndrome, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient for the treatment and / or prophylaxis of diabetic nephropathy such as diabetes mellitus, Anemia, ulcerative colitis, graft-versus-host disease or obesity, anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, But is not limited thereto.

Wherein the inflammatory disease is selected from the group consisting of gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, It may be a bacterial infection, a fungal infection, a burn, a wound due to surgical or dental surgery, PGE hyperaemia, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis or acute and chronic inflammatory disease But is not limited thereto.

The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-? Or IFN-? By an immune response and an immune response, but is not limited thereto.

The mesenchymal stem cell culture solution preferably reduces the immunoreactivity and the inflammatory response upon xenotransplantation, but is not limited thereto.

The mesenchymal stem cell culture solution preferably contains PGE2 (prostaglandin E2), more preferably 5000 pg / ml to 10000 pg / ml of PGE2, and more preferably 7900 pg / ml to 9800 pg / ml of PGE2 Is more preferable.

In a specific example of the present invention, the inventors treated the mesenchymal stem cell culture solution obtained by the above-mentioned three-dimensional culture method on the peripheral blood mononuclear cells that caused the immune response, and found that inhibition of cell proliferation of the peripheral blood mononuclear cells (See Fig. 1).

Furthermore, the inventors of the present invention have found that treatment of mesenchymal stem cell culture obtained by the above-described three-dimensional culture method on peripheral blood mononuclear cells that caused an immune response inhibits TNF-α and IFN-γ secretion in the peripheral blood mononuclear cells (See Figs. 2 and 3).

Further, the present inventors confirmed that PGE2, which is an immunoregulatory factor in the mesenchymal stem cell culture solution obtained according to the above three-dimensional culture method, was significantly increased as compared with the mesenchymal stem cell culture obtained by the two-dimensional culture method Reference).

Therefore, the mesenchymal stem cells were cultured three-dimensionally using biocompatible scaffolds in the serum-free medium according to the method of the present invention, and thus the stem cell culture medium contained a high concentration of PGE2 and excellent immunosuppressive and inflammatory cytokine secretion inhibitory effect The present invention can be usefully used in a method for producing a mesenchymal stem cell culture solution for the prevention or treatment of an immunological disease or an inflammatory disease.

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

(1) Culture of mesenchymal stem cells

The mesenchymal stem cells obtained from the tissue are suspended in a medium and cultured in a culture vessel at a concentration of 10,000 to 40,000 cells / cm 2. The substrate medium is cultured 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) Culture in expansion media

After the substrate medium is removed, the adherent cells are proliferated by culturing in a proliferation medium. The proliferation medium was DMEM or DMEM / F12 containing 10% fetal bovine serum, epidermal growth factor (EGF) at a concentration of 0.1 to 100 ng / ml and / or basic fibroblast growth factor (bFGF) at a concentration of 0.1 to 100 ng / Adherent mesenchymal stem cells are rapidly proliferating to increase the amount of cells in a short period of time.

(3) Passage culture

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 by trypsinization. For subculture, cells are diluted 1: 3 ~ 1: 4 and incubated with the growth medium in a new culture vessel. Additional subculture is possible in this way.

(4) incubation with a biocompatible scaffold

The cultured cells are washed with PBS 3 times or more to remove FBS, and cultured in a serum-free medium in the form attached to a biocompatible scaffold. Cell culture in a scaffold does not require a conventional cell culture container, and thus it is possible to cultivate large quantities in an aseptic bottle or culture bag, thereby enabling convenient cultivation at a lower cost.

The present invention also provides a composition containing a mesenchymal stem cell culture solution for preventing or treating an immunological disease or inflammatory disease produced according to the production method of the present invention.

The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-? Or IFN-? By an immune response and an immune response, but is not limited thereto.

The mesenchymal stem cell culture solution preferably reduces the immunoreactivity and the inflammatory response upon xenotransplantation, but is not limited thereto.

The mesenchymal stem cell culture solution preferably contains PGE2 (prostaglandin E2), more preferably 5000 pg / ml to 10000 pg / ml of PGE2, and more preferably 7900 pg / ml to 9800 pg / ml of PGE2 Is more preferable.

According to the production method of the present invention, the mesenchymal stem cells are cultured in a three-dimensional manner using a biocompatible scaffold in a serum-free medium. Since the cell culture medium has excellent immunosuppressive and inflammatory cytokine secretion suppressing effects, Or as a composition for preventing or treating inflammatory diseases.

The present invention also provides a composition for the manufacture of a mesenchymal stem cell culture for the treatment of an immunological disease or inflammatory disease, which comprises a biocompatible scaffold and a serum-free medium containing any one or more of bEGF or EGF as an active ingredient.

The biocompatible scaffold is preferably a natural or synthetic polymer as a cell scaffold having a surface that is adhered to a cell. Examples of the scaffold include alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) But are not limited to, poly (vinyl alcohol), polyanhydride, and the like.

The serum-free medium can be used without limitation in a basic medium known in the art, but serum-free medium is used. The basic medium may be prepared by artificial synthesis, or a commercially prepared medium may be used. Examples of commercially produced media include Dulbecco's Modified Eagle's Medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, 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 can be preferably used.

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

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

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

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

Example 1: Culture of human adipose-derived mesenchymal stem cells

Fat tissue is usually obtained by liposuction, but is not limited thereto. The adipose derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction as follows: The adipose tissue was washed 3-4 times with the same volume of KRB solution to remove blood. Then, a volume of collagenase solution such as adipose tissue was added and the mixture was reacted at 37 ° C in a water bath. This was transferred to a centrifuge tube and centrifuged at 20, 1500 rpm for 10 minutes . After centrifugation, the upper layer of fat layer was removed and the lower layer of collagenase solution was carefully removed to avoid shaking. After the substrate medium was suspended, it was centrifuged at 20 DEG C and 1500 rpm for 5 minutes. At this time, the lower layer was a stroma-vascular fraction, and the supernatant was removed. The stroma-vascular fraction was suspended in a substrate medium, inoculated into a culture vessel, and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After removing the culture medium, the cells were washed with a phosphate buffer solution, and the medium (DMEM / F12 medium containing 10% FBS) or the growth medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / The medium was grown in medium containing epidermal growth factor (EGF) at a concentration of 5 ng / ml. When the adipose-derived mesenchymal stem cells were grown to about 80 to 90% of the culture vessel, they were trypsinized and isolated into single cells. The obtained cells were diluted to 1: 3 to 1: 4 with a growth medium and subcultured.

<Example 2> Method for two-dimensional culture of human adipose-derived mesenchymal stem cells

The stem cells obtained by single-cell treatment with trypsin were washed three times with DMEM / F12 medium to remove FBS. DMEM / F12 solution was added to 1.3 x 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 500 ml of 4.0 x ¹⁰⁷ cells. After 72 hours, the cell culture medium was recovered, and the cell culture medium was recovered 2 to 11 times by adding fresh medium, followed by refrigerated storage.

Example 3: Three-dimensional culture of human adipose-derived mesenchymal stem cells with fibrin glue

The stem cells obtained by single-cell treatment with trypsin 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 占⁰⁷ / ml stem cells. The cell suspension and the fibrinogen dilution were mixed using a dual syringe and then cultured in a 3-dimensional culture in a bag or culture bag. The cell culture medium was a DMEM / F12 solution containing 1 ng / ml basic fibroblast growth factor and 5 ng / ml epidermal growth factor. After 72 hours, the cell culture medium was recovered, and the cell culture medium was recovered 2 to 11 times by adding fresh medium, followed by refrigerated storage.

Experimental Example 1 Immune response of the mesenchymal stem cell culture obtained by the three-dimensional culture method Check for inhibition

The EdU cell proliferation assay was performed to confirm the ability of the mesenchymal stem cell culture obtained in Example 2 and Example 3 to inhibit the immune response.

Specifically, 5 × 10 ⁵ peripheral blood mononuclear cells (PBMC) obtained from other donors with human leukocyte antigen (HLA) were added to 24-well plates. As a positive control, 1.0 ㎍ / ㎖ of PHA (phyto-hemagglutinin), which is a mitogen, was added to peripheral blood mononuclear cells to induce immune response of peripheral blood mononuclear cells. Then, two sets (Lot # 1 and Lot # 2) of the mesenchymal stem cell culture solution obtained by the two-dimensional culture method of Example 2 described above or the mesenchymal stem cell culture solution obtained by the three-dimensional culture method of Example 3 2) was treated at a concentration of 10% or 20%. After 3 days from the start of the reaction, the supernatant was collected 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 solution obtained by the two-dimensional culture method of Example 2, 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 since cell proliferation was inhibited 1).

Experimental Example 2 Confirmation of inhibition of inflammatory cytokine secretion of the mesenchymal stem cell culture obtained by the three-dimensional culture method

In order to confirm the inhibition of inflammatory cytokine secretion of the mesenchymal stem cell culture obtained in Example 2 and Example 3, an ELISA assay was performed.

Specifically, 5 × 10 ⁵ peripheral blood mononuclear cells were put into a 24-well plate as described in <Experimental Example 1> and activated with PHA to induce an immune response. Then, two sets (Lot # 1 and Lot # 2) of the mesenchymal stem cell culture solution obtained by the two-dimensional culture method of Example 2 described above or the mesenchymal stem cell culture solution obtained by the three-dimensional culture method of Example 3 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, 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-a and IFN- (Figs. 2 and 3).

Therefore, through the above <Experimental Example 1> and <Experimental Example 2>, the mesenchymal stem cell culture solution obtained by the three-dimensional culture method does not induce the immune response, and the secretion amount of the inflammatory cytokine To reduce the immune or inflammatory disease and to help to heal.

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

In order to confirm the secretion level of prostaglandin E2 (PGE2) which is an immunoregulatory factor in the mesenchymal stem cell culture fluid obtained in Example 2 and Example 3, in Example 2 and Example 3, Were analyzed by ELISA and the absorbance at 450 nm was measured.

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 solutions (Lot # 1 and Lot # 2) obtained by the three-dimensional culture method of Example 3 (Fig. 4).

Therefore, through the above <Experimental Examples 1 to 3>, the mesenchymal stem cell culture solution obtained by the three-dimensional culture method does not induce the immune response, and the secretion amount of the inflammatory cytokine And PGE2, which is an immunoregulatory factor, is secreted at a high concentration to increase immunoreactivity, thereby alleviating immune or inflammatory diseases and thereby helping to be cured.

Claims (9)

(a) collecting mesenchymal stem cells and culturing them in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And
(b) collecting the culture medium to obtain a mesenchymal stem cell culture solution containing PGE ₂ (prostaglandin E2)
Wherein the mesenchymal stem cell culture medium is for the prevention or treatment of immunological diseases or inflammatory diseases which inhibit the secretion of TNF-? Or IFN-? By the immune response and the immune response.
The method according to claim 1, wherein the mesenchymal stem cells are isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.
[2] The method according to claim 1, wherein the mesenchymal stem cells are cultured in a medium and a growth medium, followed by subculture.
[3] The method according to claim 1, wherein the three-dimensional culture in step (a) comprises culturing the stem cell culture medium three or more times while exchanging the medium at intervals of 3 days.
The method according to claim 1, wherein at least one of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to the serum-free medium in step (a).
2. The method of claim 1, wherein the biocompatible scaffold is a natural or synthetic polymer as a cell support having a surface that is cell adhesive.
7. The composition of claim 6 wherein the biocompatible scaffold is selected from the group consisting of alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) Gt; and / or &lt; / RTI &gt;
The method of claim 1, wherein the immune disorder is selected from the group consisting of Behcet's disease, multiple myositis, skin myositis, autoimmune hemodilution, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatomyositis, , Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, eosinophilic epidermolysis, epididymitis, glomerulonephritis, Inflammatory bowel syndrome, hemorrhagic fever, multiple sclerosis, acute myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, mucoid, malignant anemia, ulcerative colitis, graft An autoimmune disease, an autoimmune disease, an autoimmune disease, an autoimmune disease, Way.
The method of claim 1, wherein the inflammatory disease is selected from the group consisting of gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine headache, back pain, fibromyalgia, PGE hyperaemia, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, and acute and chronic inflammatory diseases, including but not limited to infections, bacterial infections, fungal infections, burns, surgical or dental surgery wounds, &Lt; / RTI &gt;

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