KR102588400B1 - 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 a mesenchymal stem cell culture medium for preventing or treating immune diseases or inflammatory diseases and a method for producing the same. According to the method of the present invention, mesenchymal stem cells are grown in three dimensions using a biocompatible scaffold in a serum-free medium. The stem cell culture medium obtained by culturing secretes PGE2, an immune regulator, at a higher concentration than the stem cell culture medium obtained by two-dimensional culture according to the conventional method, and is excellent in suppressing immune response and secretion of inflammatory cytokines, so it is effective in suppressing immune diseases or inflammatory cytokines. It can be usefully used as a composition to prevent or treat 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 preventing or treating immune diseases or inflammatory diseases that possesses significant immunosuppressive and anti-inflammatory functions by a three-dimensional culture method, and a method for producing the same.

It has been discovered that several types of progenitor cells exist in human bone marrow, and among these, progenitor cells that exhibit multipotency are called mesenchymal stem cells. Mesenchymal stem cells are known to exist in most organs of the body, including fat, liver, and muscle, as well as bone marrow. Mesenchymal stem cells have the ability to self-proliferate 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, stem cell culture media can be used for clinical purposes such as pharmaceuticals or cosmetic compositions.

However, bovine serum is typically used to culture mesenchymal stem cells, and culture media containing bovine serum are unsuitable for clinical application. In other words, for clinical application of cell culture media, the use of animal-derived proteins such as bovine serum is limited, so it is desirable to exclude bovine serum when culturing stem cells.

Since bovine serum proteins are essential for the proliferation and maintenance of stem cells, a method is required to obtain a culture medium containing a high concentration of bioactive substances while culturing mesenchymal stem cells in a serum-free medium.

Domestic Patent Publication No. 10-2009-0001163 (January 8, 2009) discloses that mesenchymal stem cells obtained from human bone marrow, umbilical cord blood, or placental tissue were cultured in serum medium, then subcultured in serum-free medium, and the cultured stem cells were A method for mass production of bFGF or TGF-β from mesenchymal stem cells is disclosed by applying physical stimulation of hypoxic culture to the cells.

Similarly, in International Publication No. WO 2007/086637 (2007. 08. 02), adult stem cells extracted from mammalian fat cells were cultured in serum medium and then subcultured in serum-free medium, and the cultured stem cells were subjected to hypoxic culture, Disclosed is a method for mass producing bFGF, VEGF, or TGF-β from mesenchymal stem cells by applying physical stimulation such as ultraviolet irradiation, nutrient deprivation, or mechanical friction and adding vitamins A, B, C, or D to the culture medium. I'm doing it.

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 maintain stem cells stably in serum-free media. There was a problem that the physiological activity of the stem cell culture medium decreased.

Meanwhile, immune diseases are diseases in which components of the mammalian immune system cause, mediate, or otherwise contribute to pathological conditions in mammals. In particular, inflammatory diseases are diseases that are attracting attention around the world, and there is no therapeutic agent that can treat the underlying disease. It is an urgent disease. Inflammation is generally a localized protective response of body tissues against 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 agents; It may be a condition associated with an immune response, such as allergies and autoimmune reactions, or oxidative stress.

Inflammation is characterized by pain, redness, swelling, heat, and ultimate loss of function of the affected area. These symptoms are the result of a complex series of interactions that occur between cells of the immune system. The cellular response results in the creation of an interactive network of several groups of inflammatory mediators: proteins (e.g. cytokines, enzymes (e.g. proteases, peroxidases), major basic proteins, and adhesion molecules (ICAMs). 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, many of these mediators of inflammation are also regulators of normal cellular activity. Thus, deficiencies in the inflammatory response can result in uncontrolled damage (i.e. infection) to the host, or in part due to chronic inflammation. Inflammatory diseases are caused by excessive production of several of the above-mentioned mediators.

Additionally, autoimmune disease, which is one of the immune diseases, is characterized by the immune system attacking its own organs and causing a spontaneous response. These reactions are due to the recognition of auto-antigens by T lymphocytes, which triggers humoral (autoantibody production) and cellular (increased cytotoxic activity of lymphocytes and macrophages) immune responses. Autoimmune diseases include rheumatic diseases, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or exacerbated immune responses due to antigens, such as asthma, drug or food allergies, etc. These diseases are all limiting and chronic diseases, and in some cases are fatal, and there is currently no effective treatment method to treat the diseases. Therefore, any drug, medicine, or medium that can alleviate or alleviate the disease during its progression can be considered an important solution for the patient's health.

Accordingly, the present inventors made efforts to develop a stem cell culture medium that can be used clinically for application as a treatment for immune or inflammatory diseases. As a result, by comparing the culture medium obtained according to existing two-dimensional culture, a biocompatible scaffold was developed in a serum-free medium. The mesenchymal stem cell culture medium obtained through 3D culture does not induce an immune response, but reduces the secretion of TNF-α and IFN-γ, which are inflammatory cytokines that play a role in increasing immune reactivity, and is an immunomodulatory factor. By confirming that phosphorus PGE2 is secreted at a high concentration, the present invention was completed by demonstrating that the culture medium obtained according to the above method can be used for the prevention or treatment of immune diseases or inflammatory diseases.

The purpose of the present invention is to provide a method for producing mesenchymal stem cell culture medium 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 preventing or treating 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 containing at least one of bEGF or EGF as an active ingredient. will be.

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

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

(b) Provides a method for producing a mesenchymal stem cell culture medium for preventing or treating immune diseases or inflammatory diseases, including the step of collecting the culture medium.

Additionally, the present invention provides a mesenchymal stem cell culture medium prepared by the above method for preventing or treating immune diseases or inflammatory diseases.

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

The stem cell culture medium obtained by three-dimensionally culturing mesenchymal stem cells using a biocompatible scaffold in a serum-free medium according to the method of the present invention contains more immunomodulatory factors than the stem cell culture medium obtained by two-dimensional culture according to the conventional method. Since PGE2 is secreted in high concentration and has excellent effects in suppressing immune responses and inflammatory cytokine secretion, it can be usefully used as a composition for preventing or treating immune diseases or inflammatory diseases.

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

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

As used herein, “mesenchymal stem cells” are capable of self-proliferation, have pluripotency, and have the following cell phenotypes: CD73 ⁺ , CD90 ⁺ , CD105 ⁺ , CD14 ^- , CD20 ^- , CD34 ^- , CD45 ^- It refers to cells that represent cells and can be isolated from bone marrow, adipose tissue, umbilical cord blood, etc., but is not limited thereto.

In this specification, “biologically active substances” collectively refer to cytokines, cell growth factors, immune regulators, etc. that can affect the function of cells or the body. Examples of bioactive substances include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), tumor growth factor-beta (TGF-beta), and insulin growth factor (IGF). It is not limited to this.

As used herein, “stem cell culture medium” refers to the cell culture supernatant obtained by culturing stem cells. Stem cell culture medium contains various bioactive substances secreted from cells during the stem cell culture process.

In this specification, a biocompatible scaffold refers to a support that is made of a material that has affinity for cells and has a so-called cell-adhesive surface, and is capable of attaching and culturing cells in three dimensions. Examples of naturally derived supports include, but are not limited to, alginate, protein, collagen, fibrin, hyaluronic acid, and cellulose. Examples of synthetic polymers include, but are not limited to, poly(alpha-hydroxy acid) series, poly(vinyl alcohol), and polyanhydride.

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

This invention

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

(b) Provides a method for producing a mesenchymal stem cell culture medium for preventing or treating immune diseases or inflammatory diseases, including 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 cultured in a matrix medium and a proliferation medium and then subcultured.

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

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

In step (b), the biocompatible scaffold is a cell scaffold having a cell adhesive surface and is preferably a natural or synthetic polymer, for example, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly(alpha-hydride). It may be hydroxy acid) series, poly(vinyl alcohol), polyanhydride, etc., but is not limited thereto.

The above immune diseases include Behçet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatomyositis, Goodficher syndrome, autoimmune meningitis, Sjogren's syndrome, and systemic erythematosus. Lupus reflexes, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre 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, graft-versus-host disease, or obesity. It may be possible, but it is not limited to this.

The inflammatory disease includes gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascial disease, viral infection, It may be bacterial infection, fungal infection, burn, surgical or dental wound, PGE excess syndrome, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, or acute and chronic inflammatory diseases. It is not limited to this.

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

The mesenchymal stem cell culture medium is preferably used to reduce 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 contains 5000 pg/ml to 10000 pg/ml of PGE2, and contains 7900 pg/ml to 9800 pg/ml of PGE2. It is more desirable.

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

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

In addition, the present inventors confirmed that PGE2, an immune regulator, in the mesenchymal stem cell culture medium obtained according to the above three-dimensional culture method was significantly increased compared to the mesenchymal stem cell culture medium obtained according to the two-dimensional culture method (Figure 4 reference).

Therefore, according to the method of the present invention, mesenchymal stem cells are 3D cultured using a biocompatible scaffold in a serum-free medium, and the stem cell culture medium contains a high concentration of PGE2 and has an excellent effect of suppressing immune response and secretion of inflammatory cytokines. By confirming, the present invention can be usefully used in a method of producing a mesenchymal stem cell culture medium for preventing or treating immune diseases or inflammatory diseases.

Cultivation of mesenchymal stem cells according to the present invention can be carried out according to the following process, and the medium used for cell culture is not limited to this.

(1) Culture of mesenchymal stem cells

Mesenchymal stem cells obtained from the corresponding tissue are suspended in matrix medium, inoculated into a culture vessel at a concentration of 10,000 to 40,000 cells/㎠, and 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 is cultured for about 24 hours.

(2) Culture in expansion media

After removing the substrate medium, adherent cells are proliferated by culturing in proliferation medium. The growth medium is DMEM or DMEM/F12 containing 10% 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/ml. It rapidly proliferates adherent mesenchymal stem cells and increases the amount of cells in a large amount of time 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 trypsin treatment. For subculture, cells are diluted 1:3 to 1:4 and cultured with proliferation medium in a new culture vessel. Additional subculture is possible in this way.

(4) Culture with biocompatible scaffolds

The cultured cells were washed three or more times with PBS to remove FBS, and then cultured in a serum-free medium in a form attached to a biocompatible scaffold. Cell culture within the scaffold does not require conventional cell culture containers, so large quantities can be cultured in sterile bottles or culture bags, which has the advantage of conveniently culturing at lower costs.

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

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

The mesenchymal stem cell culture medium is preferably used to reduce 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 contains 5000 pg/ml to 10000 pg/ml of PGE2, and contains 7900 pg/ml to 9800 pg/ml of PGE2. It is more desirable.

According to the manufacturing method of the present invention, mesenchymal stem cells are three-dimensionally cultured using a biocompatible scaffold in a serum-free medium, and the stem cell culture medium has an excellent effect of suppressing immune response and secretion of inflammatory cytokines, thereby preventing immune diseases. Alternatively, it can be usefully used as a composition for preventing or treating inflammatory diseases.

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

The biocompatible scaffold is a cell scaffold having a cell adhesive surface and is preferably a natural or synthetic polymer, for example, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly(alpha-hydroxy acid) series, It may be poly(vinyl alcohol), polyanhydride, etc., but is not limited thereto.

The serum-free medium can be any basic medium known in the art without limitation, but one to which serum is not added is used. The basic medium can be prepared by artificial synthesis, or a commercially produced medium can also 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 can be preferably used.

It is preferable that 0.5 to 2 ng/ml of bFGF is 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 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 through examples and experimental examples.

However, the following examples and experimental examples only illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

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

Adipose tissue can usually be obtained by, but is not limited to, liposuction. Adipose-derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction as follows: To remove blood, the adipose tissue was washed 3-4 times with an equal volume of KRB solution. Next, the same volume of collagenase solution as the fat tissue was added and reacted in a 37°C water bath. This was transferred to a centrifugation tube and centrifuged at 20 and 1500 rpm for 10 minutes . After centrifugation, the supernatant, the fat layer, was removed, and the lower layer, the collagenase solution, was separated carefully so as not to shake. After adding substrate medium and suspending it, it was centrifuged at 20°C and 1500 rpm for 5 minutes. At this time, what settled at the bottom was the stroma-vascular fraction, and the supernatant was removed. The stromal-vascular fraction was suspended in 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, washing with phosphate buffer solution and adding matrix medium (DMEM/F12 medium containing 10% FBS), or growth medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng/ml in matrix medium, or Proliferation was performed using a substrate medium containing epidermal growth factor (EGF) at a concentration of 5 ng/ml. When the adipose-derived mesenchymal stem cells grew to about 80-90% of the culture vessel, they were trypsinized and separated into single cells. The obtained cells were diluted 1:3 to 1:4 with proliferation medium and subcultured.

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

Stem cells obtained as single cells by trypsin treatment 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. Next, DMEM/F12 solution containing 1 ng/ml basic fibroblast growth factor and 5 ng/ml epidermal cell growth factor was added to 500 ml per 4.0 × 10 ⁷ cells. After 72 hours, the cell culture was recovered, and the cell culture was recovered 2 to 11 times more by adding new medium and stored in the refrigerator.

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

Stem cells obtained as single cells by trypsin treatment 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. The cell suspension and fibrinogen dilution were mixed using a dual syringe and then three-dimensionally cultured in a bottle or culture bag. As a cell culture medium, DMEM/F12 solution containing 1 ng/ml basic fibroblast growth factor and 5 ng/ml epidermal cell growth factor was used. After 72 hours, the cell culture was recovered, and the cell culture was recovered 2 to 11 times more by adding new medium and stored in the refrigerator.

<Experimental Example 1> Immune response of mesenchymal stem cell culture medium obtained by 3D culture method Suppression check

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

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

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

<Experimental Example 2> Confirmation of inhibition of inflammatory cytokine secretion in mesenchymal stem cell culture medium obtained by 3D culture method

In order to confirm whether the mesenchymal stem cell cultures obtained in <Example 2> and <Example 3> inhibited inflammatory cytokine secretion, 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. Next, two sets of mesenchymal stem cell culture medium obtained by the two-dimensional culture method of <Example 2> or the mesenchymal stem cell culture medium 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 the 3rd day of 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, the secretion amounts of TNF-α and IFN-γ were significantly reduced in the group treated with the mesenchymal stem cell culture medium obtained by the three-dimensional culture method of <Example 3>. This was confirmed (Figures 2 and 3).

Therefore, the mesenchymal stem cell culture medium obtained by the three-dimensional culture method through <Experimental Example 1> and <Experimental Example 2> above 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 immune diseases or inflammatory diseases by reducing them.

<Experimental Example 3> Confirmation of immune regulatory factors in mesenchymal stem cell culture medium obtained by 3D culture method

In order to confirm the level of secretion of prostaglandin E2 (PGE2), an immune regulator, 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 solution obtained, and absorbance was measured at 450 nm.

As a result, as shown in Figure 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>. (Figure 4).

Therefore, the mesenchymal stem cell culture medium obtained by the three-dimensional culture method through <Experimental Example 1> to <Experimental Example 3> above 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 relieve and heal immune diseases or inflammatory diseases by increasing immune reactivity by secreting high concentrations of PGE2, an immune regulator.

Claims (7)

(a) Mesenchymal stem cells showing a cell phenotype of CD73 ⁺ , CD90 ⁺ , CD105 ⁺ , CD14 ^- , CD34 ^- , CD45 ^- were collected and grown with at least one of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor). 3D culturing with a biocompatible scaffold in a serum-free medium supplemented with; and
(b) collecting only the culture medium to obtain a mesenchymal stem cell culture medium containing more than 5,000 pg/mL of PGE ₂ (prostaglandin E2),
Here, the mesenchymal stem cell culture medium suppresses the immune response and the secretion of TNF-α or IFN-γ by the immune response,
A pharmaceutical composition for preventing or treating immune diseases, comprising a mesenchymal stem cell culture medium as an active ingredient.
According to clause 1,
A pharmaceutical composition, characterized in that the mesenchymal stem cells in (a) are isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.
According to clause 1,
In (a), the mesenchymal stem cells are a pharmaceutical composition, characterized in that the mesenchymal stem cells are cultured in a matrix medium and a proliferation medium and then subcultured.
According to clause 1,
In (a), the three-dimensional culture is a pharmaceutical composition characterized in that stem cell culture is obtained three or more times while changing the medium at three-day intervals.
According to clause 1,
In (a), the biocompatible scaffold is a cell support having a cell-adhesive surface and is a pharmaceutical composition characterized in that it is a natural or synthetic polymer.
According to clause 5,
The biocompatible scaffold is characterized in that it is 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 made of.
According to clause 1,
Immune diseases include Behcet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatomyositis, Goodfeitzer syndrome, autoimmune meningitis, Sjogren's syndrome, and systemic erythematosus. Lupus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia , from the group consisting of 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. A pharmaceutical composition, any one of which is selected.