KR101479566B1 - Composition for improvement atopy using protein secreted from stem cells - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a composition for improving atopy using a stem cell-secreted protein, and more particularly, to a composition for culturing a stem or a bone marrow-derived stem cell cultured under a suitable medium and conditions.

Description

TECHNICAL FIELD The present invention relates to a composition for improving atopy using stem cell secretory proteins,

The present invention relates to a composition for improving atopy using a stem cell-secreted protein that uses a culture fluid of a tooth or bone marrow-derived stem cell cultured in a conditioned medium for improving atopy.

Atopic dermatitis (AD) is the most common inflammatory skin disease in infants and children. It is characterized by severe pruritus, erythema, edema, exudation, exfoliation, phlegmatization, and recurrent and chronic progression. Secondary lesions such as fusiformis are characteristic. It is also a chronic inflammatory disease of the skin that is associated with asthma allergic rhinitis and food allergy. Recently, atopic dermatitis has been increasing in adults, and it has been suggested that adults, at risk for atopic dermatitis, have facial and neck lesions, and animals, pollen and nickel allergy. The symptoms of atopic dermatitis are usually 2-6 months, and it is reported that the symptoms are most prevalent under 1 year of age and 97% occur before 5 years of age.

It is known that many cells (T lymphocytes, Langerhans cells, eosinophils, keratinocytes) and factors (cytokines and immunoglobulins, especially IgE) are involved in causing atopic dermatitis. At the early stage of atopy, IL4 and IL10 are excessively secreted in Th2-type T cells and thus IL-12 is unstable and IFN-gamma secretion is decreased.

Recently, it has been reported that bone marrow-derived stem cells have immunomodulatory functions for autoimmune diseases, and attention has been paid to reports that the immune regulatory function of oral stem cells is superior to that of bone marrow-derived stem cells.

1. Korean Patent Publication No. 2010-0105166, September 29, 2010 2. Korean Patent Publication No. 2011-0001830, 2011.01.06 3. United States Patent Publication No. 2009-0246181, October 1, 2009

It is an object of the present invention to use a stem cell culture fluid containing various growth factors and cytokines to improve atopy.

In order to achieve the above object, the present invention provides a composition for improving atopy comprising a stem cell culture fluid derived from a tooth or bone marrow as an active ingredient.

The present invention also relates to a method for culturing mesenchymal stem cells derived from a tooth or bone marrow in a conditioned medium; And

And a step of obtaining the culture solution obtained in the above step.

According to one embodiment, the mesenchymal stem cell culture fluid derived from the tooth

Dividing the cells of the isolated size, periodontal ligament, alveolar bone, apical root or gum into a serum medium containing bFGF to obtain mesenchymal stem cells from the tooth;

Culturing the mesenchymal stem cells in a serum-free medium containing bFGF and IL-b1 for 1 to 2 days to prepare a conditioned medium; And

And then obtaining a culture solution obtained from the conditioned medium.

According to another embodiment, the bone marrow-derived mesenchymal stem cell culture solution

Subculturing the isolated bone marrow-derived mesenchymal stem cells in a serum medium containing glucose and glutamine;

Culturing the mesenchymal stem cells in a serum-free medium and IL-b1 for 1 to 2 days to prepare a conditioned medium; And

And then obtaining a culture solution obtained from the conditioned medium.

The present invention relates to a culture of adult stem cells or an active ingredient thereof, particularly a stem cell culture derived from oral teeth or bone marrow, having excellent therapeutic activity for atopic regeneration. Since the composition of the present invention contains a complex of active proteins secreted by stem cells rather than stem cells, it can minimize immunological problems, safety problems, pharmaceutical problems, and individual variations that may occur when stem cells are used.

FIGS. 1 and 2 show the results before and after the treatment of the stem cell culture medium in the atopy-induced mouse model.
FIG. 3 shows the result of measurement of blood IgE concentration before and after treatment of stem cell culture in an atopy-induced mouse model.
FIG. 4 is a graph showing the results of an experiment comparing the pre-treatment (teething) and bone marrow paracrine factors before and after treatment of a stem cell culture medium in an atopy-induced mouse model.
<Explanation of Terms in Drawings>
Normal: DW treatment (without causing atopy)
Vehicle: acetone, olive oil (4: 1) (without atopy induction)
Control: distilled water application (after atopy induction)
ABCM: Culture medium obtained from bone marrow-derived stem cells (treated with IL-b1 5pg / mL)
ADCM: Cultures from Adult Dimension Stem Cells (treated with IL-b1 5 pg / mL)
ASCM: Culture medium (treated with IL-b1 5 pg / mL)
CSCM: Culture medium obtained from stromal stem cells
SHED: Teeth (teething) stem cells
BMMSC: Mesenchymal mesenchymal stem cells

Hereinafter, the configuration of the present invention will be described in detail.

The present inventors have been studying atopic skin diseases among autoimmune diseases and maintaining their activity in the human body while studying the development of a low price and a manufacturing process. The inventors of the present invention found that the oral cells or bone marrow- . Therefore, the components of the culture solution of the stem cells derived from the teeth or bone marrow in the oral cavity were analyzed, the manufacturing process was simply manufactured, and lyophilization was performed to maximize the activity of the protein. The present inventors have completed the present invention by proving that the composition of the culture solution of stem cells derived from a tooth or bone marrow has an excellent effect on the improvement of atopy through various animal experiments.

Accordingly, the present invention relates to a composition for improving atopy comprising a stem cell culture fluid derived from a tooth or bone marrow as an active ingredient.

The term "mesenchymal stem cells (MSCs)" in the present invention refers to cells that aid in the production of cartilage, bone, fat, bone marrow stroma, muscles and nerves. Cord blood, peripheral blood, other tissues, and the like, and means cells obtainable therefrom. For the purpose of the present invention, the mesenchymal stem cells of the present invention means mesenchymal stem cells derived from a tooth or bone marrow.

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In the present invention, "differentiation" means a phenomenon in which the structure or function of a cell is specialized while cells are proliferating and growing. Pluripotent mesenchymal stem cells can be differentiated into different types of progenitor cells (for example, osteoblasts, etc.) after differentiation into systematic limited progenitor cells (such as mesodermal cells) Bone cells, etc.) that perform a distinctive role in bone marrow, bone, and the like.

The mesenchymal stem cell culture fluid from the tooth or bone marrow may contain the paracrine factor shown in Fig. 4 secreted from the stem cells.

The stem cell culture medium derived from a tooth or bone marrow of the present invention is cultured in a medium supplemented with IL-b1, and the expression of IFN-y (autoimmune factor lowering substance) is increased, and the disease site of the atopy- , Physiological saline, water, or the like, or autoimmune factors such as IGE, IL-4 and IL-10 are reduced during vascular administration, and atopic wounds are cured.

The tooth-derived mesenchymal stem cell culture solution

Dividing the cells of the isolated size, periodontal ligament, alveolar bone, apical root or gum into a serum medium containing bFGF to obtain mesenchymal stem cells from the tooth;

Culturing the mesenchymal stem cells in a serum-free medium containing bFGF and IL-b1 for 1 to 2 days to prepare a conditioned medium; And

And then obtaining a culture solution obtained from the conditioned medium.

The bone marrow-derived mesenchymal stem cell culture broth

Subculturing the isolated bone marrow-derived mesenchymal stem cells in a serum medium containing glucose and glutamine;

Culturing the mesenchymal stem cells in a serum-free medium and IL-b1 for 1 to 2 days to prepare a conditioned medium; And

And then obtaining a culture solution obtained from the conditioned medium.

The mesenchymal stem cells can be obtained through purification in the tissues of the dental, periodontal ligament, alveolar bone, apical or gingival, or bone marrow, and the process of extracting the stem cells according to the characteristics of each tissue can be performed by a known method And it is now known that mesenchymal stem cells can be easily and easily obtained because human mesenchymal stem cells can be used for research purposes.

The mesenchymal stem cells used in the present invention can also be obtained by purchasing a cell line from Lonza, which can be easily cultured in a basic medium.

The essential processes involved in the culturing process of mesenchymal stem cells are disclosed in the literature, and the biopsy extracted from the source through known methods such as bone marrow, size, periodontal ligament, alveolar bone, Etc. can minimize the potential for contamination in subsequent cultures through repeated washing procedures using washing media containing common antibiotics.

The medium for the initial stage cell culture, including serum, is preferably a medium suitable for maintaining and maintaining cell types such as mesenchymal stem cells. In the present invention, DMEM (Dulbecco's Modified Eagle's Medium) and aMEM (alpha minimal essential medium), which are generally used for cell culture, are used and include sera commonly used for cell culture.

Serum is preferably supplemented with fetal bovine serum (FBS) of 0.1 to 20%, and it is advisable to add antibiotics, antifungal agents and a protector to prevent the growth of mycoplasma causing contamination.

As the antibiotic, antibiotics commonly used for cell culture such as penicillin, streptomycin or fungizone can be used. Amphotericin B is preferred as an antifungal agent, tylosin as a mycoplasma inhibitor, and gentamicin, ciprofloxacin, and azithromycin can prevent mycoplasma contamination.

If necessary, an oxidative nutrient such as glutamine and an energy metabolite such as sodium pyruvate may be further added.

According to one embodiment, the medium contains glucose and DMEM supplemented with 1 to 2 mM glutamine, 0.5 to 1 mM sodium pyruvate, 0.1 to 10% FBS, 1% antibiotic (100 IU / mL) . At this time, the concentration range of glucose may be approximately 1 g / L to 4.5 g / L. The complete serum medium provides for stable and basic cell culture conditions for the storage and maintenance of bone marrow or umbilical cord blood-derived stem cells and in vitro stabilization of cells.

The general culture conditions for the initial culture are as follows. The cells are cultured in a humidity of 90 to 95% at a temperature of 35 to 39 ° C in a 5 to 10% CO ₂ incubator under the most suitable condition for cell culture. When 5 to 10% A carbon control material such as sodium bicarbonate may be added in an amount of 0.17 to 0.22% by weight.

The medium for the stem cell passage culture of the present invention may further comprise bFGF.

During the initial incubation step, tissue fragments are preferably maintained attached to the bottom of the culture vessel and can be stimulated by short stimulation resulting from trypsin-EDTA treatment according to standard techniques of cell culture.

The cumulative population doubling time is maintained until the cells in the flask are confluent at 75-85% confluence, preferably at 80% confluence, and subcultured.

In the step cultured cells, the oral cells or bone marrow-derived stem cells are cultured in serum-free medium for 48 hours to prepare conditioned medium.

The conditional medium can be supplemented with IL-b1 at 1 to 10 pg / mL of medium to increase IFN-y.

The term "conditioned medium " as used herein refers to a medium in which cells are exchanged with a serum-free medium after reaching the logarithmic growth phase, which is the highest stage of cell division, and only the culture medium is collected. It refers to the use of an unknown growth factor, cytokine, extracted from the dividing cells in the medium. For the purpose of the present invention, the conditioned medium composition of the present invention is a composition comprising a solution obtained by removing intraoral or bone marrow-derived mesenchymal stem cells from a medium in which oral cavity or bone marrow-derived mesenchymal stem cells are cultured, And a growth factor derived from mesenchymal stem cells derived from bone marrow or bone marrow. The composition can be precipitated by ethanol precipitation and then lyophilized to produce a powdery preparation have. Therefore, the mesenchymal stem cell culture fluid derived from a tooth or bone marrow may be in the form of a lyophilized powder or in a liquid form.

The composition for improving atopy of the present invention may be a cosmetic composition.

The components contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to the culture medium of mesenchymal stem cells derived from teeth or bone marrow as an active ingredient. Examples thereof include antioxidants, stabilizers, solubilizers, vitamins, pigments And customary adjuvants such as perfumes, and carriers. In addition, the cosmetic composition may further include a skin absorption promoting substance to improve its effect.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder. When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether. When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters. In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

In addition, the composition of the present invention can be manufactured from a pharmaceutical composition.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes such as oral or parenteral routes such as oral, rectal or intravenous, muscular, subcutaneous, intra-uterine, Can be administered by injection. Preferably, it is applied by transdermal administration during parenteral administration, more preferably by topical application by application.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, The dosage of the pharmaceutical composition of the present invention can be administered in an amount of 0.1-100 mg / kg on an adult basis, once or several times a day, in the case of an oral formulation, It is preferable to apply 1 to 5 times a day in an amount of 1.0 to 3.0 ml per day and continue for 1 month or longer. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in any form suitable for pharmaceutical preparations including oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations such as ointments and creams, suppositories and sterile injectable solutions, , Dispersants, or stabilizers.

The present invention also

Culturing a mesenchymal stem cell from a tooth or bone marrow in a conditioned medium; And

And a step of obtaining the culture solution obtained in the above step.

According to one embodiment, the mesenchymal stem cell culture solution derived from the tooth

Dividing the cells of the isolated size, periodontal ligament, alveolar bone, apical root or gum into a serum medium containing bFGF to obtain mesenchymal stem cells from the tooth;

Culturing the mesenchymal stem cells in a serum-free medium containing bFGF and IL-b1 for 1 to 2 days to prepare a conditioned medium; And

And then obtaining a culture solution obtained from the conditioned medium.

According to another embodiment, the bone marrow-derived mesenchymal stem cell culture liquid

Culturing the mesenchymal stem cells derived from the separated bone marrow in a serum-free medium containing glucose and glutamine;

Culturing the mesenchymal stem cells in a serum-free medium and IL-b1 for 1 to 2 days to prepare a conditioned medium; And

And then obtaining a culture solution obtained from the conditioned medium.

The process of subculturing the stem cells and preparing the conditioned medium is as described above.

The conditional medium culture solution is collected to prepare a desired composition for atopic amelioration of the present invention.

The method for obtaining the conditional medium can be carried out by a method known in the art and can be collected by centrifugation or filtration.

The conditioned medium may be precipitated by ethanol precipitation method followed by lyophilization to produce a powdery preparation. Therefore, it may be in the form of a freeze-dried powder or a liquid form.

It is to be understood by those skilled in the art that these embodiments are only for describing the present invention more specifically and that the scope of the present invention is not limited by these embodiments.

Example 1 Obtaining Oral Stem Cells

Oral stem cell SHED was a cell isolated from a 6 - year - old boy. The extracts were extracted from collagenous lysates and then treated with collagenase (3 mg / mL) and dysphase (4 mg / mL) for 1 hour at 37 ° C. and then passed through a 70 μm cell strainer and separated into one cell. Separated cells were inactivated with aMEM containing 10% FBS and then replaced with a culture medium containing aMEM, 50 μg ascorbic acid, 10 ng bFGF, 1% penicillin-streptomycin, or amphotericin B Incubated in a 5% CO ₂ incubator under conditions of 90% humidity and about 37 ° C temperature.

For the subculture, the flask from which the culture solution had been removed was washed with PBS, cells were dropped with 0.25% Trypsin-EDTA (GIBCO), the cell suspension was centrifuged, and cell count and viability were examined. The medium used for the subculture was 50 μg ascorbic acid, 10 ng bFGF, 10% bovine serum, 1% penicillin-streptomycin or aMEM medium supplemented with amphotericin B as an antifungal agent.

When the confluency of the cells reached about 70-80%, the culture was removed from the cultured medium using a pipette, washed twice with phosphorylation buffer, and then washed with aMEM, 50 μg ascorbic acid, 10 ng bFGF , DMEM medium supplemented with penicillin-streptomycin or phonzyme, and IL-b1 (5 pg / mL) were incubated in a 5% CO ₂ incubator for 24-48 hours at a temperature of about 90% and a temperature of about 37 ° C.

Example 2 Obtaining of bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells hMSCs were purchased from human mesenchymal stem cells (Lonza, PT-2501) and were isolated from the bone marrow of a 22 year old healthy woman.

In Example 2, early passage culture of bone marrow-derived mesenchymal stem cells was performed by adding 4.5 g / L of D glucose, 584 mg / L of L-glutamine, 110 mg / L of sodium pyruvate 10% FBS, 1% penicillin-streptomycin Was added to DMEM supplemented with amphotericin B as an antifungal agent and incubated in a 5% CO ₂ incubator at a temperature of about 90% humidity and a temperature of about 37 ° C.

For the subculture, the flask from which the culture solution had been removed was washed with PBS, cells were dropped with 0.25% Trypsin-EDTA (GIBCO), the cell suspension was centrifuged, and cell count and viability were examined. The medium used for the subculture was DMEM medium supplemented with 4.5 g / L of D glucose, 584 mg / L of L-glutamine, 110 mg / L of sodium pyruvate, 1% of penicillin-streptomycin or an antifungal agent Respectively.

When the confluency of the cells reached about 70-80%, the culture was removed from the cultured medium using a pipette, washed twice with phosphorylation buffer, and then added with 4.5 g / L of D glucose, 584 mg After adding DMEM medium supplemented with L-glutamine, 110 mg / L sodium pyruvate, 1% penicillin-streptomycin or phonzyme and IL-b1 (5 pg / mL), the cells were incubated at about 90% And incubated for 24-48 hours in a 5% CO ₂ incubator under temperature conditions.

Example 3 Preparation of Conditioned Medium Powder Formulation

Specifically, bone marrow-derived mesenchymal stem cells and oral mesenchymal stem cells were cultured under the conditions of Examples 1 and 2 for 24-48 hours, and then 50 mL of the culture solution was centrifuged at 1500 rpm for 5 minutes. After centrifugation at 3000 rpm for 2-3 minutes, the remaining suspension was removed, and 100% ethanol was added 2-3 times and left at -20 ° C for one hour. Thereafter, centrifugation was carried out at 15,000 rpm at 4 DEG C for 15 minutes. The precipitate was then treated with 90% ethanol and centrifuged again at 15000 rpm for 5-10 minutes. After centrifuging the precipitate, 2.5 mL of sterilized water was added to dissolve the precipitate, and 500 μL of the solution was dispensed into a 1.8 mL tube. The solution was left in a -80 ° C. diffuser for 2-3 hours. Then, lyophilization was carried out for 8 hours and then powdered.

<Example 4> Experimental study for improvement of atopy by growth factors

(Animal culture)

Six week old male BALB / c mice were supplied from Orient Bio and used in the experiments. A total of 21 experimental animals were divided into normal group, vehicle, control group, and sample treatment group. The animals were divided into three groups: normal group, vehicle 3, control group 3, and sample 12. The samples were divided into 3 ABCM, 3 ADCM, 3 ASCM and 3 CSCM. Breeding conditions were maintained at 21 ~ 23 ℃, 40 ~ 60% humidity and 12 hours light intensity. The test animals were used for post - purification treatment for 7 days after delivery.

(Induced atopic dermatitis)

The entire back of the BALB / c mouse from the lower part of the ear to the upper part of the tail was removed and allowed to stand for 24 hours. DNCB (2,4-dinitrochlorobenzene) used for skin irritation was dissolved in a 4: 1 ratio of acetone-olive oil. First, 100 μl of a 1% DNCB solution was applied to the epilating site on day 0 and day 3 for skin sensitization. On the 7th day and the 10th day, 100 占 퐇 of a 0.2% DNCB solution was applied in the same manner as before. After the induction of atopic dermatitis, 100 μl of each test substance (ABCM, ADCM, ASCM, CSCM) was applied to the atopic dermatitis inducing site once a day for a total of 7 days. In the normal group, no treatment was performed, and the vehicle was coated with acetone-olive oil. In the control group, atopic dermatitis was induced and treated with the same amount of distilled water to evaluate the atopy improvement effect of each test substance.

(Measurement of blood IgE concentration)

Blood was collected from BALB / c mice and centrifuged at 13,000 rpm for 10 minutes to isolate only the supernatant serum. The thus obtained serum was fractionated and stored at -70 ° C, and IgE quantification was carried out by ELISA. The primary antibody and 0.1 M sodium carbonate solution (pH 9.5) were diluted 1: 250 and coated on 96-well plates at 4 ° C for at least 16 hours. The serum and assay diluent were diluted 1: 250 and reacted for 2 hours After binding the primary antibody with biotin, the absorbance was measured at 450 nm using horseradish peroxidase (HRP) and 3,3 ', 5,5'-tetramethyl benzidine (TMB).

As shown in Figs. 1 to 4, the culture medium of mesenchymal stem cells derived from a tooth or bone marrow remarkably decreased the content of IgE in the blood, and confirmed that the atopic wounds were healed in an atopy-induced mouse model.

Claims (12)

The present invention relates to a method for producing a mesenchymal stem cell, which comprises culturing a mesenchymal stem cell derived from a tooth or bone marrow in a serum-free medium, ethanol-precipitating the conditioned medium and lyophilizing the precipitate,
The conditioned medium of the mesenchymal stem cells derived from the teeth can be obtained by dividing the cells of the isolated size, the periodontal ligament, the alveolar bone, the apical root, or the gingium in a serum medium containing bFGF to obtain mesenchymal stem cells derived from the tooth, The mesenchymal stem cells are cultured in a serum-free medium containing bFGF and IL-b1 for 1 to 2 days,
The conditioned medium of bone marrow-derived mesenchymal stem cells is obtained by subculturing the mesenchymal stem cells derived from the isolated bone marrow in a serum medium containing glucose and glutamine, and the mesenchymal stem cells are cultured in serum-free medium and 1-2 ml The composition for improving atopy, which is a culture solution obtained by culturing for a day.
delete The method according to claim 1,
Wherein the powder obtained from a tooth or bone marrow-derived mesenchymal stem cell comprises the paracrine factor shown in Fig.
delete delete The method according to claim 1,
Wherein IL-b1 is contained in an amount of 1 to 10 pg per 1 mL of the medium.
delete The method according to claim 1,
Wherein the composition is a cosmetic composition.
The method according to claim 1,
Wherein the composition is a pharmaceutical composition.
delete delete delete

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