KR20220063839A - Mesenchymal stem cell culture media for promoting wound healing or skin regeneration, and preventing alopecia or activating hair growth and a method for preparing the same - Google Patents

Abstract

The present invention relates to a mesenchymal stem cell culture medium for healing wound or regenerating skin, preventing hair loss, or promoting hair growth, and a method for preparing the same, and more specifically, to a mesenchymal stem cell culture medium for healing wound or regenerating skin, and preventing hair loss, or promoting hair growth which comprises a high content of growth factors and cytokines by a 3-dimensional culture method using sodium alginate and a preparation method thereof.

Description

Mesenchymal stem cell culture media for promoting wound healing or skin regeneration, and preventing alopecia or activating hair growth and a method for preparing the wound healing or skin regeneration, and preventing hair loss or promoting hair growth same}

The present invention relates to a mesenchymal stem cell culture medium for wound healing or skin regeneration, prevention of hair loss or hair growth promotion, and a method for producing the same, and more specifically, a high content of growth factors and cytokines by a three-dimensional culture method using sodium alginate. It relates to a mesenchymal stem cell culture medium for wound healing or skin regeneration, preventing hair loss or promoting hair growth, and a method for producing the same, including Cain.

General wound healing is a continuous process that requires removal of damaged tissues and invading pathogens and remodeling of damaged tissues. ) Proliferative process in which epithelial cells around the wound move and proliferate by stimulation of growth factors secreted by leukocytes to cover the damaged area, and fibrous cells in the dermis layer to accumulate collagen to create new capillaries and fill blemishes to form epithelialization, 3 ) The inflammatory cells disappear and the temporarily created granulation tissue goes through a maturation process that matures close to the original skin tissue. This repair process includes various growth factors and cytokines - Insulin-like Growth Factor (IGF), Transforming Growth Factor beta (TGF-β), Vascular Endothelial Growth Factor ; VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), granulocyte monocyte colony stimulating factor (Granulocyte-) It proceeds through the action of Macrophage Colony Stimulating Factor (GM-CSF), Epidermal Growth Factor (EGF), and Hepatocyte Growth Factor (HGF), and also inflammatory cells, fibroblasts, keratinocytes and It proceeds through the interaction of different types of cells, such as endothelial cells.

Common wounds can be easily healed through the sequential progression of the wound healing process, and skin regeneration or wound treatment involves inflammation, re-epithelialization, granulation tissue, and fibrous tissue. It is achieved through various processes such as fibroplasia and contraction of wound tissue (Freedberg et al., J. Clin. Psychol., 57: 433-455, 2001). The series of processes of skin regeneration is achieved by the cooperation of several cells such as keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets, and the movement of these cells Complex processes such as , invasion, proliferation, and differentiation are regulated by various growth factors, cytokines, and chemokines, so the effectiveness of biological factors or chemicals is known.

However, the wound healing technology using the growth factors and cytokines requires a lot of cost to produce and isolate these proteins, and since these proteins act in a complex manner in the wound healing process, when only one type of protein is used, it is a wound healing agent provides partial wound relief, but there is a problem in actual commercialization due to the fact that it is ineffective, such as a long healing period and less than optimal response to treatment.

On the other hand, hair is ecologically derived from the epithelium as one of the skin appendages, and is formed from the second month of life. The types of hair are classified into thin hair (Vellus hair) and thick and hard terminal hair (terminal hair) according to the thickness. Hair growth is periodic and there is no temporary hair loss. Each hair follicle goes through intermittent phases of activity and rest. During the growing phase, that is, the anagen, cells of the hair bulb actively differentiate to form growing hair. 85% of human hair is in the growth phase of 3 to 6 years, 5% is in the degenerative phase where hair degeneration begins and proceeds for 2 to 3 weeks, and the remaining 10% is in the resting phase of 3 to 4 months when hair growth stops. On average, about 50 to 100 are lost per day. Causes of hair loss include heredity, nutritional disorders, endocrine disorders, tissue disorders, Demodex, head pressure, dandruff, autoimmunity, and local infection. Hair loss directly or indirectly affects hair loss, such as abnormalities in the metabolism of hormones and proteins in the hair follicles, which are closely related to hair formation, increased stress due to the increase in women's social activities as they enter the modern society, and nutritional imbalances from westernized eating habits. It is thought to be due to changes in many socio-cultural factors that can affect

Currently, hair loss prevention and treatment can be divided into medical approaches and oriental medicine approaches. Medical approaches include drug therapy, surgery, nutritional therapy, and diet. Currently, drugs that promote hair growth include Minoxidil (MXD) and Finasteride approved by the US Food and Drug Administration (FDA). 1996) Elsevier Science, 363-365). Therefore, there is a need to develop a composition for preventing hair loss and promoting hair growth that has excellent effects of preventing hair loss and promoting hair growth and has no side effects even after long-term use.

As stem cell research has recently become active, it has been revealed that stem cells can differentiate into skin cells and are more active than skin cells, so they can secrete a greater amount of various growth factors and even regulate immune responses. A method for treating diabetic wounds by transplanting them into the wound site of mice has been tried (J Diabetes Res. 2013; 2013:647107, Diabetes. 2013 Jul; 62(7):2588-94, Plast Reconstr Surg. 2011 Oct; 128(4):872-80). In addition, mesenchymal stem cells are known to secrete various growth factors and physiologically active substances during the culture process, and the cultured stem cell culture medium can be used for clinical purposes such as pharmaceuticals or cosmetic compositions.

However, bovine serum is usually used for culturing mesenchymal stem cells, and a culture medium containing bovine serum is inappropriate for clinical application. That is, since the use of animal-derived proteins such as bovine serum is limited for clinical application of cell culture, it is preferable to exclude bovine serum from culturing stem cells. Since bovine serum protein is absolutely necessary for the proliferation and maintenance of stem cells, a method for culturing mesenchymal stem cells in a serum-free medium while obtaining a culture medium containing a high concentration of physiologically active substances is required.

Accordingly, the present inventors have made efforts to develop a stem cell culture medium that has few side effects and can be clinically applied to the skin and a method for preparing the same. It was confirmed that a cell culture medium can be obtained, and growth factors and cytokines exhibiting wound healing and hair loss inhibitory effects are secreted at high concentrations in the obtained mesenchymal stem cell culture medium, and exhibit an excellent skin cell and dermal papilla cell proliferation promoting effect. . Accordingly, the present invention was completed by revealing that the culture solution obtained according to the above method can be used for wound healing or skin regeneration, and for preventing hair loss or promoting hair growth.

It is an object of the present invention to provide a method for preparing a mesenchymal stem cell culture medium containing a high content of growth factors and cytokines for wound healing or skin regeneration, and preventing hair loss or promoting hair growth.

Another object of the present invention is to provide a composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, containing the mesenchymal stem cells prepared by the above manufacturing method as an active ingredient.

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

(1) after mixing the suspension of mesenchymal stem cells and sodium alginate solution, dropping the mixed suspension into a solution containing divalent cations to form beads;

(2) After the bead formation in step (1), three-dimensional culture in a serum-free medium to which any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to obtain a mesenchymal stem cell culture medium It provides a method for preparing a mesenchymal stem cell culture medium containing growth factors and cytokines, including the step.

In addition, the present invention provides a pharmaceutical composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, containing the mesenchymal stem cells prepared by the above manufacturing method as an active ingredient.

In addition, the present invention provides a skin external preparation for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, containing the mesenchymal stem cells prepared by the above manufacturing method as an active ingredient.

In addition, the present invention provides a cosmetic composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, containing the mesenchymal stem cells prepared by the above manufacturing method as an active ingredient.

According to the method of the present invention, growth factors and cytokines exhibiting wound healing and hair loss inhibitory effects are secreted at high concentrations in the mesenchymal stem cell culture medium obtained by three-dimensional culture using sodium alginate having low viscosity, excellent skin cells and Since the dermal papilla cell proliferation promoting effect appears, the mesenchymal stem cell culture medium prepared by the manufacturing method according to the present invention can be usefully used as an active ingredient in a composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth.

In addition, the sodium alginate used in the method of the present invention is derived from a natural product and can exclude animal-derived substances in the process, so that a safe composition can be obtained.

1 is a diagram confirming the growth factors and cytokine patterns contained in a mesenchymal stem cell culture medium (3D CM) obtained by a three-dimensional culture method according to an embodiment of the present invention using an array.
2 is a view confirming the growth factors and cytokines contained in the mesenchymal stem cell culture medium (3D CM) obtained by the three-dimensional culture method according to an embodiment of the present invention using ELISA.
3 is a mesenchymal stem cell culture medium (2D CM) obtained by a two-dimensional culture method according to a comparative example of the present invention and a mesenchymal stem cell culture medium obtained by a three-dimensional culture method according to an embodiment of the present invention. It is a diagram comparing growth factors and cytokine patterns contained in (3D CM) using an array.
4 is a view confirming the content of HGF contained in the mesenchymal stem cell culture medium obtained by three-dimensional culture with sodium alginate of 2,000 CPS or more and 300 CPS or less according to a comparative example and an embodiment of the present invention.

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

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

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

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

the present invention

(1) after mixing the suspension of mesenchymal stem cells and sodium alginate solution, dropping the mixed suspension into a solution containing divalent cations to form beads;

(2) After the bead formation in step (1), three-dimensional culture in a serum-free medium to which any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to obtain a mesenchymal stem cell culture medium It provides a method for preparing a mesenchymal stem cell culture medium containing growth factors and cytokines, including the step.

In the method according to the present invention, the mesenchymal stem cells in step (1) may be isolated from fat, bone marrow, umbilical cord blood, liver or muscle, and more specifically, may be isolated from fat, but is not limited thereto. In particular, adipose-derived stem cells have the advantage of being relatively easy to access, simple to collect, and able to obtain a large amount from one individual.

In the method according to the present invention, the mesenchymal stem cells in step (1) may be subcultured after culturing in a stromal medium and a proliferation medium, and more specifically, cultured in the following steps:

a) culturing the mesenchymal stem cells in a stromal medium and then culturing in a proliferation medium; and

b) subculturing the cells in a growth medium.

The growth medium is preferably cultured by adding at least one of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) to the substrate medium.

In the above culturing method, it is preferable to culture the cells for one or more passages in the growth medium in step b), more preferably for two or more passages, but is not limited thereto.

In addition, the growth medium is preferably added to any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) to the matrix medium, specifically, the bFGF is 0.1 ~ 100 ng / ml, the EGF is 0.1 It is preferable to add ~ 100 ng/ml, the bFGF is 0.3 ~ 10 ng/ml, the EGF is 1 ~ 10 ng/ml is more preferably added, the bFGF is 0.5 ~ 5 ng/ml, the EGF is It is more preferable to add 3 to 7 ng/ml, but is not limited thereto.

In the method according to the present invention, the sodium alginate solution in step (a) preferably has a viscosity of 300 CPS or less. The sodium alginate is a natural extract, and the viscosity varies depending on the type, collection time, and method of brown algae, so the standards of the viscosity may be different for each manufacturer. For example, depending on the manufacturer, a viscosity of less than 240 CPS may be used as a low viscosity standard, a viscosity of 240 to 3500 CPS as a medium viscosity standard, a viscosity greater than 3500 CPS as a high viscosity standard, and a viscosity of less than 200 CPS as a low viscosity standard , 200 to 500 CPS may be used as the medium viscosity standard, a viscosity greater than 500 CPS may be used as a high viscosity standard, a viscosity of 300 CPS or less may be used as a low viscosity standard, and a viscosity of 2000 CPS or more may be used as the medium viscosity standard. In an embodiment of the present invention, sodium alginate sold with a viscosity of 300 CPS or less as a low viscosity standard and a viscosity of 2000 CPS or more as a medium viscosity standard was purchased and used, and mesenchymal using a sodium alginate solution having the low viscosity It was confirmed that the content of growth factors or cytokines exhibiting the efficacy of wound healing and hair growth promotion in the stem cell culture medium was significantly increased.

Accordingly, when the low viscosity standard is exceeded, for example, when a sodium alginate solution having a medium or high viscosity, specifically, a sodium alginate solution having a viscosity of 2000 CPS or more is mixed and cultured, wound healing and hair growth promotion in the mesenchymal stem cell culture medium It was newly confirmed that the efficacy of the mesenchymal stem cell culture medium may be lowered due to the small secretion of growth factors or cytokines showing efficacy.

In the method according to the present invention, the divalent cation in step (a) may be Ca ²⁺ , Mg ²⁺ , Sr ²⁺ or Ba ²⁺ , but is not limited thereto. Examples of the solution containing the divalent cation include calcium chloride (CaCl ₂ ), calcium lactate (C ₆ H ₁₀ O ₆ Ca·5H ₂ O), magnesium chloride (MgCl ₂ ), strontium chloride (SrCl ₂ ) or chloride and barium (BaCl ₂ ), but is not limited thereto.

In addition, the solution containing the divalent cation in step (a) may have a concentration of 0.5 to 3% by weight, but is not limited thereto.

In the method according to the present invention, it is preferable to add at least one of bFGF and EGF to the serum-free medium in step (2), specifically, the bFGF is 0.1 to 100 ng/ml, and the EGF is 0.1 to 100 ng/ml is preferably added, the bFGF is 0.3 to 10 ng/ml, and the EGF is 1 to 10 ng/ml is more preferably added, the bFGF is 0.5 to 5 ng/ml, and the EGF is 3 to It is more preferable to add 7 ng/ml, but is not limited thereto.

In the method according to the present invention, in the three-dimensional culture in step (2), it is preferable to obtain a culture medium three or more times, and specifically, while exchanging the medium at intervals of 2 to 4 days, more specifically at intervals of 3 days, 3 It is preferable to obtain a culture solution more than twice. In particular, in order to obtain a large amount of stem cell culture medium from a limited number of cells and to increase the content of growth factors and cytokines exhibiting wound healing and hair growth promoting efficacy contained in the culture medium, it is preferable to obtain a culture medium three or more times.

In the method according to the present invention, the growth factors and cytokines exhibit wound healing and hair growth promoting efficacy, specifically EGF, IL-6, VEGF, HGF, MIP-1α, MCP-1, MMP and TIMP. it is preferable More specifically, the EGF serves to promote cell proliferation, promote angiogenesis, promote hair growth, and switch between hair growth. The IL-6 is involved in anti-inflammatory function, hair growth inducing function. The VEGF is involved in the function of promoting angiogenesis and promoting hair growth. The HGF is involved in anti-inflammatory, cell migration and proliferation promotion, apoptosis inhibition, angiogenesis promotion, and hair growth promoting function. The MIP-1α is involved in promoting angiogenesis, promoting collagen formation, and promoting hair growth. The MCP-1 is involved in promoting cell migration and proliferation, promoting angiogenesis, and promoting hair growth. The MMP is involved in matrix protein degradation, tissue remodeling, promoting cell migration, and inhibiting fibrosis. The TIMP is involved in promoting cell proliferation and inhibiting apoptosis. In particular, the mesenchymal stem cell culture medium contains a high content compared to the mesenchymal stem cell culture medium obtained by culturing growth factors and cytokines exhibiting wound healing and hair growth promoting efficacy by a two-dimensional culture method, and thus has significantly superior efficacy. indicates

In a specific embodiment of the present invention, the present inventors have developed a growth factor and cytokine that exhibits wound healing and hair loss inhibitory efficacy in a mesenchymal stem cell culture medium obtained by three-dimensional culture using sodium alginate having low viscosity according to the method of the present invention It was confirmed that Cain was secreted at a high concentration and exhibited an excellent skin cell and dermal papilla cell proliferation promoting effect.

Therefore, the mesenchymal stem cell culture medium prepared by the manufacturing method according to the present invention can be usefully used as an active ingredient in a composition for wound healing or skin regeneration, and for preventing hair loss or promoting hair growth.

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

(1) Cultivation of mesenchymal stem cells

The mesenchymal stem cells obtained from the tissue are suspended in a stromal medium, inoculated in a culture vessel at a concentration of 10,000 ~ 40,000 cells/cm ² , and then cultured. The substrate medium is DMEM or DMEM/F12 (Dulbecco's Modified Eagle Medium/Ham's F-12 Nutrient Broth) containing 10% bovine serum, and cultured for about 24 hours.

(2) Cultivation in expansion media

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

(3) subculture

When the cells fill 80 to 90% of the bottom of the culture vessel, the growth medium is removed and the cells are removed from the culture vessel through trypsinization. For subculture, the cells are diluted 1: 3 to 1: 4 and incubated with the growth medium in a new culture vessel. In this way, additional subcultures are possible.

(4) three-dimensional culture on alginate beads

The cultured cells are washed three or more times with PBS to remove FBS, and mixed with a sodium alginate solution having a viscosity of 300 CPS or less. The mixed solution is dropped into a 0.5 to 3% divalent cation-containing solution, such as a calcium chloride solution, to form cell-alginate beads. After washing the cell-alginate beads once with sodium chloride solution, 0.1 to 100 ng/ml of EGF (epidermal growth factor) and/or 0.1 to 100 ng/ml of bFGF (basic fibroblast growth factor) containing Incubate in the form of cell-alginate beads with DMEM or DMEM/F12.

In addition, the present invention provides a pharmaceutical composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, comprising the mesenchymal stem cell culture medium prepared according to the production method of the present invention as an active ingredient.

In the present invention, the mesenchymal stem cell culture medium contains growth factors and cytokines, specifically EGF, IL-6, VEGF, HGF, MIP-1α, MCP-1, MMP and TIMP, exhibiting wound healing and hair growth promoting efficacy. It contains high content.

In the present invention, growth factors and cytokines exhibiting wound healing and hair loss inhibitory efficacy are secreted at high concentrations in the mesenchymal stem cell culture medium obtained by three-dimensional culture using sodium alginate having low viscosity according to the method of the present invention, and excellent Since it was confirmed that the skin cell and dermal papilla cell proliferation promoting effect was confirmed, the mesenchymal stem cell culture medium can be usefully used as an active ingredient in a pharmaceutical composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth.

In the present invention, when the culture solution is used as a pharmaceutical composition, the culture solution may be contained alone or may additionally contain one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, 'pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not normally cause allergic reactions or similar reactions when administered to humans.

Pharmaceutically acceptable carriers may contain various ingredients such as buffers, sterile water for injection, plain saline or phosphate buffered saline, sucrose, histidine, salts and polysorbates, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and may be administered in the form of general pharmaceutical formulations, for example, oral and parenteral various formulations during clinical administration. It can be formulated using diluents or excipients such as bulking agents, binders, wetting agents, disintegrating agents, surfactants, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the pharmaceutical composition of the present invention, for example, starch, calcium carbonate, water Cross (Sucrose) or lactose (Lactose), it can be prepared by mixing gelatin and the like.

In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In addition, when administered parenterally, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. may be administered, and the route of administration is preferably determined according to the type of disease to be applied. For example, since the pharmaceutical composition of the present invention is used for promoting hair growth or preventing and treating hair loss, it is preferably administered in a manner that is applied topically to the scalp. Therefore, the pharmaceutical composition for promoting hair growth of the present invention is preferably an external preparation for topical administration or an injection, but is not limited thereto.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, disease severity, administration time, administration route, and response sensitivity of the patient, and usually skilled A physician can readily determine and prescribe an effective dosage for the desired treatment.

In addition, the present invention provides a skin external preparation for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, comprising the mesenchymal stem cell culture medium prepared according to the production method of the present invention as an active ingredient.

In addition, the present invention provides a cosmetic composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, comprising the mesenchymal stem cell culture medium prepared according to the production method of the present invention as an active ingredient.

In the present invention, the mesenchymal stem cell culture medium contains growth factors and cytokines, specifically EGF, IL-6, VEGF, HGF, MIP-1α, MCP-1, MMP and TIMP, exhibiting wound healing and hair growth promoting efficacy. It contains high content.

In the present invention, growth factors and cytokines exhibiting wound healing and hair loss inhibitory efficacy are secreted at high concentrations in the mesenchymal stem cell culture medium obtained by three-dimensional culture using sodium alginate having low viscosity according to the method of the present invention, and excellent Since it was confirmed that the skin cell and dermal papilla cell proliferation promoting effect was confirmed, the mesenchymal stem cell culture medium can be usefully used as an active ingredient in a skin external preparation or cosmetic composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth.

In the present invention, when the culture medium is used as an external preparation for skin, a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, an interface Skin active agents, water, ionic emulsifiers, non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents or lipid vesicles It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

In addition, when the culture medium is provided as an external preparation for skin, it may be in the form of, but not limited to, an ointment, a patch, a gel, a cream, or a spray.

In the present invention, when the culture solution is used as a cosmetic, cosmetics prepared by containing the culture solution as an active ingredient may be prepared in the form of a general emulsified formulation or solubilized formulation. For example, a lotion such as a softening lotion or a nourishing lotion; emulsions such as facial lotions or body lotions; creams such as nourishing creams, moisturizing creams or eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; It may have formulations such as cleansing foam, soap, body wash, and the like.

In addition, the cosmetic is a fatty substance, an organic solvent, a solubilizer, a thickening agent, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic type in addition to the culture medium. Emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents, or lipid vesicles commonly used in the cosmetic field It may contain adjuvants.

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

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

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

Adipose tissue can usually be obtained by liposuction, but is not limited thereto. Adipose stem cells were isolated from the adipose tissue obtained by liposuction as follows: To remove blood, the adipose tissue was washed 3 to 4 times with an equal volume of KRB solution. A collagenase solution of the same volume as the adipose tissue was added and reacted in a water bath at 37°C. This was transferred to a centrifuge tube and centrifuged at 20 °C and 1200 rpm for 10 minutes. The supernatant, the fat layer, was removed, and the collagenase solution, which was the lower layer, was carefully separated so as not to be shaken. After the suspension was put in the substrate medium, centrifuged at 20 ℃, 1200 rpm for 5 minutes. At this time, what sinks below is the stromal blood vessel fraction, and the supernatant is removed. The stroma-vascular fraction was suspended in a substrate medium, inoculated into a culture vessel, and cultured at 37° C., 5% CO ₂ in an incubator for 24 hours. After removing the culture medium, washing with phosphate buffer solution, stromal medium (DMEM/F12 medium containing 10% FBS), or basic fibroblast growth factor (bFGF) 1 ng/ml or epidermal growth factor (EGF) 5 in matrix medium ng/ml, or a growth medium containing 1 ng/ml of fibroblast growth factor (bFGF) and 5 ng/ml of epidermal growth factor (EGF). When the adipose stem cells grew to about 80 to 90% of the culture vessel, they were obtained by trypsinization and separation into single cells. The obtained cells were diluted 1:3 to 1:4 with a growth medium and subcultured.

<Example 2> Method of 3D culturing of human adipose stem cells in alginate beads

Stem cells obtained as single cells by trypsinization in <Example 1> were washed 3 times with DMEM/F12 (phenol red not included) medium to remove FBS. Cells were suspended by adding a solution of DMEM/F12 (without phenol red) to 8.0 × 10 ⁷ cells/ml. The prepared cell suspension was mixed with 9.5 ml of a 2% solution of sodium alginate (Sigma-Aldrich) having a low viscosity of 300 CPS or less. The cell-sodium alginate mixture was dropped into a 1% calcium chloride solution to form cell-alginate beads. After 5 minutes of washing once with 0.15 M sodium chloride solution, DMEM/F12 (without phenol red) containing 1 ng/ml basic fibroblast growth factor (bFGF) and 5 ng/ml epidermal growth factor (EGF) was added. It was added to make 500 ml per 4 x 10 ⁷ cells. After 72 hours, the cell culture medium was recovered and a new medium was added. The cell culture medium was recovered twice more and stored in a refrigerator.

<Comparative Example 1> Method of 2-dimensional culture of human adipose-derived mesenchymal stem cells

The stem cells obtained as single cells by trypsinization in <Example 1> were washed 3 times with DMEM/F12 medium to remove FBS. DMEM/F12 solution was added to 8.0 × 10 ⁷ cells/ml stem cells, and the cells were suspended and inoculated. Then, a DMEM/F12 solution containing 1 ng/ml basic fibroblast growth factor and 5 ng/ml epidermal growth factor was added to make 500 ml per 4.0 × 10 ⁷ cells. After 72 hours, the cell culture medium was recovered and a new medium was added. The cell culture medium was recovered 2 to 11 more times and stored in a refrigerator.

<Comparative Example 2> 3D culture method in alginate beads of medium viscosity

Stem cell culture medium was recovered by three-dimensional culture (3D) in the same manner as in <Example 1> and <Example 2>, except that sodium alginate (Sigma-Aldrich) having an intermediate viscosity of 2000 CPS or higher was used, and refrigerated kept.

Here, sodium alginate is generally divided into low viscosity and medium viscosity, or low viscosity, medium viscosity, and high viscosity. However, sodium alginate is a natural extract, and the viscosity varies depending on the type of brown algae, collection time, and method, so each manufacturer It is sold under different standards of viscosity. Therefore, sodium alginate having a viscosity of 2000 CPS or more, which is the standard of manufacturer B's intermediate viscosity, was adopted as sodium alginate of moderate viscosity.

<Experimental Example 1> Growth factor and cytokine array contained in stem cell culture medium

Various growth factors and cytokines contained in the stem cell culture medium recovered in <Example 2> were analyzed using a cytokine array.

Specifically, 1 ml of the stem cell culture solution recovered in <Example 2> was added to each well of the plate containing the membrane, and reacted at room temperature for 2 hours. After washing 3 times with Washing Solution I and 2 times with Washing Solution II, 1 ml of the detection antibody solution was added, followed by reaction at 4°C overnight. After washing 3 times with Washing Solution I and 2 times with Washing Solution II, 1 ml of a horseradish-peroxidase solution bound with streptavidin was added at a time, followed by reaction at room temperature for 2 hours. After washing 3 times with Washing Solution I and 2 times with Washing Solution II, the membrane was placed on a plastic sheet and the detection solution was sprayed to develop color. After covering it well with another plastic sheet, it was put into a LAS 500 instrument and analyzed.

As a result, as shown in FIG. 1, growth factors and cytokines, EGF, which show wound healing and hair loss inhibitory efficacy in the stem cell culture medium obtained by three-dimensional culture (3D) using sodium alginate of <Example 2>, It was confirmed that the contents of IL-6, MIP-1α, MCP-1, MMP and TIMP were significantly higher.

<Experimental Example 2> Qualitative analysis of growth factors and cytokines contained in stem cell culture medium

VEGF and EGF, which are representative growth factors known to be secreted from stem cells, including growth factors and cytokines identified in <Experimental Example 1>, were analyzed by modifying the enzyme-linked immunosorbent method (ELISA).

Specifically, 100 μl of the stem cell culture solution recovered in <Example 2> was added to each well of a 96-well plate and reacted at room temperature for 2 hours. After washing 4 times with a washing solution, 100 μl of a detection antibody solution capable of recognizing a growth factor was added at a time, followed by reaction for 2 hours. After washing 4 times with a washing solution, 100 μl of a horseradish-peroxidase solution bound to streptavidin was added at a time, followed by reaction at room temperature for 20 minutes, followed by observation.

As a result, as shown in Figure 2, growth factors and cytokines, EGF, It was verified that IL-6, VEGF, HGF, MIP-1α, MCP-1, MMP and TIMP were contained.

<Experimental Example 3> Comparison of growth factors and cytokines contained in the stem cell culture medium obtained by 2D culture and the stem cell culture medium obtained by 3D culture

MIP-1 among various growth factors and cytokines contained in the stem cell culture medium recovered in <Example 2> and <Comparative Example 1> using a cytokine array as described in <Experimental Example 1> Analysis was carried out in the same manner as the method.

As a result, as shown in FIG. 3, compared with the stem cell culture medium obtained by the two-dimensional culture (2D) of <Comparative Example 1>, the three-dimensional culture (3D) using sodium alginate of <Example 2> It was confirmed that the obtained stem cell culture medium contained a high content of MIP-1α.

<Experimental Example 4> Quantification of HGF contained in stem cell culture medium

HGF, a growth factor exhibiting a representative wound healing and hair loss inhibitory effect contained in the stem cell culture medium recovered in <Example 2> and <Comparative 2>, was analyzed using an enzyme-linked immunosorbent method (ELISA, R&D system). .

Specifically, 100 μl of the stem cell culture solution recovered in <Example 2> was added to each well of a 96-well plate and reacted at room temperature for 2 hours. After washing 4 times with a washing solution, 100 μl of a detection antibody solution capable of recognizing a growth factor was added at a time, followed by reaction for 2 hours. After washing 4 times with a washing solution, 100 μl of a horseradish-peroxidase solution to which streptavidin is bound is added, followed by reaction at room temperature for 20 minutes, and a substrate solution (H ₂ O ₂ and tetramethyl benzidine) is added to develop color. made it 2 NH ₂ SO ₄ solution was added to stop the color reaction, and absorbance was measured at 450 nm.

As a result, as shown in FIG. 2, 300 CPS of <Example 2> compared to the stem cell culture obtained by three-dimensional culture (3D) with sodium alginate having an intermediate viscosity of 2000 CPS or more of <Comparative Example 2> It was confirmed that the content of HGF was significantly high in the stem cell culture solution obtained by three-dimensional culture (3D) using sodium alginate having the following low viscosity.

<Experimental Example 5> Confirmation of skin cell proliferation and dermal papilla cell proliferation promoting effect of stem cell culture medium

The effect of promoting skin cell proliferation and dermal papilla cell proliferation of the stem cell culture medium recovered in <Example 2> was confirmed.

Claims (12)

(1) after mixing the suspension of mesenchymal stem cells and sodium alginate solution, dropping the mixed suspension into a solution containing divalent cations to form beads;
(2) After the bead formation in step (1), three-dimensional culture in a serum-free medium to which any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to obtain a mesenchymal stem cell culture medium comprising steps;
The bFGF is 0.1 ~ 100 ng / ㎖, the EGF will be added 0.1 ~ 100 ng / ㎖,
A method for preparing a mesenchymal stem cell culture medium containing growth factors and cytokines.
The method of claim 1,
In the step (1), the mesenchymal stem cells are isolated from any one selected from the group consisting of fat, bone marrow, umbilical cord blood, liver and muscle, a manufacturing method.
The method of claim 1,
In the step (1), the mesenchymal stem cells,
a) culturing the mesenchymal stem cells in a stromal medium and then culturing in a proliferation medium; and
b) mesenchymal stem cells cultured through the step of subculturing the cells in a growth medium,
Here, the growth medium is a method for producing a substrate medium by adding any one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor).
4. The method of claim 3,
Characterized in culturing for two or more passages in step b), the manufacturing method.
The method of claim 1,
The sodium alginate solution in step (a) is characterized in that it has a viscosity of 300 CPS or less, the manufacturing method.
The method of claim 1,
The divalent cation in step (a) is Ca ²⁺ , Mg ²⁺ , Sr ²⁺ , Ba ²⁺ , characterized in that any one selected from the group consisting of, the manufacturing method.
The method of claim 1,
The solution containing the divalent cation in step (a) is characterized in that it has a concentration of 0.5 to 3% by weight, the manufacturing method.
The method of claim 1,
The three-dimensional culture in step (2) is characterized in that to obtain a stem cell culture medium three or more times while exchanging the medium at intervals of 2 to 4 days, the manufacturing method.
The method of claim 1,
The growth factors and cytokines are EGF, IL-6, VEGF, HGF, MIP-1α, MCP-1, MMP and TIMP, characterized in that the production method.
A pharmaceutical composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, comprising the mesenchymal stem cell culture medium prepared according to any one of claims 1 to 9 as an active ingredient.
10. A skin external preparation for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, comprising the mesenchymal stem cell culture medium prepared according to any one of claims 1 to 9 as an active ingredient.
10. A cosmetic composition for wound healing or skin regeneration, and preventing hair loss or promoting hair growth, comprising the mesenchymal stem cell culture medium prepared according to any one of claims 1 to 9 as an active ingredient.

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