KR102029358B1 - Cosmetic composition comprising a stem-cell culture fluid, and method for preparing the stem-cell cultee fluid - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising a stem cell culture and a method for producing the stem cell culture. More specifically, the present invention relates to a composition for wound treatment, skin improvement and hair loss prevention and treatment comprising a stem cell culture solution obtained by three-dimensional culture of stem cells under hypoxic conditions as an active ingredient.

Description

Cosmetic composition comprising a stem cell culture medium and a method for producing the stem cell culture medium TECHNICAL FIELD

The present invention relates to a cosmetic composition comprising a stem cell culture and a method for producing the stem cell culture. More specifically, the present invention relates to a composition for wound treatment, skin improvement and hair loss prevention and treatment comprising a stem cell culture medium obtained by three-dimensionally culturing stem cells under low oxygen and high carbon dioxide conditions as an active ingredient.

Recently, stem cell technology has emerged as a new chapter in the treatment of intractable disease. Previously, organ transplantation and gene therapy have been suggested for the treatment of intractable disease in humans. As interest in pluripotent stem cells, which have the ability to form all organs through proliferation and differentiation, has been raised, it has been recognized that stem cells can fundamentally solve organ damage as well as treat most diseases. The application of stem cells has been suggested in a variety of ways, from the treatment of almost all organ regeneration to the treatment of Parkinson's disease, various cancers, diabetes and spinal cord injury.

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These adult stem cells were first isolated from adult bone marrow (Y. Jiang et al., Nature, 418: 41, 2002) and subsequently identified in other adult tissues (CM Verfaillie, Trends Cell Biol., 12: 502, 2002). In other words, bone marrow is the most widely known source of stem cells, but pluripotent stem cells can also be obtained from fat, umbilical cord, skin, blood vessels, muscle and brain, etc. (JG Toma et al., Nat. Cell Biol., 3: 778, 2001; M. Sampaolesi et al, Science, 301: 487, 2003; Y. Jiang et al., Exp. Hematol., 30: 896, 2002). An important point in understanding adult stem cells is that adult stem cells are present in small amounts in each tissue. It stays silent for years without dividing or proliferating until the tissue is diseased or damaged and the adult stem cells are activated. Scholars are investigating how adult stem cells can proliferate through cell culture and induce differentiation into specific cells that can be used when our bodies are hurt or diseased.

The difficulty in acquiring these adult stem cells is that adult stem cells are very rarely present in adult tissues, and these cells are difficult to culture without induction of differentiation, making it difficult to culture the cells without specifically screened medium. However, among the adult stem cells, adipose stem cells can be easily obtained by simple liposuction, and the cell yield is more than 500 times higher than that of bone marrow-derived stem cells, which are representative adult stem cells, and contain abundant growth factors and cytokines. In view of the most valuable adult stem cells (Rubio D, 65: 3035-3039, 2005). Despite the potential benefits of stem cells, there are some limitations to the clinical application of stem cells. First, they have a relatively short lifespan of transplanted stem cells. That is, most of the transplanted cells are known to be lost within a few days after transplantation (Greene AK, 16: 99-102, 2003; Rubio D, 3: e1398-, 2008). First of all, it is possible that malignant transformation of stem cells is possible (Orlic D, 98: 10344-10349, 2001; Trento C, 140: w13121-, 2010). One of the ways to overcome the limitations of such stem cell treatment is to use stem cell secretome, a stem cell secretion, in place of stem cells. Stem cell cultures are a total collection of proteins secreted from stem cells, including cytokines, hormones, growth factors, and the like. In recent studies, stem cell culture has attracted attention as a therapeutic component of stem cells, which suggests that stem cells have the ability to repair damaged organs by the various protein components secreted in response to the damaged microenviroment. Facts have been revealed (Baglio SR, 3: 359-, 2012; Zhang W, 12: 9-18, 2002).

On the other hand, there is a big difference in the therapeutic effect of stem cells depending on the conditions under which stem cells are cultured. This suggests that the secretion amount and composition of stem cell culture may vary depending on the culture condition of stem cells. Conventional experiments with stem cells are always under normal oxygen partial pressure (21% oxygen). However, oxygen incorporation is known to be very low when the in vivo environment, ie, stem cells, actually works in the body (Marcos A, 69: 1375-1379, 2000). Liu et al. (Kiuchi T, 67: 321-327, 1999) and Rosova et al. (Banas A, 26: 2705-2712, 2008) provide a hypoxic environment, such as the in vivo environment. Differentiation and angiogenesis were induced and reported to increase the therapeutic effect of stem cells. In addition, in the case of mesenchymal stem cells, adherence culture is carried out in two dimensions in a flask with a flat bottom because of the property of survival by attachment. In the conventional method, stem cells are simply two-dimensionally cultured, or two-dimensional cultures are carried out under hypoxic conditions in order to increase the content of VEGF, and stem cells are not considered stable in a serum-free medium. There was a problem that the physiological activity of the culture solution is lowered. That is, in two-dimensional culture, even if given low oxygen conditions, there is a limit to increase the content of VEGF and the like contained in the culture solution.

Accordingly, the present inventors have made diligent efforts to prepare mesenchymal stem cell culture medium containing a high content of cytokines and cell growth factors. As a result, the stem cell proliferation rate is increased when stem cells are cultured under hypoxic conditions in three-dimensional culture. It was confirmed that the content of cytokines and cell growth factors increased. In addition, the present inventors confirmed that the stem cell proliferation rate significantly increased and the content of cytokines and cell growth factors in the stem cell culture significantly increased when the stem cells were cultured three-dimensionally under low oxygen conditions and high carbon dioxide conditions. It was completed.

It is an object of the present invention to provide a pharmaceutical composition for wound treatment, skin improvement and hair loss prevention and treatment comprising a stem cell culture solution obtained by three-dimensional culture of stem cells under hypoxic conditions as an active ingredient.

The present invention also relates to a food composition for wound treatment, skin improvement and hair loss prevention and treatment comprising a stem cell culture solution obtained by three-dimensional culture of stem cells under hypoxic conditions as an active ingredient.

The present invention also relates to a cosmetic composition for wound treatment, skin improvement and hair loss prevention and treatment comprising a stem cell culture solution obtained by three-dimensional culture of stem cells under hypoxic conditions as an active ingredient.

As used herein, the term "stem cells" refers to proliferating stem cells. The stem cells may be bone marrow stem cells, umbilical cord stem cells or adipose-derived stem cells, may be stem cells derived from the human body or animal or plant, for example, may be human adipose derived stem cells, but is not limited thereto.

As used herein, the term "mesenchymal stromal cells (MSCs)" is excellent in self-renewal and can be differentiated into various cells such as liver, bone, cartilage, fat, blood vessels, heart, nervous system, etc. Refers to adult stem cells.

The term "hypoxia" as used herein may refer to a low oxygen partial pressure condition compared to 21% oxygen partial pressure, which is a normal normal oxygen condition. The low oxygen condition may be 5 to 15%, 8 to 12%, for example, 10%.

As used herein, the term "spheroid" refers to a cell mass known as a 3D cell mass (3DCM), which refers to a spherical colony formed by gathering a plurality of daughters formed by division of an individual.

According to the first embodiment, the present invention

a) culturing stem cells in serum-free medium; And

b) to provide a pharmaceutical composition for wound treatment, skin improvement and hair loss prevention or treatment containing the stem cell culture solution as an active ingredient comprising the step of culturing the stem cells in a three-dimensional spheroid under hypoxic conditions.

In the pharmaceutical composition according to the present invention, the stem cells are characterized in that the mesenchymal stem cells.

In the pharmaceutical composition according to the present invention, the stem cells are characterized in that the human skin fibroblasts (HDF), human skin keratinocytes (HEK) or human hair follicle cells (HDP).

In the pharmaceutical composition according to the invention, the serum-free medium is characterized in that it comprises a vitamin or an analog thereof. Vitamins or analogs thereof included in the serum-free medium of the present invention may include one or more substances selected from the group consisting of vitamins A, B, C, D and vitamin E. In one embodiment, vitamin A (2 uM), vitamin C (100 μg / ml) and vitamin D (50 nM) were used.

In the pharmaceutical composition according to the present invention, the serum-free medium is fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor β-l (TGF-1), It comprises at least one material selected from the group consisting of platelet-derived growth factor (PDGF), Vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) or IFG-1 (insulin-like growth factor). .

In the pharmaceutical composition according to the present invention, the low oxygen condition is characterized in that the oxygen partial pressure of 1% to 15%.

In the pharmaceutical composition according to the invention, step b) is characterized in that it is carried out in the presence of 5% to 15% of carbon dioxide.

The pharmaceutical composition according to the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods. Carriers, excipients and diluents which may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like can be used, but are not limited thereto.

The pharmaceutical composition according to the present invention may be used in the form of oral dosage forms, external preparations, suppositories, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. Can be. For example, when formulated, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, in the pharmaceutical composition of the present invention. , Sucrose, lactose, gelatin and the like can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , Sublingual or rectal. Oral or parenteral release is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical compositions of the present invention depend on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, extent of disease, drug form, route of administration and duration, and may be appropriately selected by those skilled in the art and may be 0.0001 to 50 mg / day. It may be administered at kg or 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical compositions according to the invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

According to the second embodiment, the present invention

a) culturing stem cells in serum-free medium; And

b) to provide a food composition for wound treatment, skin improvement and hair loss prevention or treatment containing stem cell culture solution as an active ingredient comprising the step of culturing the stem cells in a three-dimensional spheroid under hypoxic conditions.

In the food composition according to the present invention, the stem cells are characterized in that the mesenchymal stem cells.

In the food composition according to the present invention, the stem cells are characterized in that the human skin fibroblasts (HDF), human skin keratinocytes (HEK) or human hair follicle cells (HDP).

In the food composition according to the present invention, the serum-free medium is characterized in that it comprises a vitamin or an analog thereof. Vitamins or analogs thereof included in the serum-free medium of the present invention may include one or more substances selected from the group consisting of vitamins A, B, C, D and vitamin E. In one embodiment, vitamin A (2 uM), vitamin C (100 μg / ml) and vitamin D (50 nM) were used.

In the food composition according to the present invention, the serum-free medium is fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor β-l (TGF-β 1), PDGF (platelet-derived growth factor), VEGF (Vascular endothelial growth factor), HGF (hepatocyte growth factor) or IFG-1 (insulin-like growth factor) characterized in that it comprises one or more materials selected from the group consisting of.

In the food composition according to the present invention, the low oxygen condition is characterized in that the oxygen partial pressure of 1% to 15%.

In the food composition according to the invention, the step b) is characterized in that it is carried out in the presence of 5% to 15% of carbon dioxide.

The food composition according to the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, breads and the like. When the food composition is prepared in the form of a drink, there is no particular limitation other than the containing the food composition in the ratio indicated, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general drinks. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavourant include natural flavourant (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavourant (saccharin, aspartame, etc.). Food composition of the various flavors, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as colorants, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, Stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.

According to the third embodiment, the present invention

a) culturing stem cells in serum-free medium; And

b) to provide a cosmetic composition for wound treatment, skin improvement and hair loss prevention or treatment containing a stem cell culture solution as an active ingredient comprising the step of culturing the stem cells in a three-dimensional spheroid under hypoxic conditions.

In the cosmetic composition according to the present invention, the stem cells are characterized in that the mesenchymal stem cells.

In the cosmetic composition according to the present invention, the stem cells are characterized in that the human skin fibroblasts (HDF), human skin keratinocytes (HEK) or human hair follicle cells (HDP).

In the cosmetic composition according to the invention, the serum-free medium is characterized in that it comprises a vitamin or an analog thereof. Vitamins or analogs thereof included in the serum-free medium of the present invention may include one or more substances selected from the group consisting of vitamins A, B, C, D and vitamin E. In one embodiment, vitamin A (2 uM), vitamin C (100 μg / ml) and vitamin D (50 nM) were used.

In the cosmetic composition according to the present invention, the serum-free medium is FBS (fetal bovine serum), bFGF (basic fibroblast growth factor), EGF (epidermal growth factor), TGF-β 1 (transforming growth factor β-l), PDGF (platelet-derived growth factor), VEGF (Vascular endothelial growth factor), HGF (hepatocyte growth factor) or IFG-1 (insulin-like growth factor) characterized in that it comprises one or more materials selected from the group consisting of.

In the cosmetic composition according to the present invention, the low oxygen condition is characterized in that the oxygen partial pressure of 1% to 15%.

In the cosmetic composition according to the invention, the step b) is characterized in that it is carried out in the presence of 5% to 15% of carbon dioxide.

In the cosmetic composition according to the present invention, the cosmetic composition is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence Selected from the group consisting of nutrition essences, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, face washes, treatments, essences, beauty packs, ointments, gels, linens, solutions, patches and sprays It is characterized in that the formulation of any one.

In the cosmetic composition according to the present invention, the cosmetic composition is antibiotics, binders, disintegrants, diluents, lubricants, stabilizers, preservatives, fragrances, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments and It is characterized by comprising an additive selected from the group consisting of preservatives.

The stem cell culture according to the present invention promotes cell migration to the wound site and has excellent wound healing or wound healing promoting activity. While excellent in skin permeability, it is excellent in skin improvement effects such as skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, and is safe without irritation or side effects on the skin. In addition, the stem cell culture according to the present invention is excellent in the hair growth and hair growth effect is excellent in the prevention and treatment of hair loss, it is safe without side effects even when ingested or applied to the skin.

Figure 1 shows the stem cell culture screening test results according to Example 1 of the present invention.
Figure 2 shows the results of the cell proliferation test under hypoxia and three-dimensional culture conditions according to Example 3 of the present invention.
Figure 3 shows the wound recovery test results according to Example 4 of the present invention.
Figure 4 shows the culture medium active ingredient analysis results according to Example 5 of the present invention.
Figure 5 shows the culture medium effective test results in the wound animal model according to Example 6 of the present invention.
Figure 6 shows the culture medium effective test results in the wound animal model according to Example 6 of the present invention.

Hereinafter, various examples are presented to help understand the invention. The following examples are merely provided to more easily understand the invention, but the protection scope of the invention is not limited to the following examples.

<Example>

Example 1. Stem cell culture screening test

In order to select the optimal media components according to the various culture components, 200 human skin fibroblasts (HDF), human skin keratinocytes (HEK), and human adipose stem cells (ADSC) were placed in 96-well plates (SPL). Inoculated with 100 μl of DMEM (Gibco) medium containing 10% FBS (Gibco), and incubated in a humidified culture conditions of 37 ℃, 5% CO ₂ . After 24 hours, the supernatant was removed and washed three times by adding 200 μl of PBS (Gibco). PBS was removed and six cultures were added to each of 100 μl according to Table 1 below, followed by incubation for 72 hours at 37 ° C. and humidified culture conditions of 5% CO ₂ .

Default medium Furtherance Media 1 DMEM 10% FBS (fetal bovine serum) Media 2 α-MEM 5% FBS Media 3 α-MEM 5% FBS, Vitamin A (2 uM), Vitamin C (100 μg / ml), Vitamin D (50 nM) Media 4 α-MEM 1% human albumin, vitamin C (100 μg / ml), vitamin D (50 nM) Media 5 α-MEM 1% human albumin, insulin (1 mg / mL), transferrin (0.55 mg / mL), Lipid concentrate (0.1%), vitamin A (2 uM) Media 6 α-MEM 1% human albumin, insulin (1 mg / mL), transferrin (0.55 mg / mL), Lipid concentrate (0.1%), vitamin A (2 uM), vitamin C (100 μg / ml), vitamin D (50 nM)

After incubation for 72 hours, 10 μl of CCK-8 (Cell counting kit-8, Sigma) reagent was added to the wells, and further incubated for 2 hours, and then the absorbance at 450 nm was measured using a microplate reader (Eon, BioTek). The results are shown in FIG.

As a result, when cultured with Media 3, not only skin cells but also adipose stem cells showed high proliferation rate (FIG. 1a). In the case of human adipose stem cells, media 3 culture showed the highest cell proliferation rate (FIG. 1b). Based on the results of this experiment, when stem cells were cultured with Media 3 culture, it was determined that the incubation period could be shortened when 3D spheroid formation and finally, the culture optimized for skin regeneration could be produced.

Example 2. Low Oxygen and Three-Dimensional Culture

2-1. Spheroid manufacturers

Cultured stem cells were recovered by treatment with enzymes (TrypLE EXPRESS, Gibco), and 6X10 ⁸ cells were counted and dispensed into 3 x ^{10 8} 50ml tubes (SPL). After washing with the addition of physiological saline (0.9%) to the divided cell pellet, centrifuged for 5 minutes at 1500rpm and the supernatant was removed. The washing process was repeated twice. 30 ml of spheroid preparation medium (Table 1 Media 3) was added to the cell pellet of each tube, and the cells were suspended to a concentration of 5 × ¹⁰ 7/5 ml, and 5 ml each was dispensed into 12 50 ml tubes. 25 ml of 30% F-127 hydrogel solution was added to 5 ml of cell suspension (5 × ¹⁰ 7/5 ml) and mixed with the cells to a total of 30 ml. 30 ml of the solution mixed with the cells was slowly dispensed into an ultra-low adhesion flask (ULA T75 flask, Corning) and incubated for 1 hour for each spheroid formation condition according to Table 2 below.

Experimental group cell Spheroid Formation Conditions Spheroid Culture Production Conditions ADS 1 Adipose derived stem cells Normoxia 37 ° C, 20% O ₂ , 5% CO ₂ ADS 2 Adipose derived stem cells Hypoxia 2% 37 ° C, 1% O ₂ , 5% CO ₂ ADS 3 Adipose derived stem cells Hypoxia 2% 37 ° C, 2% O ₂ , 10% CO ₂ UCS 1 Umbilical Cord Stem Cells Normoxia 37 ° C, 20% O2, 5% CO2 UCS 2 Umbilical Cord Stem Cells Hypoxia 2% 37 ° C, 1% O ₂ , 5% CO ₂ UCS 3 Umbilical Cord Stem Cells Hypoxia 2% 37 ° C, 2% O ₂ , 10% CO ₂

After 1 hour, when the solution was gelated, 5 ml of the spheroid preparation medium at 37 ° C. was added, and then spheroids were prepared by incubating for 5 days at 37 ° C. humidified culture conditions.

2-2. Spheroid Recovery

Each experimental group was incubated for 10 minutes at room temperature for the F-127 gel containing a spheroid, and then physiological saline was added to the same volume and recovered in 50 ml tubes. After centrifugation at 800 rpm for 5 minutes to remove the supernatant, 50 ml of physiological saline is added to wash the spheroid. The washing process was repeated twice.

2-3. Spheroid Culture Production

Each recovered spheroid was dispensed into 3L polycarbonate Erlenmeyer flasks (Corning) for each experimental group, and cultured under the culture medium production conditions (Table 2) for each experimental group. The spheroid culture medium used at this time was Media 6 of Table 1, and the medium volume was 2L. Spheroid culture production was shaken for 10 days at 25 rpm in a humidified culture conditions of 37 ℃, 5% CO ₂ . The spheroid culture was recovered in 2L total every 2 days and replaced with fresh spheroid culture medium. The culture broth recovered 5 times for 10 days was homogenized in a 10L bottle and refrigerated.

Example 3. Cell Proliferation Test

In order to select the optimal hypoxic and three-dimensional culture conditions, 200% of human parietal fibroblasts (HDF), human skin keratinocytes (HEK), and human hair follicle cells (HDP) in a 96-well plate (SPL), each 10% FBS ( 100 μl of DMEM (Gibco) medium containing Gibco) was incubated at 37 ° C. and humidified culture conditions of 5% CO ₂ . After 24 hours, the supernatant was removed and washed three times by adding 200 μl of PBS (Gibco). PBS was removed and six cultures produced under the conditions shown in Table 2 were added to each 100 μl, followed by incubation for 72 hours at 37 ° C. and 5% CO ₂ humidified culture conditions. After 72 hours of incubation, 10 μl of CCK-8 (Cell counting kit-8, Sigma) reagent was added to the wells, and further incubated for 2 hours, and then the absorbance at 450 nm was measured using a microplate reader (Eon, BioTek). Is shown in FIG. 2.

 As a result, cultures produced by hypoxic cultures increased HDF proliferation by 34% (fat stem cells) and 23% (cord stem cells), and HEK was 21% (fat stem cells) and 23% (normal oxygen partial pressure). Umbilical cord stem cells) and HDP were found to proliferate 59% (fat stem cells) and 12% (umbilical stem cells). In addition, as the skin cell proliferation rate was higher in 5% carbon dioxide culture conditions (ADS2, UCS2), it was confirmed that not only oxygen partial pressure but also carbon dioxide partial pressure was involved in skin cell proliferation rate.

Example 4 In vitro Wound Recovery Test

Insert human skin and hair follicle cells into a 24-well plate (SPL) for a wound repair assay (Ichrabi), and DMEM (Gibco) containing 5x10 ³ cells containing 100 μl of 10% FBS (Gibco). Inoculated with medium and incubated for 24 hours at 37 ℃, humidified culture conditions of 5% CO2. After incubation for 24 hours, the insert was removed using tweezers to make a scratch in the center of the well plate, and then the supernatant was removed and washed three times by adding 1 ml of PBS (Gibco). Remove PBS and add 1 ml of medium of each condition (<Table 2>), incubate in humidified culture condition of 37 ℃, 5% CO2 for 24 hours, and check the cell regeneration rate by microscopic observation every 6 hours. A picture was taken and shown in FIG. 3.

As a result, the culture solution (ADS2, ADS3, UCS2, UCS3) produced by the hypoxic culture than the normal oxygen partial pressure conditions (ADS1, USC1) was shown to increase the wound repair ability of HDF, HEK and HDP. In addition, it was confirmed that the wound recovery ability is superior to 10% carbon dioxide culture conditions than 5% carbon dioxide culture conditions.

Example 5. Analysis of active ingredient in culture

In order to identify the components of the ADS1 spheroid culture medium and the ADS3 spheroid culture solution, the analysis was performed by Proteome Profiler Human Cytokine Array (R & D systems, ARY005B), and the results are shown in FIG. 4.

As a result, the expression of MIP-1 (Grophage inflammatory protein-1), G-CSF, IL-1b and IL-1r was increased when ASC spheroids were cultured at low O ₂ and high CO ₂ concentrations. And decreased expression of SDF-1. MIP-1α (CCL3) and MIP-1β (CCL4) are important cytokines in the immune response to infection and inflammation. They activate granulocytes (neutrophils, eosinophils and basophils), and IL-1 from fibroblast and macrophage. IL-6, a cytokine that promotes TNF-a secretion. In addition, G-CSF promotes the migration of hematopoietic stem cells (HSC), IL-1 is a cytokine that induces inflammatory responses to infection, and IL-1r is an antagonist for IL-1α and IL-1β to regulate the inflammatory response. It plays a role. SDF-1 is a cytokine that induces chemotaxis of lymphocytes and MSCs, and is known to induce endothelial progenitor cells (EPCs) from bone marrow, especially during adult angiogenesis.

Example 6 Culture Effectiveness Test in Wound Animal Model

After punching the skin with a 5mm biopsy punch on both sides of the hairless mouse, six wound animal models were prepared for each treatment group (Table 2) .Then, Vaseline 0.01g (control) on the left and 0.01 Vaseline on the right. 10 ul mixer of the g + test culture was applied every two days. After treatment of each test material, the wound area is first attached to Tegaderm ^tm of 1.2cmX1.2cm and additionally attached to Tegaderm ^tm of 3cm x 6cm to prevent falling. It was. The width of the wound area of each group was recorded at two-day intervals, and photographs of the wound area of each group were taken on days 0, 4, 10, and 14 (FIG. 5). Before sacrifice of each group of mice, blood was collected from the heart, incubated in a 1.5ml tube for 30 minutes, centrifuged at 13000 rpm for 15 minutes, and serum of the supernatant was taken to perform cytokine array (FIG. 6).

As a result, the wound area was reduced by more than 50% in the group treated with the adipose stem cell spheroid culture medium produced under 1% oxygen and 10% carbon dioxide, and there was no significant difference in the other groups. In addition, the analysis of serum cytokine in each group showed that IL-5, CCL-17, TNF-a and CXCL-2 were increased in the serum of ADS1 and ADS-3 treated mice. IL-5 is a pro-inflammatory cytokine that induces the infiltration of eosinophils into the wound site and increases the action of eliminating the infection [Leitch VD, et al., Immunol Cell Biol. 87 (2009) 131] CCL-17 is known to promote the healing of fibroblasts and promote wound healing. Kato T. et al., Exp. Dermatol. 20 (2011) 669] TNF-a is a cytokine involved in the early stages of wound healing [Ritsu, M. et al., J Dermatol Dermatol Surgery, 21 (2017) 14]. It is known to be involved cytokine (Ding, et al., Adv Wound Care (New Rochelle) 4 (2015) 673).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (9)

A cosmetic composition for wound improvement comprising stem cell culture as an active ingredient, wherein the stem cell culture is:
a) culturing stem cells in serum-free medium; And
b) the stem cells are prepared by the method for producing a stem cell culture solution comprising the step of culturing the three-dimensional spheroid under hypoxic conditions,
The hypoxic condition is characterized in that the oxygen partial pressure of 1% to 15% and carbon dioxide partial pressure of 5% to 15%, cosmetic composition.
The method of claim 1,
Stem cells are mesenchymal stem cells, characterized in that the cosmetic composition.
The method of claim 1,
The stem cell is characterized in that the human parvofibroblast (HDF), human keratinocytes (HEK) or human hair follicle cells (HDP), cosmetic composition.
The method of claim 1,
Vitamin or analogs included in the serum-free medium is characterized in that it comprises one or more substances selected from the group consisting of vitamin A, B, C, D and vitamin E, cosmetic composition.
The method of claim 1,
The serum-free medium is fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor β-l (TGF-β 1), platelet-derived growth factor (PDGF), and VEGF. (Vascular endothelial growth factor), characterized in that it comprises one or more substances selected from the group consisting of hepatocyte growth factor (HGF) or IFG-1 (insulin-like growth factor), cosmetic composition.
delete delete The method of claim 1,
The cosmetic composition is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing Foam, cleansing lotion, cleansing cream, body lotion, body cleanser, facial cleanser, treatment, essence, cosmetic pack, and a spray formulation, characterized in that any one selected from the group consisting of, cosmetic composition.
The method of claim 1,
The cosmetic composition is an additive selected from the group consisting of antibiotics, binders, disintegrants, diluents, lubricants, stabilizers, preservatives, fragrances, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments and preservatives It characterized in that it comprises a cosmetic composition.

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