KR20150100260A - Non-antibiotics serum-free mesenchymal stem cells medium, method for preparing the same, and cosmetic composition comprising the same - Google Patents

Abstract

The present invention relates to an antibiotic-free and serum-free mesenchymal stem cell culture medium, a preparing method thereof and a cosmetic composition using the same. More specifically, provided is a method for preparing a safer mesenchymal stem cell culture medium by using an antibiotic-free and serum-free basal culture medium, and a cosmetic composition including the same is effective in improving skin wrinkles.

Description

TECHNICAL FIELD The present invention relates to a non-antibiotic serum-free serum-free mesenchymal stem cell culture solution, a method for producing the same, and a cosmetic composition using the same. BACKGROUND ART [0002] NON- ANTIBIOTICS SERUM-FREE MESENCHYMAL STEM CELLS MEDIUM, METHOD FOR PREPARING THE SAME, AND COSMETIC COMPOSITION COMPRISING THE SAME

The present invention relates to a non-antibiotic-free serum-free mesenchymal stem cell culture, a method for producing the same, and a cosmetic composition using the same. More specifically, the present invention relates to a method for producing safer mesenchymal stem cell culture medium using a non-antibiotic serum-free basic culture medium, and a cosmetic composition effective for improving skin wrinkles containing the same.

Stem cells are cells that can self-proliferate and differentiate into cells of other tissues and exist in many tissues of our body. Among adult stem cells, the most accessible and abundant stem cells can be obtained from adipose tissue. Recently, adipose tissue has been shown to regenerate various tissues such as epithelial cells, chondroblasts, osteogenic cells, neuronal cells, muscle cells as well as adipose tissue as a source of multipotential stem cells (M. Brzoska et al., BBRC, 330 Brie et al., Biomaterials, 25: 3211, 2004, pp. 142, 2005. Y. Cao et al., BBRC, 332: 370, 2005. KM Safford et al., BBRC, 294: 371, 2005; Fujimura et al., BBRC, 333: 116, 2005).

Since the regeneration ability and differentiation ability of stem cells are suitable for use in the treatment such as regeneration of damaged tissues, studies for applying them are currently being carried out in various fields such as medicine and cosmetics. However, in order to produce products using human stem cell culture solution in Korea, it can be used only after passing the raw material stability test. Therefore, it is required to produce safer human adipose stem cell culture liquid by using a non-antibiotic serum-free basic culture medium, and it is possible to produce a cosmetic which is effective for skin improvement and harmless to human body when used.

It is an object of the present invention to provide a mesenchymal stem cell culture solution obtained by culturing mesenchymal stem cells in a non-antibiotic serum-free culture medium and a method for producing the same, and to provide a mesenchymal stem cell culture solution safe for human use.

Another object of the present invention is to provide a cosmetic composition comprising the mesenchymal stem cell culture solution so as to be effective for skin improvement and harmless to human body.

The mesenchymal stem cell culture solution of the present invention is obtained by culturing mesenchymal stem cells in a serum-free antibiotic medium containing a rust-out serum substitute, and then recovering the supernatant of the medium.

The serum-free non-antibiotic medium may be DMEM or DMEM / F12 medium.

The mesenchymal stem cells may be derived from any one of human adipose tissue, bone marrow tissue, amnion tissue, decidual tissue, umbilical cord tissue and placental tissue.

The mesenchymal stem cell culture medium may have a skin irritation index of less than 1 in a skin irritation test according to a Draize test.

The mesenchymal stem cell culture medium may have an intraocular mucosal irritation index of less than 0.1 in a mucosal membrane stimulation test according to a Draize test.

The mesenchymal stem cell culture medium may have a photosensitizing index of less than 0.1 in a photosensitizing skin reaction test according to an Adjuvant Strip test.

The mesenchymal stem cell culture medium may have a phototoxicity index of less than 0.1 in a phototoxic skin reaction test according to the Morikawa test.

The mesenchymal stem cell culture medium may have a sensitization index of less than 0.1 in the skin sensitization test according to the Maximization test.

The cosmetic composition of the present invention comprises the mesenchymal stem cell culture solution.

The method for culturing mesenchymal stem cells according to the present invention comprises the steps of preparing mesenchymal stem cells, preparing a serum-free antibiotic medium containing a rust-out serum substitute, and culturing the mesenchymal stem cells in the serum- Culturing the mesenchymal stem cells, and recovering the supernatant from the medium in which the mesenchymal stem cells have been cultured.

The method may further include a step of pre-treating the mesenchymal stem cells with a culture inhibitor before culturing the mesenchymal stem cells in the serum-free antibiotics medium.

The serum-free non-antibiotic medium may be DMEM or DMEM / F12 medium.

The mesenchymal stem cells may be derived from any one of human adipose tissue, bone marrow tissue, amnion tissue, decidual tissue, umbilical cord tissue and placental tissue.

The method of the present invention for producing a mesenchymal stem cell culture solution can be used to produce a human-free antibiotic-free serum-free mesenchymal stem cell culture, and the cosmetic composition of the present invention is more safe for the human body and effective for improving skin wrinkles .

Brief Description of the Drawings Fig. 1 is an actual micrograph showing the state of mesenchymal stem cells before and after the recovery of human adipose-derived non-serum-free serum-free mesenchymal stem cell culture media.
FIG. 2 is a fluorescence microscope photograph (Scale bars: 200 μm) showing the expression of a fluorescent substance when irradiated with mesenchymal stem cell markers (CD90 and CD105) to examine human adipose tissue-derived mesenchymal stem cells.
FIG. 3 is a graph showing the content of procollagen according to the concentration of human adipose tissue-derived non-antibiotic serum-free mesenchymal stem cell culture liquid.
4 is a graph showing VEGF (vascular endothelial growth factor) content according to the concentration of human adipose tissue-derived non-antibiotic serum-free mesenchymal stem cell culture fluid.
5 is a graph showing the total protein content according to the concentration of human adipose tissue-derived non-antibiotic serum-free mesenchymal stem cell culture liquid.
Fig. 6 shows the degree of improvement of the eye wrinkles by skin aging mitigation of cosmetic compositions containing human non-antibiotic serum-free mesenchymal stem cell culture derived from human adipose tissue.
Fig. 7 shows the rate of improvement in transdermal water loss by a skin barrier strengthening clinical trial of a cosmetic comprising a human adipose-derived non-serum-derived mesenchymal stem cell culture.

Stem cells are undifferentiated cells that can divide for a long period of time and self-renew, and are capable of differentiating into various types of cells given certain conditions. Stem cells can be classified into embryonic stem cells isolated from inner cell mass of blastocyst and adult stem cells isolated from adult tissue. Although there are limited disadvantages to embryonic stem cells, many therapeutic agents have been studied in adult stem cells, which have no ethical problems and no side effects. Specifically, in the present invention, adult stem cells are used, and stem cells isolated from commercially available stem cells or living tissues can be used.

The present invention provides a mesenchymal stem cell culture obtained by culturing mesenchymal stem cells in a serum-free antibiotic medium containing a green-out serum substitute, and recovering the supernatant of the medium.

The mesenchymal stem cells include adult stem cells as well as embryonic stem cells derived from mammals, and adult stem cells may be derived from adult stem cells of all tissues. For example, adult stem cells may be selected from bone marrow, cord blood, blood, liver, skin, gastrointestinal tract, placenta, nerve, adrenal, epithelial and uterine, It may be a stem cell isolated from a tissue such as a fat tissue, a bone marrow, an amniotic tissue, a decidual tissue, an umbilical cord tissue, or a placental tissue .

Preferably, the mesenchymal stem cells may be stem cells derived from human adipose tissue. Human adipose-derived stem cells are a kind of adult stem cells isolated from human adipose tissue. The acquisition of adipose tissue is advantageous in that a sufficient amount of stem cells can be easily obtained and cultured, which can be supplementarily obtained in a liposuction process, a delivery process, or a laparotomy in a patient's surgical procedure, which are conventionally performed, and It is more safe than bone marrow harvesting. In a specific embodiment of the present invention, a mesenchymal stem cell culture broth was prepared using human adipose tissue-derived mesenchymal stem cells, but the present invention is not limited thereto.

In the present invention, the term 'mesenchymal stem cell culture solution' means a supernatant part of a mesenchymal stem cell culture solution obtained by culturing mesenchymal stem cells in a medium, and the term 'basic culture solution' or ' The term " culture solution " refers to a culture solution for putting stem cells into culture.

The culture medium for the stem cell culture may be any of the basic media known in the art without limitation, but serum and antibiotics are not added. The basic medium may be prepared by artificial synthesis, or a commercially prepared medium may be used. Examples of commercially produced media include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Isocove's Modified Dibbecco's Medium, but the present invention is not limited thereto, and DMEM or DMEM / F12 can be preferably used.

The serum-free, non-antibiotic, mesenchymal stem cell culture solution prepared according to the present invention can be used as a cosmetic composition in its entirety, but it can be generally manufactured in combination with additives used in the production of a cosmetic composition, 0.01 to 80 parts by weight based on 100 parts by weight of the resin. When the amount of the culture medium of mesenchymal stem cells of the present invention is less than 0.01 part by weight based on the total weight of the composition, sufficient wrinkle improvement and skin improvement effect can not be expected. If the amount exceeds 80 parts by weight, skin allergic reaction This is to prevent this. Preferably 1 to 20 parts by weight.

The cosmetic composition may further contain, in addition to the serum-free anti-microbial mesenchymal stem cell culture of the present invention, a lipid, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, , A perfume, a surfactant, water, an ionic or nonionic emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, a wetting agent, a essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent, Or any other ingredient conventionally used in external preparations for dermatological purposes. The components can also be introduced in amounts commonly used in the field of dermatology.

Wetting agents that may be used in the cosmetic composition of the present invention may include wheat protein, hair keratin amino acid, sodium peroxycinelcarboic acid, panthenol, tocopherol (vitamin E) and dimethicone or mixtures thereof. Sodium chloride may also be present, especially if the hair keratin amino acid is included as a wetting agent. The wetting agent is typically used in an amount of about 0.01 to 10% by weight, preferably about 0.05 to 1.5% by weight, more preferably about 0.01 to 1% by weight of the composition.

The antioxidant that can be used in the cosmetic composition of the present invention can be used both as an enzyme-type antioxidant and as a non-enzyme-type antioxidant. Examples of natural enzyme-type antioxidants include peroxide dismutase (SOD), catalase and glutathione peroxidase. Suitable non-enzymatic antioxidants include vitamin E (e.g., tocopherol), vitamin C (ascorbic acid), carotenoids, echinacoside, caffeoyl derivatives, oligomeric proanthocyanidins, (E.g., grape seed extract), silymarin (e.g., Milk Thistle extract, Silie Boom Mariannium), ginkgo biloba and green tea polyphenols or mixtures thereof. Carotenoids are potent antioxidants and include beta-carotene, canthaxanthin, zeaxanthin, lycopene, lutein, crocetin and capsanthin, and the like. Preferred antioxidants are vitamin E, vitamin C or carotenoids. Antioxidants, when used, are used in amounts sufficient to inhibit or alleviate the effect of free-radicals in the skin. A typical use amount of the antioxidant is about 0.001 to 1% by weight of the composition, and a preferable amount is about 0.01 to 0.5% by weight.

The cosmetic composition of the present invention may contain other adjuvants conventionally used in the art. Examples of such adjuvants include perfumes, dyes (including dyes that simultaneously dye hair or add color), solvents, opacifiers, pearlescent agents (e.g., esters of fatty acids and polyols, magnesium salts of fatty acids, and zinc (For example, sodium chloride, potassium chloride, ammonium chloride and sodium sulfate), fatty acid alkylol amides, cellulose derivatives, natural gums, plant extracts (for example, aloe vera leaf extract, Natural or synthetic polypeptides, yolk, lecithin, lanolin or lanolin derivatives, fats, oils (such as sunflower seed oil, barley seed oil, jojoba oil, etc.), albumin derivatives (e.g., gelatin), collagen hydrolyzate Oil, camellia oil, avocado oil, etc.), fatty alcohols, silicones, diodermants, antimicrobials, anti-seborrheic agents, keratolytic agents (for example, sulfur, salicylic acid Sulfur, salicylic acid, selenium sulfide, antimicrobial agents, benzoyl peroxide, chlorhexidine, iodine, triclosan, antifungal agents and enzymes), or a combination of these substances (such as, for example, benzoyl peroxide, selenium sulfide, PG, And mixtures thereof.

The cosmetic composition containing the cosmetic composition of the present invention can be produced in the form of a general emulsified formulation and a solubilized formulation. For example, creams, essences, cosmetic creams, sprays, gels, packs, sunscreens, make-up bases, liquids such as lotions such as lotion, facial lotion, body lotion, A powder, a cleansing lotion, a makeup removing agent such as a cleansing oil, a cleaning agent such as a cleansing foam, a soap, a body wash and the like.

The method for culturing mesenchymal stem cells according to the present invention comprises the steps of preparing mesenchymal stem cells, preparing a serum-free antibiotic medium containing a rust-out serum substitute, and culturing the mesenchymal stem cells in the serum- Culturing the mesenchymal stem cells, and recovering the supernatant from the medium in which the mesenchymal stem cells have been cultured.

The method may further include a step of pre-treating the mesenchymal stem cells with a culture inhibitor before culturing the mesenchymal stem cells in the serum-free antibiotics medium. This inhibits the growth of cells with culture inhibitors when the mesenchymal stem cells are inoculated into the culture dish and the number of cells in the culture dish is 90%, that is, about 2 days after inoculation into the culture dish. Mitomycin-C (Sigma) may be used as the culture inhibitor.

The serum-free non-antibiotic medium may be DMEM or DMEM / F12 medium.

The mesenchymal stem cells may be derived from any one of human adipose tissue, bone marrow tissue, amnion tissue, decidual tissue, umbilical cord tissue and placental tissue.

Hereinafter, the present invention will be described more specifically by way of examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

1. Preparation of human adipose tissue-derived mesenchymal stem cell culture

i) Culture of adipose tissue cells

Adipose tissue was obtained from subcutaneous fat in the laparotomy area of the consenting patients. For cell culture, the adipose tissue was placed in a 0.1% collagenase type I (Invitrogen) solution, which was a cell-separating enzyme, and reacted in a shaking incubator at 36 ° C for 1 hour to separate into cell clumps or cells. For the cultivation of the separated cells, a culture solution (90% Dlabco's Modified Eagle's Medium (DMEM) Nutrient Mixture F-12 (1: 1, Gibco # 11320), 10% fetal bovine serum (Hyclone) The cells were washed twice with 1% nonessential amino acids, and basic fibroblast growth factor (bFGF, KOMA Biotech, Inc.), 75 ug / mL potassium penicillin G and 50 ug / mL streptomycin sulphate] Was removed and placed in a new culture medium and inoculated at a concentration of approximately 1 x 10 ⁶ on a 100 mm diameter culture dish. Initially incubated for 7 to 10 days to the passages (primary cell c㎕ture) grown cells can be put in a single cell separation, an enzyme for TrypLE (Gibco) solution separated for 5 minutes and 1 x 10 ⁶ cells in Lt; / RTI > in a fresh culture dish. This procedure was repeated 3 to 5 times to massively proliferate adipose tissue cells.

ii) Acquisition of mesenchymal stem cell culture fluids from human adipose tissue cells

Cell CD271 microbead kit (MACS, Mitenyl Biotech GmbH, Germany) was used to obtain mesenchymal stem cells from mass proliferated adipose tissue cells. Immunocytochemistry was performed using CD90 (ab133350, Abcam, 1: 100), CD105 (ab114052, Abcam, 1: 100) antibody to verify that the obtained cells were mesenchymal stem cells.

iii) Preparation of human adipose tissue-derived mesenchymal stem cell culture

When adipose tissue-derived mesenchymal stem cells are inoculated into a culture dish at 30,000 cells / cm ² and the number of cells in the culture container is about 90%, that is, when cells are grown in the culture container for about 2 days, Cells were cultured in RPMI 1640 medium supplemented with 90% Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, serum and phenol red, and then incubated with 10 μg / ml mitomycin-C (Sigma) (BFGF, KOMA Biotech, Inc)) was added to the culture medium. The culture medium was replaced with a mixture of F-12 (1: 1, Gibco # 21041), 20% serum substitute (Knockout SR, Gibco), 1% nonessential amino acids, and 8 ng / After the incubation for 5 days after the replacement, the supernatant was recovered daily and filtered with a 0.22 탆 filter. The prepared human adipose tissue-derived mesenchymal stem cell culture concentrate was stored at -80 ° C.

iv) Isolation of human adipose tissue-derived mesenchymal stem cells

FIG. 1 is a photomicrograph showing the state of proliferation of human adipose tissue-derived mesenchymal stem cells before and after the recovery of the mesenchymal stem cell culture fluid.

As shown in FIG. 1, when the isolated mesenchymal stem cells were proliferated and observed under an inverted microscope, their adhered appearance was similar to fibroblasts.

To confirm the presence of mesenchymal stem cells, immunocytochemistry was performed by using an antibody with a fluorescent substance.

FIG. 2 is a fluorescence microscope photograph (Scale bars: 200 μm) showing the expression of a fluorescent substance when irradiated with mesenchymal stem cell markers (CD90 and CD105) to examine human adipose tissue-derived mesenchymal stem cells.

As shown in FIG. 2, a significant amount of fluorescent substance expressed by CD90 and CD105 was detected and confirmed to be mesenchymal stem cells.

2. Investigation of effective substance content of human adipose tissue-derived mesenchymal stem cell culture

As a representative active material of human adipose tissue-derived mesenchymal stem cell culture media, procollagen (PIP) EIA kit, # MK101, TAKARA, and VEGF Quantikine ELISA kit, # DVE00, R & D systems ), Total protein (Pierce BCA Protein Assay Kit, # 23227, Thermo).

i) Procollagen content analysis

For the analysis of procollagen content, 100 μl of the antibody-peroxidase binding solution and 20 μl of the sample or standard solution to be tested were reacted at 37 ° C. for 3 hours in a microtitre plate coated with the antibody. After the reaction time has elapsed, the reaction solution is removed, washed 4 times with PBS, and the water is completely removed. 100 μl of the substrate solution is added and reacted at room temperature for 15 minutes. 100 μl of the reaction stop solution is further added, The absorbance was examined within 30 minutes. The standard solution was diluted to 0, 10, 20, 40, 80, 160, and 320 ng / ml, and the absorbance was examined to confirm the standard curve.

FIG. 3 is a graph showing the content of procollagen in cultured human non-antibiotic serum-free mesenchymal stem cells derived from adipose tissue.

The samples were diluted to 1/5 and the final 1%, 3%, 5% and 100% values were measured. The values of procollagen were 70 ng / ml, 320 ng / ml, 340 ng / ml and 1700 ng / ml Respectively.

ii) Analysis of vascular endothelial growth factor (VEGF) content

For the analysis of vascular endothelial growth factor, 50 μl of the diluted solution was added to the antibody-coated microtiter plate, and 200 μl of the sample or standard solution was added and reacted at room temperature for 2 hours. After this time, the reaction solution was removed, washed 3 times with PBS, and the water was completely removed. Then, 200 μl of the antibody-conjugated solution was added and reacted at room temperature for 2 hours. After the reaction time has elapsed, the reaction solution is again removed, washed 3 times with PBS, and the water is completely removed. Then 200 μl of substrate solution is added and reacted at room temperature for 25 minutes. Then, 50 μl of the reaction stop solution is added, The absorbance of the reader was measured at a wavelength of 450 nm within 30 minutes. The standard solution was diluted to 0, 15, 30, 60, 120, 250, 500, and 1,000 pg / ml, and the absorbance was checked to confirm the standard curve.

FIG. 4 is a graph showing VEGF (vascular endothelial growth factor) content of a human adipose tissue-derived serum-free serum-free mesenchymal stem cell culture.

VEGF levels were 130 pg / ml, 410 pg / ml, and 750 pg / ml, respectively, when the samples were diluted to 1/5 and the final 1%, 3%, 5% , And 6050 pg / ml, respectively.

iii) Analysis of total protein content

For total protein content, 25 μl of each sample and standard solution was added to 100 μl of microplate, 200 μl of working reagent was added, and the mixture was shaked for 30 seconds in a shaker and incubated at 37 ° C for 30 minutes. After cooling the plate at room temperature, the absorbance was measured at 562 nm in reader.

FIG. 5 is a graph showing the total protein content of human non-antibiotic-serum-free mesenchymal stem cell culture derived from adipose tissue.

The total protein contents of samples 1%, 3%, 5% and 100% were 290 mg / ml, 540 mg / ml, 820 mg / ml and 1,900 mg / ml.

3. Safety test of human adipose tissue-derived mesenchymal stem cell culture

i) skin irritation test

The skin irritation of the mesenchymal stem cell culture of the present invention was tested according to the Draize test. Young and healthy white rabbits (2.0-3.0 kg) were used and the dose was 0.5 ml. The application area is skin of hair clippings, 2.5cm × 2.5cm, 2 skin areas and 2 skin areas. The test method was applied to the skin of 2.5cm in length and then covered with a gauze. The gauze was fixed with a tape. To prevent evaporation of the test material, the test material was covered with a non-permeable, Respectively. After 24 hours of application of the test substance, mortality, general symptoms, weight change and skin irritation were evaluated for 72 hours.

(Table 1) Evaluation of skin reaction

As a result of the evaluation, there were no deaths due to the application of the test substance during the test period, and no abnormal symptoms were observed in all test animals. Body weight measurements showed normal body weight gain in all animals. The skin irritation index (PII) was calculated to be 0.3 in the Draize calculation method based on the result of observing the skin irritation of the applied part after application of the test material according to the above Table 1. The mesenchymal stem cell culture of the present invention, It was judged as polar irritant.

ii) mucous membrane stimulation test

 The skin irritation of the mesenchymal stem cell culture of the present invention was tested according to the Draize test. Young and healthy white rabbits (2.0 ~ 3.0kg) were used, and both eyes of the test animals were examined by preliminary examination 24 hours before the start of the test, and it was confirmed that there was no damage to the cornea such as eye damage. The test substance was instilled in one eye of 9 rabbits, and after 20 to 30 seconds, 3 eyes were washed with 20 ml of aseptic physiological saline solution on both eyes for 1 minute, and the remaining 6 eyes I did not wash it. Mortality, general symptoms, weight changes and mucosal irritation were evaluated for 72 hours after administration of the test substance, in contrast to the other eyes to which the test substance was not administered.

 (Table 2) Grade of ocular lesion

As a result of the evaluation, there were no deaths due to the application of the test substance during the test period, and no abnormal symptoms were observed in all test animals. Body weight measurements showed normal body weight gain in all animals. The calculated acute mucosal irritation index (IAOI) was calculated to be 0.0, based on the skin irritation of the applied part after application of the test material according to the above Table 2. The mesenchymal stem cell culture solution of the present invention, Respectively.

iii) Photosensitization test

The method of testing the optical sensitization is known as Adjuvant and Strip Test, Harber Test, Morikawa Test, Horio Test, Jordan Test, Kochever Test, Maurer Test and Vinson Test. In this example, the adjuvant and strip test was conducted using adjuvant to increase the induction of sensitization of the test substance, and guinea pig was used as an experimental subject. The test subjects were divided into the test substance administration group and the control group, and the lamp alone of the UV-A region was used as the light source. After application of the mesenchymal stem cell culture of the present invention, the mortality, general symptoms, body weight change for 72 hours, and skin reaction at 24, 48 and 72 hours were evaluated according to the judgment criteria of the adjuvant strip test.

As a result of the evaluation, no mortality was observed during the test period, and no abnormal abnormalities due to the application of the test substance were observed. All the body weight measurements during the test period showed a general weight gain. The test material was applied to the test material treatment zone at a concentration of 1,500 μg / ml for 2 hours, irradiated with UVA (320 to 420 nm) to a light intensity of 10 J / cm ^{2 for} a certain period of time, The skin response was observed at 24 hours, 48 hours, and 72 hours, and the positive control group had a photo-allergic index of 1.8, 0.8, and 0.5, respectively. On the other hand, in the case of the test substance administration group and the negative control group, the skin reaction was not observed in the evaluation at 24 hours, 48 hours and 72 hours, and the light sensitivity index was calculated as 0.0.

iv) Phototoxicity test

Ison test, Ljunggren test, Morikawa test, Sams test and Stott test are known to test the phototoxicity. Morikawa test was conducted in this embodiment, and guinea pig was used as an experimental target. The test subjects were divided into the test substance administration group and the control group, and the lamp alone of the UV-A region was used as the light source. After applying the mesenchymal stem cell culture of the present invention, the mortality, general symptoms, body weight change for 72 hours, skin reaction at 24, 48 and 72 hours were evaluated according to Morikawa test judgment criteria.

As a result of the evaluation, no mortality was observed during the test period, and no abnormal abnormalities due to the application of the test substance were observed. All the body weight measurements during the test period showed a general weight gain. The test material was applied to the test material treatment zone at a concentration of 1,500 μg / ml for 2 hours, irradiated with UVA (320 to 420 nm) to a light intensity of 15 J / cm ^{2 for} a certain period of time, The skin reaction was observed at 24 hours, 48 hours, and 72 hours, and the phototoxic index of the positive control group was evaluated as 5.2, 4.7, and 3.7, respectively. On the other hand, in the test substance administration group and the negative control group, skin reaction was not observed at 24 hours, 48 hours and 72 hours, and the phototoxicity index was 0.0.

v) Skin Sensitization Test

As a method to test whether a skin sensation is made, Adjuvant and Patch Test, Buehler Test, Draize Test, Freund's Complete Adjuvant Test, Open Epicutaneous Test, Optimization Test, and Split Adjuvant Test are known. In this example, evaluation was made according to the criteria of the Maximization test, which is the most widely used method for testing whether or not the skin of a test substance is inspected.

Guinea pigs were used for about one week, and females were either not pregnant or had no experience of pregnancy. The test subjects were divided into the test substance group, the solvent control group and the positive control group. Three kinds of the following samples were injected intramuscularly into the skin of the back part of the epilating test animal (about 2 x 4 cm) Respectively.

1) Emulsion (1: 1 mixture) of distilled water (or physiological saline) and Freund's Complete Adjuvant (FCA)

2) Test substance

3) Test substances and emulsions of FCA (1: 1 mixture)

After 1 week of the first sensitization, patches (2 x 4 cm) containing the test substance were attached to the site where the test substance was injected intracutaneously, and the second sensitization was induced by occlusion for 48 hours. After 2 weeks of occlusion, the test animals were sacrificed for 24 hours with a patch (2 x 2 cm) containing the test material removed and the upper abdomen or lateral abdomen (other than the sensitization site) removed.

As a result of evaluation, no dead animals were observed in the negative control group and the positive control group during the test period. However, in the test substance-administered group, 1 individual exhibited weakening on the day of induction of secondary sensitization and died on day 1 (8 days after induction of primary sensitization) . No abnormal abnormalities were observed due to the application of the test substance. During the test period, the body weight was measured, and normal weight gain was observed in all test animals in the test substance-administered group. As a result of evaluating the skin reaction at 24 hours and 48 hours after inducing the test substance to a concentration of 1,500 μg / ml, the sensitization index (average skin reaction score) of the first sensitization and the second sensitization was 2.0 and 1.4 in the positive control group The frequency index (sensitization rate) was calculated as 100% and 100%, respectively. On the other hand, the sensitization index and the frequency index were calculated as 0.0 and 0.0% in both the first and second test substances, respectively.

4. Origin of human fat tissue Mesenchymal stem cells  Containing culture medium Cosmetics  Composition clinical evaluation

According to one embodiment of the present invention, a cosmetic composition containing a culture medium of mesenchymal stem cells derived from human adipose tissue is manufactured, and the resulting cosmetic product is topically applied to the skin of a human body to improve the elasticity, wrinkles and moisturizing properties of photoaged skin And the like were tested and evaluated. The average age was 45.6 years, with 30 to 2, 40 to 16, and 50 to 3 persons for each age group. To evaluate the degree of improvement of wrinkles, skin compactness was evaluated using PRIMOS, skin elasticity was evaluated using a cutometer, and skin moisture was evaluated using a corneometer. The evaluations were evaluated 3 times (0 weeks, 2 weeks, 4 weeks) over 4 weeks.

i) Wrinkle improvement rate evaluation

(Table 3)

Table 3 above shows the wrinkle improvement rate of subjects in a skin aging mitigation clinical trial of a cosmetic composition containing human non-antibiotic serum-free mesenchymal stem cell culture derived from human adipose tissue.

FIG. 6 is a graph showing the degree of improvement of wrinkles in the eye caused by skin aging mitigation of a cosmetic composition containing human non-antibiotic serum-free mesenchymal stem cell culture derived from human adipose tissue.

Results of measurement using PRIMOS (Phaseshift Rapid In-Vivo Measurement of Skin) showed statistically significant improvement of eye wrinkles after 2 weeks of use and 4 weeks after use of product (p <0.05).

ii) Evaluation of elasticity improvement rate

(Table 4)

Table 4 shows the improvement rate of the subject's elasticity in the skin aging mitigation clinical trial of the cosmetic comprising the human adipose tissue-derived non-serum-free serum-free mesenchymal stem cell culture liquid.

The R2 (total elasticity) increased 3.3% after 2 weeks of product use and 4.9% after 4 weeks of product use, and R5 (pure elasticity) increased to 4.9% after 2 weeks of product use with cutometer (Courage and Khazaka Electronic, Germany) %, And 25.1% after 4 weeks of using the product, respectively, statistically significant improvement of the elasticity was observed (p <0.05).

iii) Evaluation of moisturizing improvement rate

(Table 5)

Table 5 shows the subject's moisturizing improvement rate in the skin aging mitigation clinical trial of the cosmetic comprising the human adipose tissue-derived non-antibiotic serum-free mesenchymal stem cell culture liquid.

As a result of using the Corneometer (Courage and Khazaka Electronic, Germany), the moisturizing effect was increased by 7.7% after 2 weeks of use and by 12.9% after 4 weeks of using the product, and a statistically significant improvement was observed (p <0.05) .

iv) Evaluation of skin regeneration ability

In one embodiment of the present invention, a cosmetic preparation containing a culture medium of human adipose-derived non-antibiotic serum-free mesenchymal stem cells is prepared, and the resulting cosmetic product is topically applied to the skin of a human body to test for barrier strengthening And evaluated. The average age was 27.3 years. To assess skin regeneration, transdermal water loss (TEWL) using a Tewameter was measured. The evaluation was evaluated twice a day over two weeks.

Fig. 7 shows the rate of improvement in transdermal water loss by a skin barrier strengthening clinical trial of a cosmetic comprising a human adipose-derived non-serum-derived mesenchymal stem cell culture.

According to the evaluation method, when the product application area and the nonwashing area were measured by the apparatus, the improvement rate of the transdermal water loss (TEWL) after one week of use was 89.95% for the application area and 86.05% for the nonwashing area. (TEWL) improvement rate was 97.45% for the application area and 93.57% for the non-dermal area. The difference was statistically significant (p <0.05).

Claims (13)

A mesenchymal stem cell culture solution obtained by culturing mesenchymal stem cells in a serum-free antibiotic medium containing a substitute for a green-out serum, and recovering the supernatant of the medium.
The method according to claim 1,
The serum-free antibiotic-free medium is DMEM or DMEM / F12 medium.
The method according to claim 1,
Wherein the mesenchymal stem cell is derived from any one of human adipose tissue, bone marrow tissue, amniotic membrane tissue, decidual tissue, umbilical cord tissue and placental tissue.
The method according to claim 1,
Wherein the mesenchymal stem cell culture medium has a skin irritation index of less than 1 in a skin irritation test according to a Draize test.
The method according to claim 1,
Wherein the mesenchymal stem cell culture medium has a mucosal irritation index of less than 0.1 in a mucosal membrane stimulation test according to a Draize test.
The method according to claim 1,
The mesenchymal stem cell culture medium has a photosensitizing index of less than 0.1 in a photosensitizing skin reaction test according to an Adjuvant and Strip test.
The method according to claim 1,
The mesenchymal stem cell culture medium has a phototoxicity index of less than 0.1 in a phototoxic skin reaction test according to the Morikawa test.
The method according to claim 1,
The mesenchymal stem cell culture medium has an index of sensitization of less than 0.1 in the skin sensitization test according to the Maximization test.
9. A cosmetic composition comprising a mesenchymal stem cell culture solution according to any one of claims 1 to 8.
Preparing mesenchymal stem cells;
Preparing a serum-free antibiotic medium comprising a green-out serum replacement agent;
Culturing the mesenchymal stem cells in the serum-free antibiotics medium; And
Recovering the supernatant from the medium in which the mesenchymal stem cells have been cultured;
/ RTI &gt; cells. &Lt; / RTI &gt;
11. The method of claim 10,
Before the step of culturing mesenchymal stem cells in the serum-free antibiotics medium,
Further comprising the step of pre-treating the mesenchymal stem cells with a culture inhibitor.
11. The method of claim 10,
Wherein the serum-free antibiotic-free medium is DMEM or DMEM / F12 medium.
11. The method of claim 10,
Wherein the mesenchymal stem cell is derived from any one of human adipose tissue, bone marrow tissue, amniotic membrane tissue, decidual tissue, umbilical cord tissue and placental tissue.

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