A skin care composition containing stem cell extract and its preparation method and application

Abstract

The present invention proposes a skin care composition containing stem cell extracts, and a preparation method and application thereof, which belong to the field of cosmetic technology. The extracted urine-derived stem cells and antler stem cells are mixed and cultured in a DMEM culture medium to which epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 are added, and then the culture fluid is separated to obtain mixed exosomes, mixed stem cells and extracellular matrix, and the mixed stem cells are prepared into stem cell liposomes, which are embedded in titanium dioxide / silicon dioxide microspheres with the mixed exosomes, added to the extracellular matrix, and evenly dispersed to obtain a skin care composition containing stem cell extracts. The skin care composition containing stem cell extracts obtained by the present invention can significantly prolong the storage period of stem cells and exosomes, improve their repair and improvement of the skin, and the obtained composition has good anti-aging, whitening, skin elasticity promotion, anti-wrinkle and other effects, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a skin care composition containing stem cell extracts, and a preparation method and application thereof. Background Art

[0002] Aging is the phenomenon that the morphology, structure and function of the whole body gradually decline during the life process. The most obvious external manifestation of human aging is the skin, which is a complex organ that extends throughout the body. With age, the cycle of skin metabolism will gradually lengthen. Starting from the age of 25, as the rate of skin metabolism gradually slows down, the elastic fibers in the dermis of the skin begin to break and deform, and aging cells continue to form and stay on the epidermis for a long time. They cannot be replaced by new cells, which causes the elasticity and moisture of the skin to continue to decrease, resulting in a series of problems such as sagging skin, wrinkles, and loss of skin luster. With the improvement of living standards, people's pursuit of skin care is getting higher and higher. In addition to medical cosmetic surgery, most people prefer to use high-quality skin care products to gently keep their skin young. Therefore, preventing and delaying skin aging has become a research hotspot in the skin care industry.

[0003] Cell aging is the root cause of human aging. Aging cells have a reduced ability to remove free radicals in the body. Free radicals and reactive oxygen species react with proteins in the skin, causing them to denature and lose their original functions. These proteins include collagen and elastin in the dermis, leading to sagging skin and other problems.

[0004] Mesenchymal stem cells are a type of adult stem cell with significant plasticity and multidirectional differentiation potential. They exist in tissues such as bone marrow, fat, synovium, bones, muscles, lungs, liver, pancreas, as well as amniotic fluid and umbilical cord blood. Under certain conditions, they can differentiate into osteocytes, chondrocytes, adipocytes, etc. Studies have shown that mesenchymal stem cells have multidirectional differentiation capabilities, can exert immunomodulatory and paracrine effects, and help repair damaged tissues. These characteristics of mesenchymal stem cells make them increasingly popular in the field of skin care products.

[0005] Existing skin care products that add mesenchymal stem cell extracts have many problems, such as the inability of mesenchymal stem cell extracts to maintain activity and stability, which increases the production cost of the product, and the inability to fully exert the anti-aging effect when used, which only becomes a publicity gimmick. Therefore, how to make stem cell extracts play a better role in skin care products is a problem that those skilled in the art need to solve. Summary of the invention

[0006] The purpose of the present invention is to provide a skin care composition containing stem cell extracts and its preparation method and application, which can significantly prolong the storage period of stem cells and exosomes, improve their repair and improvement of the skin, and the prepared composition has good anti-aging, whitening, skin elasticity promotion, anti-wrinkle and other effects, and has broad application prospects.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The present invention provides a method for preparing a skin care composition containing stem cell extracts. The extracted urine-derived stem cells and antler stem cells are mixed and cultured in a DMEM culture medium to which epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 are added, and then the culture fluid is separated to obtain mixed exosomes, mixed stem cells and extracellular matrix. The mixed stem cells are prepared into stem cell liposomes, which are embedded in titanium dioxide / silicon dioxide microspheres with the mixed exosomes, added into the extracellular matrix, and evenly dispersed to obtain a skin care composition containing stem cell extracts.

[0009] As a further improvement of the present invention, the following steps are included:

[0010] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0011] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it, add the precipitate to the urine-derived stem cell basal medium, resuspend it, and culture it. When the fusion rate of adherent cells reaches 80-90%, subculture the cells, centrifuge it, resuspend the precipitate in cell freezing solution, and freeze it;

[0012] S3. Extraction of antler stem cells: Take the tip of the antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium for culture, and when the fusion rate of adherent cells reaches 80-90%, digest it, centrifuge it, precipitate it, resuspend it in cell freezing solution, and freeze it;

[0013] S4 mixed culture: thawing the frozen cells in step S2 and step S3, centrifuging, resuspending the precipitate in culture medium, and mixed culturing to obtain a mixed culture solution;

[0014] S5. Extraction of mixed exosomes: The mixed culture fluid is centrifuged, and the precipitate and supernatant are collected separately. The precipitate is mixed stem cells, which are resuspended in fresh culture medium to obtain a stem cell suspension. The supernatant is separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain mixed exosomes and extracellular matrix;

[0015] S6. Preparation of stem cell liposomes: dissolving lecithin and cholesterol in a mixed solution, adding stem cell suspension dropwise, stirring at a constant temperature, rotary evaporation, ultrasonic dispersion, and freeze drying to obtain stem cell liposomes;

[0016] S7. Preparation of loaded titanium dioxide / silicon dioxide microspheres: adding tetrabutyl titanate and alkyl orthosilicate to an organic solvent to obtain an oil phase; adding a porogen and a surfactant to water, adding stem cell liposomes and mixed exosomes, and ultrasonically dispersing them uniformly to obtain an aqueous phase; dropping the aqueous phase into the oil phase, emulsifying, adjusting the pH value of the solution, stirring the reaction, centrifuging, washing, and drying to obtain loaded titanium dioxide / silicon dioxide microspheres;

[0017] S8. Preparation of a skin care composition: Add the loaded titanium dioxide / silicon dioxide microspheres into the extracellular matrix and disperse them evenly by ultrasonication to prepare a skin care composition containing stem cell extracts.

[0018] As a further improvement of the present invention, the amount of epidermal growth factor added in step S1 is 3-5 μg / L, the amount of cholera toxin added is 25-35 μg / L, the amount of penicillin added is 80-120 U / mL, the amount of streptomycin added is 0.8-1.2 g / L, the amount of transferrin added is 3-5 mg / L, the amount of IL-2 added is 1-2 mg / L, and the amount of IL-15 added is 0.7-1.5 mg / L; the speed of centrifugation in step S2 is 1000-2000 r / min, the time is 10-20 min, the urine-derived stem cell basal medium is composed of a mixture of 3 / 4 DMEM medium and 1 / 4 Ham's F-12 medium, and the culture conditions are 36-38°C, 4-6 v / v% CO 2 The cells are cultured under the following conditions, the cells are subcultured to the 3rd to 4th generation, and the cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 5-7:2-4:1.

[0019] As a further improvement of the present invention, the antler culture medium in step S3 is a DMEM culture medium containing 8-12wt% fetal bovine serum, and the culture conditions are 36-38°C, 4-6v / v% CO 2 The conditions of the mixed culture are as follows: the digestion is to add trypsin to a system content of 0.1-0.2wt%, the digestion is 1-3min, the centrifugal speed is 1000-2000r / min, and the time is 10-20min; the thawing in step S4 is thawing at room temperature, the centrifugal speed is 1000-2000r / min, and the time is 10-20min, and the mixed culture conditions are 36-38°C, 4-6v / v%CO 2 Culture under the same conditions for 3-5 days.

[0020] As a further improvement of the present invention, the centrifugal speed in step S5 is 1000-2000 r / min, the time is 10-20 min, and the cell content of the stem cell suspension is 10 4 -10 5 / mL, the ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 3-5°C, 3000-4000r / min for 15-20min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, 3-5°C, 12000-15000r / min centrifuged for 20-30min to remove large vesicles and proteins, the supernatant is filtered with a 0.22μm filter membrane, and finally centrifuged at 3-5°C, 30000-35000r / min for 60-70min, the precipitate is resuspended in PBS with pH=6.7-7, and centrifuged again at 3-5°C, 30000-35000r / min for 60-70min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=6.7-7 and stored in a -80°C refrigerator.

[0021] As a further improvement of the present invention, the mass ratio of lecithin, cholesterol and stem cell suspension in step S6 is 10-12:7-10:20-30, the mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:3-5, and the constant temperature stirring temperature is 35-37°C and the time is 20-30min.

[0022] As a further improvement of the present invention, the mass ratio of tetrabutyl titanate, alkyl orthosilicate, porogen, surfactant, stem cell liposomes, and mixed exosomes in step S7 is 10-14:3-5:1-2:0.5-1:8-10:3-5, the pH value of the adjustment solution is 9-10, the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate, the porogen is hexadecyltrimethylammonium chloride or hexadecyltrimethylammonium bromide, and the surfactant is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

[0023] As a further improvement of the present invention, the mass ratio of the loaded titanium dioxide / silicon dioxide microspheres to the extracellular matrix in step S8 is 10-12:25-30.

[0024] The present invention further protects a skin care composition containing stem cell extracts obtained by the above preparation method.

[0025] The present invention further protects the use of the above-mentioned skin care composition containing stem cell extracts in the preparation of anti-aging, moisturizing, whitening and ultraviolet radiation protection cosmetics.

[0026] The present invention has the following beneficial effects:

[0027] Human urine-derived stem cells are a type of stem cells derived from urine. They are non-invasive, have simple extraction and separation steps, are highly safe, have good biocompatibility, and are not prone to allergies. They promote wound healing by secreting growth factors and extracellular vesicles (such as exosomes), enhance wound angiogenesis, promote epithelial regeneration, improve the deposition and remodeling of collagen fibers, help promote skin metabolism, thereby improving the skin microenvironment and playing a good anti-aging role for the skin.

[0028] Deer antler stem cells can significantly promote the healing speed of skin wounds, inhibit the transformation of fibroblasts into myofibroblasts, reduce the occurrence of fibrosis, can scavenge free radicals, have antioxidant function, and significantly promote the proliferation of skin cells, thus showing good application prospects in anti-skin aging.

[0029] The present invention adds transferrin to the culture medium to provide an iron source. Iron is a trace element necessary for cell growth and proliferation, and plays an important role in DNA synthesis and cell respiration. Transferrin transports and releases iron ions into cells by binding to transferrin receptors on the cell surface. These iron ions bind to ferritin, which then plays the function of maintaining cell growth and proliferation. It can tightly bind to iron ions and reduce the generation of free radicals catalyzed by free iron ions, thereby protecting cells from oxidative stress and protein damage, thereby promoting the healthy and stable proliferation of stem cells.

[0030] The IL-2 added in the present invention can promote the proliferation of stem cells and the secretion of factors such as IFN-γ, TNF-β and TGF-β. IL-15 can stimulate the proliferation of stem cells, enhance the proliferation signal, weaken the apoptosis signal, increase the secretion of immunoglobulins, enrich the generation of stem cell secretion products, and the produced products have rich cell activity. The addition of the two has a synergistic effect, enhances the cell activity of stem cells, and improves their survival ability.

[0031] The present invention can solve the limitations of mesenchymal stem cells from a single tissue source in repairing tissues by co-culturing two types of stem cells. The various cytokines secreted by mesenchymal stem cells from different tissue sources can promote each other's proliferation and improve cell consistency. At the same time, the secretion amount of stem cell secretion products is increased. The obtained mixed exosomes have a longer circulation half-life, can be internalized into cells faster, release cytokines and bioactive molecules, express immunosuppressive cell surface markers, transfer RNA and microRNA, play a role in reducing inflammation and promoting skin collagen regeneration response, and its effect is better than that of a single exosome.

[0032] The present invention embeds stem cells through liposomes, which can improve the survival rate of stem cells and extend their shelf life on the one hand, and on the other hand, the liposome structure is self-assembled by phospholipids, which have a polar phosphate head and a hydrophobic lipid tail, and can pass through the skin barrier into the bottom layer of the skin muscle, thereby better exerting a regulatory effect.

[0033] The present invention prepares titanium dioxide / silicon dioxide porous microspheres through a sol-gel reaction, and embeds stem cell liposomes and mixed exosomes in the microspheres. On the one hand, the microspheres are white, which has a certain promoting effect on skin whitening. At the same time, they also have a good anti-ultraviolet radiation effect and reduce the production of melanin. On the other hand, the microspheres can protect the stem cell liposomes and mixed exosomes, increase their shelf life, and avoid their inactivation. After being applied to the skin, the active substances have better affinity with the skin, slowly flow out of the microspheres to contact the skin, and pass through the skin barrier, thereby exerting better anti-aging, whitening, skin elasticity promotion, anti-wrinkle and other effects.

[0034] The skin care composition containing stem cell extracts prepared by the present invention can significantly prolong the storage period of stem cells and exosomes, and improve their repair and improvement of the skin. The prepared composition has good anti-aging, whitening, skin elasticity promotion, anti-wrinkle and other effects, and has broad application prospects. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example 1

[0036] This embodiment provides a method for preparing a skin care composition containing stem cell extracts, comprising the following steps:

[0037] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 (interleukin-2) and IL-15 (interleukin-15) are added to DMEM (Dulbecco's modification of Eagle's medium) to prepare a culture medium;

[0038] The amount of epidermal growth factor added is 3 μg / L, the amount of cholera toxin added is 25 μg / L, the amount of penicillin added is 80 U / mL, the amount of streptomycin added is 0.8 g / L, the amount of transferrin added is 3 mg / L, the amount of IL-2 added is 1 mg / L, and the amount of IL-15 added is 0.7 mg / L;

[0039] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 1000r / min for 20min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 36℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 80%, the cells were subcultured to the third generation, centrifuged at 1000 r / min for 20 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0040] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0041] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 5:2:1;

[0042] S3. Extraction of antler stem cells: Take the tip of the second antler, split it longitudinally to obtain the reserve mesenchyme (RM) tissue, mince it, add it to the antler culture medium, and incubate it at 36°C, 5v / v%CO 2 When the fusion rate of adherent cells reached 80%, trypsin was added to a system content of 0.1wt%, digested for 1min, centrifuged at 1000r / min for 10min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0043] The antler culture medium is a DMEM culture medium containing 8wt% fetal bovine serum;

[0044] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 5:2:1;

[0045] S4. Mixed culture: The frozen cells in step S2 and step S3 were mixed at a volume ratio of 1:1 and thawed at room temperature. The mixture was centrifuged at 1000 rpm for 10 min and the pellet was resuspended in culture medium at 36°C and 5 v / v% CO. 2 The mixed culture solution was obtained by culturing for 3 days under the above conditions;

[0046] S5. Extraction of mixed exosomes: The mixed culture solution was centrifuged at 1000 r / min for 20 min, and the precipitate and supernatant were collected separately. The precipitate was mixed stem cells, which were resuspended in fresh culture medium to obtain a stem cell suspension with a cell content of 10 4 -10 5 / mL, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain mixed exosomes and extracellular matrix;

[0047] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 3000 r / min for 15 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C, 12000 r / min for 20 min, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 30000 r / min for 60 min, the precipitate is resuspended with PBS (phosphate buffered saline) with pH=6.7, and centrifuged again at 4°C, 30000 r / min for 60 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended with PBS with pH=6.7 and frozen in a -80°C refrigerator;

[0048] S6. Preparation of stem cell liposomes: 10 g of lecithin and 7 g of cholesterol were dissolved in 200 mL of the mixed solution, 20 g of the stem cell suspension was added dropwise, the mixture was stirred at 35°C for 20 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0049] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:3;

[0050] S7. Preparation of loaded titanium dioxide / silica microspheres: 10 g of tetrabutyl titanate and 3 g of methyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 1 g of hexadecyltrimethylammonium chloride and 0.5 g of Tween-20 were added to 100 mL of water, 8 g of stem cell liposomes and 3 g of mixed exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 9, the reaction was stirred for 3 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0051] S8. Preparation of a skin care composition: 10 g of loaded titanium dioxide / silicon dioxide microspheres were added to 25 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts. Example 2

[0052] This embodiment provides a method for preparing a skin care composition containing stem cell extracts, comprising the following steps:

[0053] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0054] The amount of epidermal growth factor added is 5 μg / L, the amount of cholera toxin added is 35 μg / L, the amount of penicillin added is 120 U / mL, the amount of streptomycin added is 1.2 g / L, the amount of transferrin added is 5 mg / L, the amount of IL-2 added is 2 mg / L, and the amount of IL-15 added is 1.5 mg / L;

[0055] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 2000r / min for 10min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 38℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 90%, the cells were subcultured to the fourth generation, centrifuged at 2000 r / min for 10 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0056] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0057] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 7:4:1;

[0058] S3. Extraction of antler stem cells: Take the tip of the second antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium, and incubate it at 38°C, 5v / v%CO 2 When the fusion rate of adherent cells reached 90%, trypsin was added to a system content of 0.2wt%, digested for 3min, centrifuged at 2000r / min for 10min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0059] The antler culture medium is a DMEM culture medium containing 12wt% fetal bovine serum;

[0060] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 7:4:1;

[0061] S4. Mixed culture: The frozen cells in step S2 and step S3 were mixed at a volume ratio of 1:1, thawed at room temperature, centrifuged at 2000 rpm for 10 min, and the pellet was resuspended in culture medium at 38°C and 5 v / v% CO. 2Under the same conditions, the culture was carried out for 5 days to obtain a mixed culture solution;

[0062] S5. Extraction of mixed exosomes: The mixed culture solution was centrifuged at 2000 r / min for 10 min, and the precipitate and supernatant were collected separately. The precipitate was mixed stem cells, which were resuspended in fresh culture medium to obtain a stem cell suspension with a cell content of 10 4 -10 5 / mL, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain mixed exosomes and extracellular matrix;

[0063] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 4000 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, centrifuged at 4°C, 15000 r / min for 30 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 35000 r / min for 70 min, the precipitate is resuspended in PBS with pH=7, and centrifuged again at 4°C, 35000 r / min for 70 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=7 and frozen in a -80°C refrigerator;

[0064] S6. Preparation of stem cell liposomes: 12 g of lecithin and 10 g of cholesterol were dissolved in 200 mL of the mixed solution, 30 g of the stem cell suspension was added dropwise, the mixture was stirred at 37°C for 30 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0065] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:5;

[0066] S7. Preparation of loaded titanium dioxide / silica microspheres: 14 g of tetrabutyl titanate and 5 g of tetraethyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 2 g of hexadecyltrimethylammonium bromide and 1 g of Tween-40 were added to 100 mL of water, 10 g of stem cell liposomes and 5 g of mixed exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 10, the reaction was stirred for 5 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0067] S8. Preparation of a skin care composition: 12 g of loaded titanium dioxide / silicon dioxide microspheres were added to 30 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts. Example 3

[0068] This embodiment provides a method for preparing a skin care composition containing stem cell extracts, comprising the following steps:

[0069] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0070] The amount of epidermal growth factor added is 4 μg / L, the amount of cholera toxin added is 30 μg / L, the amount of penicillin added is 100 U / mL, the amount of streptomycin added is 1 g / L, the amount of transferrin added is 4 mg / L, the amount of IL-2 added is 1.5 mg / L, and the amount of IL-15 added is 1 mg / L;

[0071] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 1500r / min for 15min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, the cells were subcultured to the fourth generation, centrifuged at 1500 r / min for 15 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0072] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0073] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0074] S3. Extraction of antler stem cells: Take the tip of the second antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium, and incubate it at 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, trypsin was added to a system content of 0.15wt%, digested for 2min, centrifuged at 1500r / min for 15min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0075] The antler culture medium is a DMEM culture medium containing 10wt% fetal bovine serum;

[0076] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0077] S4. Mixed culture: The frozen cells in step S2 and step S3 were mixed at a volume ratio of 1:1, thawed at room temperature, centrifuged at 1500 rpm for 15 min, and the pellet was resuspended in culture medium at 37°C and 5 v / v% CO. 2 The mixed culture solution was prepared by culturing for 4 days under the above conditions;

[0078] S5. Extraction of mixed exosomes: The mixed culture solution was centrifuged at 1500 r / min for 15 min, and the precipitate and supernatant were collected separately. The precipitate was mixed stem cells, which were resuspended in fresh culture medium to obtain a stem cell suspension with a cell content of 10 4 -10 5 / mL, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain mixed exosomes and extracellular matrix;

[0079] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C, 13000 r / min for 25 min, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 32000 r / min for 65 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C, 32000 r / min for 65 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=6.8 and frozen in a -80°C refrigerator;

[0080] S6. Preparation of stem cell liposomes: 11 g of lecithin and 8 g of cholesterol were dissolved in 200 mL of the mixed solution, 25 g of the stem cell suspension was added dropwise, the mixture was stirred at 36°C for 25 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0081] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:4;

[0082] S7. Preparation of loaded titanium dioxide / silica microspheres: 12 g of tetrabutyl titanate and 4 g of tetraethyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 1.5 g of hexadecyltrimethylammonium bromide and 0.7 g of Tween-85 were added to 100 mL of water, 9 g of stem cell liposomes and 4 g of mixed exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 9.5, the reaction was stirred for 4 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0083] S8. Preparation of a skin care composition: 11 g of loaded titanium dioxide / silicon dioxide microspheres were added to 27 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts.

[0084] Comparative Example 1

[0085] Compared with Example 3, the difference is that the cryopreserved cells in Step S2 are not added in Step S4, but the stem cell suspension and the urine-derived stem cell cryopreservation solution are mixed in a volume ratio of 1:1.

[0086] The details are as follows:

[0087] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0088] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, the added amount of transferrin is 4 mg / L, the added amount of IL-2 is 1.5 mg / L, and the added amount of IL-15 is 1 mg / L;

[0089] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 1500r / min for 15min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, the cells were subcultured to the fourth generation, centrifuged at 1500 r / min for 15 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0090] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0091] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0092] S3. Extraction of antler stem cells: Take the tip of the second antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium, and incubate it at 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, trypsin was added to a system content of 0.15wt%, digested for 2min, centrifuged at 1500r / min for 15min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -105 / mL, frozen;

[0093] The antler culture medium is a DMEM culture medium containing 10wt% fetal bovine serum;

[0094] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0095] S4. Culture: Thaw the frozen cells in step S2 at room temperature, centrifuge at 1500 rpm for 15 min, resuspend the pellet in culture medium, and incubate at 37°C, 5 v / v% CO 2 Under the conditions, the culture was cultured for 4 days to obtain the culture fluid;

[0096] S5. Extraction of exosomes: The mixed culture solution was centrifuged at 1500 r / min for 15 min, and the precipitate and supernatant were collected separately. The precipitate was stem cells, which were resuspended in fresh culture medium to obtain a stem cell suspension with a cell content of 10 4 -10 5 / mL, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain exosomes and extracellular matrix;

[0097] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C, 13000 r / min for 25 min, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 32000 r / min for 65 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C, 32000 r / min for 65 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=6.8 and frozen in a -80°C refrigerator;

[0098] S6. Preparation of stem cell liposomes: 11 g of lecithin and 8 g of cholesterol were dissolved in 200 mL of a mixed solution, 25 g of a stem cell mixture (stem cell suspension and the thawed cell cryopreservation solution in step S2) was added dropwise, stirred at 36°C for 25 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0099] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:4;

[0100] S7. Preparation of loaded titanium dioxide / silica microspheres: 12 g of tetrabutyl titanate and 4 g of tetraethyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 1.5 g of hexadecyltrimethylammonium bromide and 0.7 g of Tween-85 were added to 100 mL of water, 9 g of stem cell liposomes and 4 g of exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 9.5, the reaction was stirred for 4 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0101] S8. Preparation of a skin care composition: 11 g of loaded titanium dioxide / silicon dioxide microspheres were added to 27 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts.

[0102] Comparative Example 2

[0103] Compared with Example 3, the difference is that the cryopreserved cells in step S3 are not added in step S4, but the stem cell suspension and the antler stem cell cryopreservation solution are mixed in a volume ratio of 1:1.

[0104] The details are as follows:

[0105] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0106] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, the added amount of transferrin is 4 mg / L, the added amount of IL-2 is 1.5 mg / L, and the added amount of IL-15 is 1 mg / L;

[0107] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 1500r / min for 15min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, the cells were subcultured to the fourth generation, centrifuged at 1500 r / min for 15 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0108] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0109] The cell cryopreservation solution is a mixed solution of medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0110] S3. Extraction of antler stem cells: Take the tip of the second-branched antler, longitudinally split it to obtain the reserve mesenchymal tissue, crush it, add it to the antler medium, and culture it at 37 °C under 5 v / v% CO 2 conditions. When the fusion rate of adherent cells reaches 85%, add trypsin to the system to a content of 0.15 wt%, digest for 2 min, centrifuge at 1500 r / min for 15 min, resuspend the precipitate in the cell cryopreservation solution, and the cell content is 10 4 -10 5 cells / mL, and cryopreserve;

[0111] The antler medium is DMEM medium containing 10 wt% fetal bovine serum;

[0112] The cell cryopreservation solution is a mixed solution of medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0113] S4. Culture: Thaw the cryopreserved cells in step S2 at room temperature, centrifuge at 1500 r / min for 15 min, resuspend the precipitate in the medium, and culture it at 37 °C under 5 v / v% CO 2 conditions for 4 d to obtain a culture solution;

[0114] S5. Extraction of exosomes: Centrifuge the mixed culture solution at 1500 r / min for 15 min, collect the precipitate and supernatant respectively. The precipitate is stem cells, resuspend it in fresh medium to obtain a stem cell suspension, and the cell content is 10 4 -10 5 cells / mL. The supernatant is separated by ultra-high speed centrifugation combined with an ultrafiltration tube method to obtain exosomes and extracellular matrix;

[0115] The method of ultra-high speed centrifugation combined with an ultrafiltration tube method is as follows: Centrifuge the cell supernatant at 3500 r / min for 20 min at 4 °C to remove cell debris, etc. Concentrate the supernatant with an ultrafiltration tube, centrifuge at 13000 r / min for 25 min at 4 °C to remove large vesicles and proteins, filter the supernatant with a 0.22 μm filter membrane, and finally centrifuge at 32000 r / min for 65 min at 4 °C. Resuspend the precipitate with PBS at pH = 6.8, centrifuge at 32000 r / min for 65 min again at 4 °C. The obtained precipitate is exosomes, and the supernatant is extracellular matrix. Resuspend it with PBS at pH = 6.8 and cryopreserve it in an -80 °C refrigerator;

[0116] S6. Preparation of stem cell liposomes: 11 g of lecithin and 8 g of cholesterol were dissolved in 200 mL of a mixed solution, 25 g of a stem cell mixture (stem cell suspension and the thawed cell cryopreservation solution in step S3) was added dropwise, stirred at 36°C for 25 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0117] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:4;

[0118] S7. Preparation of loaded titanium dioxide / silica microspheres: 12 g of tetrabutyl titanate and 4 g of tetraethyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 1.5 g of hexadecyltrimethylammonium bromide and 0.7 g of Tween-85 were added to 100 mL of water, 9 g of stem cell liposomes and 4 g of exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 9.5, the reaction was stirred for 4 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0119] S8. Preparation of a skin care composition: 11 g of loaded titanium dioxide / silicon dioxide microspheres were added to 27 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts.

[0120] Comparative Example 3

[0121] Compared with Example 3, the difference is that the cryopreserved cells in step S2 are not added in step S4, and the urine-derived stem cell cryopreservation solution is not added to the stem cell suspension.

[0122] The details are as follows:

[0123] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0124] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, the added amount of transferrin is 4 mg / L, the added amount of IL-2 is 1.5 mg / L, and the added amount of IL-15 is 1 mg / L;

[0125] S2. Extraction of antler stem cells: Take the tip of the second antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium, and incubate it at 37°C, 5v / v%CO 2When the fusion rate of adherent cells reached 85%, trypsin was added to a system content of 0.15wt%, digested for 2min, centrifuged at 1500r / min for 15min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0126] The antler culture medium is a DMEM culture medium containing 10wt% fetal bovine serum;

[0127] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0128] S3. Culture: The frozen cells from step S2 were mixed at a volume ratio of 1:1, thawed at room temperature, centrifuged at 1500 rpm for 15 min, and the pellet was resuspended in culture medium at 37°C, 5 v / v% CO 2 Under the conditions, the culture was cultured for 4 days to obtain the culture fluid;

[0129] S4. Extraction of exosomes: The culture medium was centrifuged at 1500 r / min for 15 min, and the precipitate and supernatant were collected separately. The precipitate was stem cells, which were resuspended in fresh culture medium to obtain a stem cell suspension with a cell content of 10 4 -10 5 / mL, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain exosomes and extracellular matrix;

[0130] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C, 13000 r / min for 25 min, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 32000 r / min for 65 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C, 32000 r / min for 65 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=6.8 and frozen in a -80°C refrigerator;

[0131] S5. Preparation of stem cell liposomes: 11 g of lecithin and 8 g of cholesterol were dissolved in 200 mL of the mixed solution, 25 g of the stem cell suspension was added dropwise, the mixture was stirred at 36°C for 25 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0132] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:4;

[0133] S6. Preparation of loaded titanium dioxide / silica microspheres: 12 g of tetrabutyl titanate and 4 g of tetraethyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 1.5 g of hexadecyltrimethylammonium bromide and 0.7 g of Tween-85 were added to 100 mL of water, 9 g of stem cell liposomes and 4 g of exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 9.5, the reaction was stirred for 4 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0134] S7. Preparation of a skin care composition: 11 g of loaded titanium dioxide / silicon dioxide microspheres were added to 27 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts.

[0135] Comparative Example 4

[0136] Compared with Example 3, the difference is that the cryopreserved cells in step S3 are not added in step S4, and the antler stem cell cryopreservation solution is not added to the stem cell suspension.

[0137] The details are as follows:

[0138] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0139] The amount of epidermal growth factor added is 4 μg / L, the amount of cholera toxin added is 30 μg / L, the amount of penicillin added is 100 U / mL, the amount of streptomycin added is 1 g / L, the amount of transferrin added is 4 mg / L, the amount of IL-2 added is 1.5 mg / L, and the amount of IL-15 added is 1 mg / L;

[0140] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 1500r / min for 15min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, the cells were subcultured to the fourth generation, centrifuged at 1500 r / min for 15 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0141] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0142] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0143] S3. Culture: The frozen cells in step S2 were mixed at a volume ratio of 1:1, thawed at room temperature, centrifuged at 1500 rpm for 15 min, and the pellet was resuspended in culture medium at 37°C and 5 v / v% CO. 2 Under the conditions, the culture was cultured for 4 days to obtain the culture fluid;

[0144] S4. Extraction of exosomes: The mixed culture solution was centrifuged at 1500 r / min for 15 min, and the precipitate and supernatant were collected separately. The precipitate was stem cells, which were resuspended in fresh culture medium to obtain a stem cell suspension with a cell content of 10 4 -10 5 / mL, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain exosomes and extracellular matrix;

[0145] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C, 13000 r / min for 25 min, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 32000 r / min for 65 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C, 32000 r / min for 65 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=6.8 and frozen in a -80°C refrigerator;

[0146] S5. Preparation of stem cell liposomes: 11 g of lecithin and 8 g of cholesterol were dissolved in 200 mL of the mixed solution, 25 g of the stem cell suspension was added dropwise, the mixture was stirred at 36°C for 25 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0147] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:4;

[0148] S6. Preparation of loaded titanium dioxide / silica microspheres: 12 g of tetrabutyl titanate and 4 g of tetraethyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 1.5 g of hexadecyltrimethylammonium bromide and 0.7 g of Tween-85 were added to 100 mL of water, 9 g of stem cell liposomes and 4 g of exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 9.5, the reaction was stirred for 4 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0149] S7. Preparation of a skin care composition: 11 g of loaded titanium dioxide / silicon dioxide microspheres were added to 27 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts.

[0150] Comparative Example 5

[0151] Compared with Example 3, the difference is that IL-2 is not added in step S1.

[0152] The details are as follows:

[0153] S1. Preparation of culture medium: adding epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, and IL-15 to DMEM culture medium to prepare a culture medium;

[0154] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, the added amount of transferrin is 4 mg / L, and the added amount of IL-15 is 2.5 mg / L.

[0155] Comparative Example 6

[0156] Compared with Example 3, the difference is that IL-15 is not added in step S1.

[0157] The details are as follows:

[0158] S1. Preparation of culture medium: adding epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, and IL-2 to DMEM culture medium to prepare a culture medium;

[0159] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, the added amount of transferrin is 4 mg / L, and the added amount of IL-2 is 2.5 mg / L.

[0160] Comparative Example 7

[0161] Compared with Example 3, the difference is that IL-2 and IL-15 are not added in step S1.

[0162] The details are as follows:

[0163] S1. Preparation of culture medium: adding epidermal growth factor, cholera toxin, penicillin, streptomycin, and transferrin to DMEM culture medium to prepare a culture medium;

[0164] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, and the added amount of transferrin is 4 mg / L.

[0165] Comparative Example 8

[0166] Compared with the embodiment 3, the difference is that step S6 is not performed.

[0167] The details are as follows:

[0168] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0169] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, the added amount of transferrin is 4 mg / L, the added amount of IL-2 is 1.5 mg / L, and the added amount of IL-15 is 1 mg / L;

[0170] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 1500r / min for 15min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, the cells were subcultured to the fourth generation, centrifuged at 1500 r / min for 15 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0171] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0172] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0173] S3. Extraction of antler stem cells: Take the tip of the second antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium, and incubate it at 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, trypsin was added to a system content of 0.15wt%, digested for 2min, centrifuged at 1500r / min for 15min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0174] The antler culture medium is a DMEM culture medium containing 10wt% fetal bovine serum;

[0175] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0176] S4. Mixed culture: The frozen cells in step S2 and step S3 were mixed at a volume ratio of 1:1, thawed at room temperature, centrifuged at 1500 rpm for 15 min, and the pellet was resuspended in culture medium at 37°C and 5 v / v% CO. 2 The mixed culture solution was prepared by culturing for 4 days under the above conditions;

[0177] S5. Extraction of mixed exosomes: The mixed culture solution was centrifuged at 1500 r / min for 15 min, and the precipitate and supernatant were collected separately. The precipitate was mixed stem cells, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain mixed exosomes and extracellular matrix;

[0178] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C, 13000 r / min for 25 min, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 32000 r / min for 65 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C, 32000 r / min for 65 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=6.8 and frozen in a -80°C refrigerator;

[0179] S6. Preparation of loaded titanium dioxide / silica microspheres: 12 g of tetrabutyl titanate and 4 g of tetraethyl orthosilicate were added to 200 mL of ethyl acetate to obtain an oil phase; 1.5 g of hexadecyltrimethylammonium bromide and 0.7 g of Tween-85 were added to 100 mL of water, 9 g of mixed stem cells and 4 g of mixed exosomes were added, and ultrasonic dispersion was performed at 1000 W for 15 min to obtain an aqueous phase; the aqueous phase was added dropwise to the oil phase, emulsified at 8000 r / min for 15 min, the pH value of the solution was adjusted to 9.5, the reaction was stirred for 4 h, centrifuged, washed, and dried to obtain loaded titanium dioxide / silica microspheres;

[0180] S7. Preparation of a skin care composition: 11 g of loaded titanium dioxide / silicon dioxide microspheres were added to 27 g of extracellular matrix, and ultrasonically dispersed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts.

[0181] Comparative Example 9

[0182] Compared with the embodiment 3, the difference is that step S7 is not performed.

[0183] The details are as follows:

[0184] S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium;

[0185] The added amount of epidermal growth factor is 4 μg / L, the added amount of cholera toxin is 30 μg / L, the added amount of penicillin is 100 U / mL, the added amount of streptomycin is 1 g / L, the added amount of transferrin is 4 mg / L, the added amount of IL-2 is 1.5 mg / L, and the added amount of IL-15 is 1 mg / L;

[0186] S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it at 1500r / min for 15min, add the precipitate to the urine-derived stem cell basal medium and resuspend it in 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, the cells were subcultured to the fourth generation, centrifuged at 1500 r / min for 15 min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0187] The urine-derived stem cell basal culture medium is composed of a mixture of 3 / 4 DMEM culture medium and 1 / 4 Ham's F-12 culture medium;

[0188] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0189] S3. Extraction of antler stem cells: Take the tip of the second antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium, and incubate it at 37℃, 5v / v%CO 2 When the fusion rate of adherent cells reached 85%, trypsin was added to a system content of 0.15wt%, digested for 2min, centrifuged at 1500r / min for 15min, and the precipitate was resuspended in cell freezing solution. The cell content was 10 4 -10 5 / mL, frozen;

[0190] The antler culture medium is a DMEM culture medium containing 10wt% fetal bovine serum;

[0191] The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 6:3:1;

[0192] S4. Mixed culture: The frozen cells in step S2 and step S3 were mixed at a volume ratio of 1:1, thawed at room temperature, centrifuged at 1500 rpm for 15 min, and the pellet was resuspended in culture medium at 37°C and 5 v / v% CO. 2 The mixed culture solution was prepared by culturing for 4 days under the above conditions;

[0193] S5. Extraction of mixed exosomes: The mixed culture solution was centrifuged at 1500 r / min for 15 min, and the precipitate and supernatant were collected separately. The precipitate was mixed stem cells, which were resuspended in fresh culture medium to obtain a stem cell suspension with a cell content of 10 4 -10 5 / mL, and the supernatant was separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain mixed exosomes and extracellular matrix;

[0194] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C, 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C, 13000 r / min for 25 min, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C, 32000 r / min for 65 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C, 32000 r / min for 65 min. The precipitate obtained is exosomes, and the supernatant is the extracellular matrix, which is resuspended in PBS with pH=6.8 and frozen in a -80°C refrigerator;

[0195] S6. Preparation of stem cell liposomes: 11 g of lecithin and 8 g of cholesterol were dissolved in 200 mL of the mixed solution, 25 g of the stem cell suspension was added dropwise, the mixture was stirred at 36°C for 25 min, rotary evaporated, ultrasonically dispersed at 1000 W for 10 min, and freeze-dried to obtain stem cell liposomes;

[0196] The mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:4;

[0197] S7. Mixture: 9 g of stem cell liposomes and 4 g of mixed exosomes were mixed to obtain a mixture;

[0198] S8. Preparation of a skin care composition: 11 g of the mixture was added to 27 g of an extracellular matrix, and ultrasonic dispersion was performed at 1000 W for 15 min to prepare a skin care composition containing stem cell extracts.

[0199] Comparative Example 10

[0200] Compared with Example 3, the difference is that no mixed exosomes are added in step S7.

[0201] The details are as follows:

[0202] S7. Preparation of loaded titanium dioxide / silica microspheres: Add 12 g of tetrabutyl titanate and 4 g of tetraethyl orthosilicate into 200 mL of ethyl acetate to obtain an oil phase; add 1.5 g of hexadecyltrimethylammonium bromide and 0.7 g of Tween-85 into 100 mL of water, add 13 g of stem cell liposomes, and ultrasonically disperse at 1000 W for 15 min to obtain an aqueous phase; add the aqueous phase dropwise into the oil phase, emulsify at 8000 r / min for 15 min, adjust the pH value of the solution to 9.5, stir the reaction for 4 h, centrifuge, wash, and dry to obtain loaded titanium dioxide / silica microspheres.

[0203] Test Example 1 DPPH free radical scavenging rate test

[0204] Preparation of DPPH working solution: 0.0082 g DPPH powder was dissolved in 100 mL ethanol and stored at 4°C away from light.

[0205] Preparation of the test substance: The skin care compositions containing stem cell extracts prepared in Examples 1-3 and Comparative Examples 1-10 were prepared into 10 mg / L solutions. Ascorbic acid was used as a positive control.

[0206] Take 1mL DPPH working solution and add 200μL test substance, react for 30min in the dark, and the absorbance reading at a wavelength of 517nm is A 1 Take 1mL of DPPH working solution and add 200μL of distilled water. Protect from light and react for 30min. The absorbance reading at a wavelength of 517nm is A 2 Take 1mL of ethanol and add 200μL of the test substance, react for 30min in the dark, and the absorbance reading at a wavelength of 517nm is A 3 . 10 mg / L ascorbic acid was used as the positive control group.

[0207] Clearance rate (%) = [( A 2 + A 3 - A 1 ) / A 2 ]×100%

[0208] The results are shown in Table 1.

[0209] Table 1

[0210]

[0211] It can be seen from the above table that the skin care compositions containing stem cell extracts prepared in Examples 1-3 of the present invention have good antioxidant properties.

[0212] Test Example 2

[0213] Female volunteers aged 22-49 were selected on a voluntary basis and randomly divided into 13 groups, each with 6 people, including Examples 1-3 and Comparative Examples 1-10. No products irrelevant to the test were applied during the test, and the samples were applied on both sides of the face of the subjects. The sample group used the skin care composition containing stem cell extracts prepared in Examples 1-3 and Comparative Examples 1-10. Once a day for 10 days, at the same time before and after the test, the subjects washed the test area with clean water, wiped it dry, and sat quietly for 20 minutes in a constant environment of 23±2°C and a relative humidity of 50±5%, and then tested.

[0214] 1. Skin moisturizing test

[0215] The stratum corneum moisture content was measured before the start of the test, and the stratum corneum moisture content of the subjects was tested 1 hour after using the sample on the 10th day, and the skin moisture content change rate (%) was calculated. The average value of 5 times was taken.

[0216] 2. Skin elasticity test

[0217] Before the start of the experiment, the skin elasticity and roughness parameters were tested. On the 10th day, 1 hour after using the sample, the stratum corneum moisture content of the subjects was tested and the skin moisture content change rate (%) was calculated. The average value of 5 times was taken.

[0218] Change rate (%) = (skin test value after use - skin test value before use) / skin test value before use × 100%

[0219] The results are shown in Table 2.

[0220] Table 2

[0221]

[0222] It can be seen from the above table that the skin care compositions containing stem cell extracts prepared in Examples 1-3 of the present invention have good moisturizing, anti-aging and anti-wrinkle properties.

[0223] Test Example 1 Human skin patch and allergy test

[0224] Test samples: skin care compositions containing stem cell extracts prepared in Examples 1-3 and Comparative Examples 1-10 of the present invention.

[0225] Experimental method: Volunteers aged 22-50 were selected on a voluntary basis and randomly divided into 13 groups, with 6 people in each group. Among them, the subject's right forearm flexor side was marked with a 5cm×5cm area and medical disinfection was used to create an injury in the marked area. The method was: 36-needle microneedle introduced nutrient solution for 30s, and a cotton ball soaked in physiological saline was used to gently wipe the residual liquid to keep the marked area dry; the test samples were placed in the 1-13 spot tester wells, respectively, with a dosage of 15μL. Bathing was prohibited during the test, and the normal reaction was negative. The test samples were distributed to the subjects and acted on the face, neck and back of the ears of the subjects. The skin condition of the subjects was observed after 15 minutes. The experimental results are shown in Table 3.

[0226] Table 3

[0227]

[0228] As can be seen from the above table, the patch reactions of the skin care compositions containing stem cell extracts prepared in Examples 1-3 of the present invention and Comparative Examples 1-10 are negative reactions, and they are safe to use without any allergic reactions.

[0229] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a skin care composition containing stem cell extracts, characterized in that: The following steps are involved: S1. Preparation of culture medium: epidermal growth factor, cholera toxin, penicillin, streptomycin, transferrin, IL-2 and IL-15 were added to DMEM medium to prepare a culture medium; S2. Extraction of urine-derived stem cells: Collect fresh urine, filter it, centrifuge it, add the precipitate to the urine-derived stem cell basal medium, resuspend it, and culture it. When the fusion rate of adherent cells reaches 80-90%, subculture the cells, centrifuge it, resuspend the precipitate in cell freezing solution, and freeze it; S3. Extraction of antler stem cells: Take the tip of the antler, split it longitudinally to obtain the reserve mesenchymal tissue, mince it, add it to the antler culture medium for culture, and when the fusion rate of adherent cells reaches 80-90%, digest it, centrifuge it, precipitate it, resuspend it in cell freezing solution, and freeze it; S4 mixed culture: thawing the frozen cells in step S2 and step S3, centrifuging, resuspending the precipitate in culture medium, and mixed culturing to obtain a mixed culture solution; S5. Extraction of mixed exosomes: The mixed culture fluid is centrifuged, and the precipitate and supernatant are collected separately. The precipitate is mixed stem cells, which are resuspended in fresh culture medium to obtain a stem cell suspension. The supernatant is separated by ultra-high-speed centrifugation combined with ultrafiltration tube method to obtain mixed exosomes and extracellular matrix; S6. Preparation of stem cell liposomes: dissolving lecithin and cholesterol in a mixed solution, adding stem cell suspension dropwise, stirring at a constant temperature, rotary evaporation, ultrasonic dispersion, and freeze drying to obtain stem cell liposomes; S7. Preparation of loaded titanium dioxide / silicon dioxide microspheres: adding tetrabutyl titanate and alkyl orthosilicate to an organic solvent to obtain an oil phase; adding a porogen and a surfactant to water, adding stem cell liposomes and mixed exosomes, and ultrasonically dispersing them uniformly to obtain an aqueous phase; dropping the aqueous phase into the oil phase, emulsifying, adjusting the pH value of the solution, stirring the reaction, centrifuging, washing, and drying to obtain loaded titanium dioxide / silicon dioxide microspheres; S8. Preparation of a skin care composition: Add the loaded titanium dioxide / silicon dioxide microspheres into the extracellular matrix and disperse them evenly by ultrasonication to prepare a skin care composition containing stem cell extracts.

2. The preparation method according to claim 1, characterized in that: In step S1, the amount of epidermal growth factor added is 3-5 μg / L, the amount of cholera toxin added is 25-35 μg / L, the amount of penicillin added is 80-120 U / mL, the amount of streptomycin added is 0.8-1.2 g / L, the amount of transferrin added is 3-5 mg / L, the amount of IL-2 added is 1-2 mg / L, and the amount of IL-15 added is 0.7-1.5 mg / L; the speed of centrifugation in step S2 is 1000-2000 r / min, the time is 10-20 min, and the urine-derived stem cell basal medium is 3 / 4 DMEM medium, 1 / 4 Ham's The cells are cultured at 36-38°C and 4-6 v / v% CO2. The cells are subcultured to the 3rd or 4th generation. The cell freezing solution is a mixture of culture medium: fetal bovine serum: dimethyl sulfoxide in a volume ratio of 5-7:2-4:

1.

3. The preparation method according to claim 1, characterized in that: The antler culture medium in step S3 is a DMEM culture medium containing 8-12wt% fetal bovine serum, the culture conditions are 36-38°C, 4-6v / v%CO2, the digestion is adding trypsin to a system content of 0.1-0.2wt%, digestion for 1-3min, the centrifugal speed is 1000-2000r / min, and the time is 10-20min; the thawing in step S4 is room temperature melting, the centrifugal speed is 1000-2000r / min, the time is 10-20min, and the mixed culture conditions are 36-38°C, 4-6v / v%CO2 for 3-5d.

4. The preparation method according to claim 1, characterized in that: The centrifugal speed in step S5 is 1000-2000 r / min, the time is 10-20 min, and the cell content of the stem cell suspension is 10 4 -10 5 / mL, the ultrahigh-speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 3-5°C, 3000-4000r / min for 15-20min to remove cell debris, the supernatant is concentrated by ultrafiltration tube, 3-5°C, 12000-15000r / min is centrifuged for 20-30min to remove large vesicles and proteins, the supernatant is filtered with a 0.22μm filter membrane, and finally 3-5°C, 30000-35000r / min is centrifuged for 60-70min, the precipitate is resuspended in PBS with pH=6.7-7, 3-5°C, 30000-35000r / min is centrifuged again for 60-70min, the obtained precipitate is exosomes, the supernatant is extracellular matrix, it is resuspended with PBS with pH=6.7-7, and stored in a -80°C refrigerator.

5. The preparation method according to claim 1, characterized in that: In step S6, the mass ratio of lecithin, cholesterol and stem cell suspension is 10-12:7-10:20-30, the mixed solution is a mixed solution of dichloromethane and ethanol in a volume ratio of 10:3-5, the temperature of the constant temperature stirring is 35-37°C, and the time is 20-30min.

6. The preparation method according to claim 1, characterized in that: In step S7, the mass ratio of tetrabutyl titanate, alkyl orthosilicate, porogen, surfactant, stem cell liposomes, and mixed exosomes is 10-14:3-5:1-2:0.5-1:8-10:3-5, the pH value of the adjustment solution is 9-10, the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate, the porogen is hexadecyltrimethylammonium chloride or hexadecyltrimethylammonium bromide, and the surfactant is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the loaded titanium dioxide / silicon dioxide microspheres to the extracellular matrix in step S8 is 10-12:25-30.

8. A skin care composition containing stem cell extracts obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the skin care composition containing stem cell extracts as claimed in claim 8 in the preparation of anti-aging, moisturizing, whitening and UV radiation protection cosmetics.

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