Preparation method and application of stem cell exosome for hair care and regeneration

Abstract

The invention relates to a preparation method and application of a stem cell exosome for hair care and regeneration, and belongs to the technical field of stem cell exosomes. During preparation of the exosome, insulin, L-ascorbic acid 2-phosphoric acid, insulin-like growth factors, epidermal growth factors, resveratrol and ligustrazine are added into an MEMS alpha culture solution containing 10% of FBS; the proliferation speed of the mesenchymal stem cells is effectively increased, the multiplication time is effectively shortened, the yield of the prepared exosome is high, the VEGF content is high, the prepared exosome is stored in the form of freeze-dried powder, the structure and biological activity of the original exosome can be kept for a long time, the exosome can be transported at normal temperature, and the survival rate of the exosome is increased. And the exosome can be preserved for at least 12 months at 20-15 DEG C. According to the exosome prepared by the invention, an in-vitro rat hair follicle cell proliferation promoting experiment shows that the rat proliferation promoting capacity is stronger, and the exosome is found to be capable of obviously promoting the growth of hair when being used for treating wounds of scalded dogs.

Description

Technical Field

[0001] The present invention relates to a preparation method and an application, and particularly to a preparation method and an application of stem cell exosomes for hair care and regeneration in the technical field of stem cell exosomes. Background Art

[0002] Alopecia is clinically mainly divided into non-scarring (non-scarring) alopecia and scarring alopecia. The most common forms are androgenetic alopecia (AGA) and alopecia areata (AA). By the age of 50, AGA affects 70% of men and 50% of women globally, and the incidence of AA is approximately 2%. Alopecia not only affects appearance but also mental health. However, the treatment options for alopecia are limited. Finasteride and minoxidil are the most widely recognized drugs for treating AGA. However, finasteride is a potential teratogen and is generally prohibited for use in women of childbearing age. The effect of topical treatment is limited by poor adhesion, and it may occasionally have an adverse effect on sexual function. The efficacy of minoxidil lotion is limited by poor solubility. Solutions greater than 5% are unstable, and percutaneous absorption reaches saturation after twice-daily use. When the solvent evaporates, minoxidil crystallizes (or forms a powder) on the scalp, resulting in the loss of the active substance and also having an adverse cosmetic effect on the hair.

[0003] Exosomes are lipid bilayer vesicles with a diameter of 30 - 150 nanometers and have the function of influencing cell signal transduction and gene expression (such as cytokines, growth factors). In vitro studies have found that mesenchymal stem cell exosomes can promote the proliferation and migration of dermal papilla cells (DPCs) and upregulate the expression levels of hair follicle markers alkaline phosphatase (ALP), versican, and α-smooth muscle actin (α-SMA). In vivo studies have demonstrated that mesenchymal stem cell exosomes increase the number of hair follicles and the dermal thickness in hair-deprived model mice, and accelerate hair regeneration and growth rate, and increase hair density, with a better effect than minoxidil. Moreover, in a clinical trial, after 12 weeks of treatment with exosomes in 39 patients with mild to moderate AGA, there was a significant improvement in hair density and thickness.

[0004] The invention patent with the publication number CN116763822A discloses an exosome mixture for promoting hair growth, a preparation method thereof, and an application. The invention provides a novel and effective solution for promoting hair growth. The exosome mixture can stimulate hair follicle regeneration, regulate immune response, and promote angiogenesis, thereby creating a favorable environment for hair growth.

[0005] The invention patent with the publication number CN118384089A discloses a method for promoting hair growth and umbilical cord mesenchymal stem cell-derived exosomes used therein. The exosome composition in this invention has a promoting effect on hair growth, which can make the hair length longer, the hair diameter and hair density increase. The exosomes of this invention can also promote the proliferation of hair follicle cells by increasing the stemness of hair follicle stem cells.

[0006] Since preclinical studies and clinical trials have both proven the safety and effectiveness of exosome therapy in the treatment of hair loss, exosomes are expected to be a new treatment method for hair loss. However, the yield and quality of exosomes depend on factors such as the proliferation rate of mesenchymal stem cells, the amount of exosomes produced, and the content of active ingredients such as VEGF in exosomes. Therefore, it is necessary to optimize the culture system to improve the yield and quality of exosomes to meet the needs of hair loss treatment.

[0007] Therefore, we propose a preparation method and application of stem cell exosomes for hair care and regeneration. Summary of the Invention

[0008] Aiming at the above-mentioned existing technologies, the technical problem to be solved by this invention is: how to obtain exosomes with high yield and high VEGF content to solve the limitations of existing hair loss treatment drugs and the problems of insufficient exosome yield and quality, and achieve efficient hair care and regeneration.

[0009] To solve the above problems, this invention provides a preparation method of stem cell exosomes for hair care and regeneration, including the following steps:

[0010] S1. Add insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol, and tetramethylpyrazine to the MEMα culture medium containing 10% FBS, and culture mesenchymal stem cells of the 3rd to 5th generations. When the cell density reaches 80%-90%, aspirate the original culture medium, wash twice with PBS, and replace it with a culture medium without FBS and continue to culture in an incubator at a temperature of 37°C and a carbon dioxide concentration of 5% for 40-50 hours, and then collect the culture medium;

[0011] S2. Centrifuge the collected culture medium at a relative centrifugal force of 300g for 15 min to remove cells and cell debris, then transfer the supernatant to a 0.22 μm filter membrane for filtration, and then centrifuge at a relative centrifugal force of 1500-2500g for 30 min, and then collect the concentrate;

[0012] S3. Centrifuge the collected concentrate at a temperature of 4°C and a relative centrifugal force of 15000-20000g for 30 min - 2h, discard the supernatant, add an appropriate amount of PBS or sterile injection water, and pipette the precipitate to obtain an exosome resuspension, and then prepare exosome lyophilized powder or conduct subsequent experiments.

[0013] As a further improvement of the present application, in step S1, after adding insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol, and ligustrazine to the MEMα culture medium containing 10% FBS, the concentration of insulin in the MEMα culture medium is 50 μM, the concentration of Asc-2-P is 200 μM, the concentration of IGF is 100 ng / ml, the concentration of EGF is 10 nM, the concentration of resveratrol is 1 μM, and the concentration of ligustrazine is 80 μM.

[0014] As a further improvement of the present application, the mesenchymal stem cells are isolated from the fat, umbilical cord, placenta, and bone marrow of healthy mammalian individuals.

[0015] As a further improvement of the present application, the proliferation rate of the mesenchymal stem cells cultured in step S1 is faster than that of the mesenchymal stem cells cultured only in the MEMα culture medium containing 10% FBS.

[0016] As a further improvement of the present application, the number of exosomes produced by the mesenchymal stem cells cultured in step S1 is more than that of the exosomes produced by the mesenchymal stem cells cultured only in the MEMα culture medium containing 10% FBS.

[0017] As a further improvement of the present application, the VEGF content in the exosomes produced by the mesenchymal stem cells cultured in step S1 is more than that of the VEGF content in the exosomes produced by the mesenchymal stem cells cultured only in the MEMα culture medium containing 10% FBS.

[0018] As a further improvement of the present application, the exosomes are stored in the form of freeze-dried powder.

[0019] As a further improvement of the present application, the specific method for preparing the freeze-dried exosome powder in step S4 is as follows:

[0020] A1. Prepare an exosome mixture in a ratio of exosomes 80 - 90%, mannitol 1 - 10%, and trehalose 1 - 10%. First, weigh an appropriate amount of mannitol according to the ratio, place it in an appropriate amount of compound electrolyte injection solution, stir until the mannitol is completely dissolved, then add an appropriate amount of trehalose to the solution according to the ratio, stir until the trehalose is completely dissolved, let the solution stand overnight to fully swell, and then perform high-temperature sterilization treatment on the swollen solution to obtain the exosome adjuvant. Take an appropriate amount of exosome resuspension according to the ratio and mix it with the prepared exosome adjuvant to obtain the exosome mixture;

[0021] A2. Transfer the exosome mixture to a freeze-dryer with a vacuum degree < 80 mT, and perform freeze-drying treatment at -50°C for 36 - 48 h to obtain the freeze-dried exosome powder.

[0022] A cosmetic product containing stem cell exosomes, which includes hair washing products, scalp care products, and hair care products.

[0023] The beneficial effects of the present invention are as follows:

[0024] When preparing exosomes in the present invention, by adding insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol, and tetramethylpyrazine to MEMα culture medium containing 10% FBS, the proliferation rate of mesenchymal stem cells is effectively increased, and the doubling time is effectively shortened, resulting in a high yield of exosomes prepared by the present invention and a high VEGF content. Moreover, the exosomes prepared by the present invention are stored in the form of freeze-dried powder, which can maintain the original structure and biological activity of exosomes for a long time, and can be transported at room temperature and stored at 20 - 15°C for at least 12 months. When in use, the exosome freeze-dried powder can be dissolved with a solvent solution. Description of the Drawings

[0025] Figure 1 It is a flowchart of the preparation method of stem cell exosomes for hair care and regeneration in the first embodiment of the present application;

[0026] Figure 2 It is an analysis chart of the doubling time of mesenchymal stem cells in the second embodiment of the present application;

[0027] Figure 3 It is a transmission electron microscope image of mesenchymal stem cell exosomes in the second embodiment of the present application;

[0028] Figure 4 It is an analysis chart of the diameter of mesenchymal stem cell exosomes in the second embodiment of the present application;

[0029] Figure 5 It is a detection chart of marker proteins for identifying mesenchymal stem cell exosomes by Western Blot in the second embodiment of the present application;

[0030] Figure 6 It is a detection chart of acetylcholinesterase activity of mesenchymal stem cell exosomes in the second embodiment of the present application;

[0031] Figure 7 It is an analysis chart of the VEGF content in mesenchymal stem cell exosomes in the second embodiment of the present application;

[0032] Figure 8 It is a chart showing the effect of mesenchymal stem cell exosomes on the proliferation of rat hair follicle cells in the second embodiment of the present application;

[0033] Figure 9 It is a detection chart of the combing force of mesenchymal stem cell exosomes in the second embodiment of the present application;

[0034] Figure 10 This is the soothing detection diagram of mesenchymal stem cell exosomes in the second implementation mode of this application;

[0035] Figure 11 This is the gentle detection diagram of mesenchymal stem cell exosomes in the second implementation mode of this application;

[0036] Figure 12 This is the moisturizing detection diagram of mesenchymal stem cell exosomes in the second implementation mode of this application;

[0037] Figure 13 This is the diagram of mesenchymal stem cell exosomes in the second implementation mode of this application for wound repair and hair growth promotion. Specific implementation mode

[0038] The following will give a detailed description of the two implementation modes of this application with reference to the accompanying drawings.

[0039] The first implementation mode:

[0040] Please refer to Figure 1 , a preparation method of stem cell exosomes for hair care and regeneration, including the following steps:

[0041] S1. Add insulin, L-ascorbic acid 2-phosphate (Asc-2-P), insulin-like growth factor (IGF1), epidermal growth factor (EGF), resveratrol, and ligustrazine to MEMα culture medium containing 10% FBS, and culture mesenchymal stem cells of the 3rd to 5th generation. When the cell density reaches 80%-90%, aspirate the original culture medium, wash twice with PBS, change to a culture medium without FBS, and continue to culture in an incubator at 37°C and a carbon dioxide concentration of 5% for 40-50 hours, and then collect the culture medium;

[0042] S2. Centrifuge the collected culture medium at a relative centrifugal force of 300g for 15 min to remove cells and cell debris, then transfer the supernatant to a 0.22 μm filter membrane for filtration, and then centrifuge at a relative centrifugal force of 1500-2500g for 30 min, and then collect the concentrate;

[0043] S3. Centrifuge the collected concentrate at 4°C and a relative centrifugal force of 15000-20000g for 30 min-2 h, discard the supernatant, add an appropriate amount of PBS or sterile injection water, and pipette the precipitate to obtain an exosome resuspension, and then prepare exosome freeze-dried powder or conduct subsequent experiments.

[0044] In step S1, after adding insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol, and ligustrazine to the MEMα culture medium containing 10% FBS, the concentration of insulin in the MEMα culture medium is 50 μM, the concentration of Asc-2-P is 200 μM, the concentration of IGF is 100 ng / ml, the concentration of EGF is 10 nM, the concentration of resveratrol is 1 μM, and the concentration of ligustrazine is 80 μM.

[0045] Mesenchymal stem cells are isolated from the fat, umbilical cord, placenta, and bone marrow of healthy mammalian individuals.

[0046] The proliferation rate of the mesenchymal stem cells cultured in step S1 is faster than that of the mesenchymal stem cells cultured only with the MEMα culture medium containing 10% FBS.

[0047] The number of exosomes produced by the mesenchymal stem cells cultured in step S1 is more than that of the exosomes produced by the mesenchymal stem cells cultured only with the MEMα culture medium containing 10% FBS.

[0048] The content of VEGF in the exosomes produced by the mesenchymal stem cells cultured in step S1 is more than that of the VEGF in the exosomes produced by the mesenchymal stem cells cultured only with the MEMα culture medium containing 10% FBS.

[0049] The exosomes are stored in the form of freeze-dried powder.

[0050] The specific method for preparing the exosome freeze-dried powder in step S4 is as follows:

[0051] A1. Prepare an exosome mixture in the ratio of exosomes 80 - 90%, mannitol 1 - 10%, and trehalose 1 - 10%. First, weigh an appropriate amount of mannitol according to the ratio, place it in an appropriate amount of compound electrolyte injection, stir until the mannitol is completely dissolved, then add an appropriate amount of trehalose to the solution according to the ratio, stir until the trehalose is completely dissolved, let the solution stand overnight to fully swell, and then perform high-temperature sterilization treatment on the swollen solution to obtain the exosome excipient. Take an appropriate amount of the exosome suspension according to the ratio and mix it with the prepared exosome excipient to obtain the exosome mixture;

[0052] A2. Transfer the exosome mixture to a freeze-dryer with a vacuum degree < 80 mT, and perform freeze-drying treatment at -50°C for 36 - 48 h to obtain the exosome freeze-dried powder.

[0053] A cosmetic containing stem cell exosomes, the cosmetic includes hair washing products, scalp care products, and hair care products.

[0054] When preparing exosomes in the present invention, by adding insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol, and ligustrazine to MEMα culture medium containing 10% FBS, the proliferation rate of mesenchymal stem cells is effectively increased, and the doubling time is effectively shortened, resulting in a high yield of exosomes prepared by the present invention and a high VEGF content. Moreover, the exosomes prepared by the present invention are stored in the form of freeze-dried powder, which can maintain the original structure and biological activity of exosomes for a long time, and can be transported at room temperature and stored at 20 - 15°C for at least 12 months. When in use, the exosome freeze-dried powder can be dissolved with a solvent solution.

[0055] The second embodiment:

[0056] Preparation of canine adipose mesenchymal stem cells:

[0057] 1. The adipose tissue is derived from healthy beagle dogs less than 2 years old. Bilateral inguinal fat is taken in a sterile environment, and visible blood vessels and fibrous components in the fat are removed.

[0058] 2. The adipose tissue specimen is repeatedly rinsed several times with a sterile PBS rinse solution containing 2% antibiotics (penicillin / streptomycin) prepared to remove bloody fluid and fascia components to obtain pure adipose tissue.

[0059] 3. The adipose tissue is cut into a milky state with tissue scissors, and then 0.1% type I collagenase is added, and digestion is carried out by shaking at 37°C for 30 min to uniformly digest the tissue.

[0060] 4. Centrifugation is carried out at a relative centrifugal force of 2000g for 10 min, the supernatant is discarded, MEMα culture medium containing 10% fetal bovine serum is added, and it is placed in an incubator at 37°C and a carbon dioxide concentration of 5% for adherent culture.

[0061] 5. After 24 h, the medium is changed. The non-adherent cells and other impurities are washed away with PBS, and then fresh culture medium is added. Thereafter, the medium is changed every three days. When the cell density reaches 80%, digestion and passage are carried out with 0.05% trypsin.

[0062] Detection of the doubling time of mesenchymal stem cells:

[0063] The doubling time of mesenchymal stem cells is detected by the direct counting method. The mesenchymal stem cells are inoculated into a six-well plate at a density of 1×10^4 / cm 2 and cultured with MEMα culture medium containing 10% FBS and MEMα culture medium containing 10% FBS and added with insulin, L-ascorbic acid 2-phosphate (Asc-2-P), insulin-like growth factor (IGF1), epidermal growth factor (EGF), resveratrol, and ligustrazine for the 3rd to 5th generation mesenchymal stem cells. Each group has 3 replicates.

[0064] When the cell density reaches 70%-80%, aspirate the original culture medium, wash twice with PBS, digest the cells with 0.05% trypsin, count with a hemocytometer, and passage and culture at the same density;

[0065] The calculation formula for the cell doubling time is t×lg2 / (lgnt - lgn0), where t is the time, n0 is the initial cell number, and nt is the final cell number. Calculate the cell doubling times of the P3, P4, and P5 generations;

[0066] Please refer to Figure 2 , the doubling time of mesenchymal stem cells cultured with the multi-component culture medium is shorter than that of the control group, indicating that the proliferation ability of mesenchymal stem cells cultured with the added multi-component complex is stronger than that of the control group.

[0067] Preparation of mesenchymal stem cell exosomes:

[0068] 1. Add insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol, and ligustrazine to the MEMα culture medium containing 10% FBS, and culture the mesenchymal stem cells of the 3rd to 5th generations. When the cell density reaches 80%-90%, aspirate the original culture medium, wash twice with PBS, change to the culture medium without FBS, and continue to culture in an incubator at 37°C and a carbon dioxide concentration of 5% for 40-50 hours, and then collect the culture medium;

[0069] 2. Centrifuge the collected culture medium at a relative centrifugal force of 300g for 15 min to remove cells and cell debris, then transfer the supernatant to a 0.22 μm filter membrane for filtration, and then centrifuge at a relative centrifugal force of 1500-2500g for 30 min, and then collect the concentrate;

[0070] 3. Centrifuge the collected concentrate at a temperature of 4°C and a relative centrifugal force of 15000-20000g for 30 min - 2 h, carefully discard the supernatant, add an appropriate amount of PBS or sterile injection water, and gently pipette the precipitate to obtain an exosome resuspension;

[0071] 4. Take an appropriate amount of the exosome resuspension, use a BCA protein assay kit to detect the exosome concentration, and store the remaining exosomes at -80°C for later use;

[0072] 5. Prepare the exosome excipients:

[0073] Exosomes 80-90%;

[0074] Mannitol 1-10%;

[0075] Trehalose 1-10%;

[0076] Taking the preparation of 100 ml of exosome excipient solution as an example: Weigh 1 - 10 g of mannitol, dissolve it by stirring with compound electrolyte injection, then add 1 - 10 g of trehalose and stir until the trehalose is completely dissolved. Let the solution stand overnight to allow it to swell fully, and then perform high-temperature sterilization on the swollen solution to obtain the exosome excipient. Take an appropriate amount of the corresponding exosome resuspension according to the ratio, and mix it with the prepared exosome excipient to obtain the exosome mixture;

[0077] The exosome solution is freeze-dried at -50 °C for 36 - 48 h in a vacuum condition (a freeze-dryer with a vacuum degree < 80 mT) to obtain the exosome freeze-dried powder;

[0078] In step 4, the BCA test result shows that the protein content in the exosomes is 11.03 g / L.

[0079] Analysis of the morphology and particle size of mesenchymal stem cell exosomes:

[0080] 1. Please refer to Figure 3 , take an appropriate amount of the exosome PBS resuspension, and use a transmission electron microscope (TEM) to observe the morphology of canine mesenchymal stem cell exosomes. It can be observed that the exosome vesicles have a disc-shaped structure with clear edges and a complete structure;

[0081] 2. Please refer to Figure 4 , take an appropriate amount of the exosome PBS resuspension, and use a nanoparticle Coulter counter to detect the particle size of canine mesenchymal stem cell exosomes. According to the results, the original supernatant particle content is calculated to be 1.77E12 pcs / mL, and the average particle size is 72 nm, which is in line with the size of exosomes.

[0082] Western Blot identification of the marker proteins of mesenchymal stem cell exosomes:

[0083] Preparation of SDS-PAGE gel;

[0084] 1. Dilute the sample stock solution to the required loading amount, add the loading buffer and load the sample for electrophoresis;

[0085] 2. After electrophoresis, transfer the bands on the gel to a PVDF membrane;

[0086] 3. Place the transferred PVDF membrane in a TBST blocking incubation box containing 5% skim milk powder and incubate for 1 h;

[0087] 4. After blocking, place the membrane in the CD9, CD63, and GAPDH primary antibody dilution solutions and incubate with shaking at 4 °C overnight;

[0088] 5. Incubate with the corresponding secondary antibody at room temperature for 1 - 2 h;

[0089] 6. Place the membrane in an imager, add ECL luminescent solution for chemiluminescence reaction, and take pictures.

[0090] Please refer to Figure 5 , and identify the expression of exosome marker proteins CD9 and CD63 by Western Blot method. The results show that the extract of the present invention expresses two exosome marker proteins, further proving that the extract is mesenchymal stem cell exosomes.

[0091] Detection of the secretion amount of mesenchymal stem cell exosomes:

[0092] Use acetylcholinesterase activity to assay exosomes:

[0093] 1. Take 10 μL of the isolated exosomes and mix them in 90 μL of PBS.

[0094] 2. Add 100 μL of the diluted exosomes into a flat-bottom 96-well plate.

[0095] 3. Add 100 μL of Amplex Red reagent / HRP / alkaline phosphatase / choline oxidase / lecithin working solution to each well.

[0096] 4. After incubating in the dark at 37 °C for 30 minutes, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the emitted light at 590 nm and correct the normalized protein concentration.

[0097] 5. Plot and analyze the ratio of mesenchymal stem cell exosomes obtained from the medium supplemented with the multi-component complex to the non-supplemented complex group.

[0098] 6 Please refer to Figure 6 , and the amount of exosomes isolated by culturing with the multi-component complex is significantly higher than that of the control group.

[0099] Detection of VEGF in exosomes - ELISA detection:

[0100] 1. Add 50 μL of incubation buffer to each well.

[0101] 2. Add 100 μL of the standard product to each well of the standard product wells, mix well, and incubate at room temperature for 2 h.

[0102] 3. Discard the solution and wash the wells 4 times with 1X washing buffer.

[0103] 4. Add 100 μL of VEGF biotin-labeled antibody to each well and incubate at room temperature for 1 h.

[0104] 5. Discard the solution and wash the wells 4 times with 1X washing buffer.

[0105] 6. Add 100 μL of 1X streptavidin-HRP solution to each well and incubate at room temperature for 30 minutes.

[0106] 7. Aspirate the solution and wash the wells 4 times with 1X wash buffer.

[0107] 8. Add 100 μL of stabilized chromogen to each well and incubate in the dark at room temperature for 30 minutes.

[0108] Please refer to Figure 7 , and it was detected that the VEGF content in the exosomes isolated by culturing with the multi-component complex was significantly higher than that in the control group.

[0109] Detection of the effect of exosomes on the proliferation of rat hair follicle cells by CCK-8 method:

[0110] 1. Inoculate 1×10^4 / cm of rat hair follicle cell suspension (100 μl / well) in a 96-well plate coated with rat tail collagen type I (2 - 5 μg / cm2). Divide it into 3 groups, and use hair follicle cell medium, hair follicle cell medium + 10 μL of isolated exosomes (produced by culturing in MEMα without complex addition), and hair follicle cell medium + 10 μL of isolated exosomes (produced by culturing in MEMα with multi-component complex addition) for cell proliferation detection, with 6 replicates for each detection. 2 2. When the culture plate is incubated in the incubator for 24 h, 48 h, and 72 h, add 10 μl of CCK-8 solution to each well and incubate the culture plate in the incubator for 2 h.

[0111] 3. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0112] 4. Calculate the cell proliferation rate of each treatment group relative to the control group, draw a curve, and analyze the dynamic changes of cell proliferation and the effect of different exosomes on proliferation.

[0113] Please refer to

[0114] Figure 8 , at 48 h, the proliferation rate of the exosome group was significantly higher than that of the control group, and after treatment with exosomes obtained by culturing with the multi-component complex, the proliferation of rat hair follicle cells was significantly faster than that of exosomes obtained without the complex. At 72 h, the difference between the two was more significant.

[0115]

[0116]

[0116] 1. In vitro standard human hair tresses (damaged hair quality): length 35 cm - 50 cm, about 25 g / strand;

[0117] 2. Comb (hair combing device, model XJ810): The tooth density is moderate (tooth spacing 0.9 - 1.1 mm), the tooth length is 2.0 - 3.0 cm, the comb length is not less than 8 cm (excluding the handle), and the same specification and material of the comb are used during the test;

[0118] 3. Preparation before the test: Randomly divide the in vitro real hair into an experimental group and a control group, with 5 bundles in each group. Measure the combing force of the hair, and measure each bundle of hair at least 3 times;

[0119] 4. Test determination: According to the usage instructions of the tested sample, pre-treat the hair in the experimental group. After wetting the hair bundle with tap water, take an appropriate amount of this product and evenly apply it on the hair bundle. Knead and wash it along the hair to the end of the hair, ensuring that it does not become messy and is evenly sampled for several minutes. After rinsing with water for 30 s, test it. The control group is treated with clean water; after it dries naturally, measure the combing force of the hair again in the same way.

[0120] Please refer to Figure 9 and Table 1. The average combing force of the experimental sample group is better than that of the blank control group, and the P value of the statistical difference < 0.05. As shown in the following table, it shows a significant difference, proving that this mesenchymal stem cell exosome has a hair care effect.

[0121] Table 1: Statistical results and difference analysis of the experimental group and the control group before and after using mesenchymal stem cell exosomes

[0122]

[0123] Note: In the significance analysis, P < 0.05 indicates a significant difference; P ≥ 0.05 indicates no significant difference.

[0124] Detection of the soothing effect of mesenchymal stem cell exosomes:

[0125] Judge whether the test sample has a soothing effect by using the hyaluronidase inhibition rate. The higher the hyaluronidase inhibition rate of the test sample, the better the soothing effect of the test sample;

[0126] 1. Put mesenchymal stem cell exosomes at different concentrations into tubes, and add appropriate concentrations of hyaluronidase solution and sodium hyaluronate solution respectively;

[0127] 2. After fully shaking each tube, add absolute ethanol and place it at room temperature for 30 min;

[0128] 3. Measure the absorbance value at 530 nm and calculate the hyaluronidase inhibition rate;

[0129] 4. Please refer to Figure 10As shown in Table 2, mesenchymal stem cell exosomes have an obvious inhibitory effect on hyaluronidase, and with the increase of the sample concentration, the inhibition rate of hyaluronidase shows a linear increase, proving that mesenchymal stem cell exosomes have a significant soothing effect.

[0130] Table 2: Results of hyaluronidase inhibition rate

[0131]

[0132]

[0133] Gentle detection of mesenchymal stem cell exosomes:

[0134] By detecting the changes in the toxicity of the chorioallantoic membrane of chicken embryos, the potential irritation of mesenchymal stem cell exosomes was evaluated. This method is applicable to the screening and detection of the toxicity of cosmetics and cosmetic raw materials. Mesenchymal stem cell exosomes were contacted with the chorioallantoic membrane of chicken embryos, and after acting for a period of time, the changes in the toxicity effect indicators of the chorioallantoic membrane (such as bleeding, coagulation, and vascular lysis) were observed, and then combined scoring was performed. The final scoring ES value: 0 ≤ ES ≤ 12, no / low irritation; 12 < ES < 16, moderate irritation; ES ≥ 16, strong irritation.

[0135] 1. Select fertilized eggs of White Leghorn chickens and incubate them in an incubator at a temperature of 37.5°C ± 0.5°C and a relative humidity of 55%-70%.

[0136] 2. The experiment was divided into three groups: the exosome experimental group, the negative control group with 0.9% sodium chloride solution, and the positive control group with 0.1 mol / L sodium hydroxide solution. Each group had at least 6 embryos, and in addition, 1 chicken embryo each was set for the positive substance (reference substance) and the solvent / carrier control.

[0137] 3. Take out the 10-day-old embryos, use forceps to peel off the upper part of the eggshell in the air chamber, expose the white eggshell membrane, drip an appropriate amount of 0.9% sodium chloride solution to moisten the eggshell membrane, carefully remove the eggshell membrane with forceps, record the situation of the chorioallantois with a photographing device, coat the exosomes, 0.9% sodium chloride solution, and 0.1 mol / L sodium hydroxide solution on the surface of the plastic film to form a thin layer, cover it on the chorioallantoic membrane, and after acting for several minutes, record the degree of vascular damage of the chorioallantois with a photographing device and score according to the degree of vascular damage.

[0138] 4. Please refer to Figure 11 , and the test score detection proves that mesenchymal stem cell exosomes are gentle and non-irritating.

[0139] Moisturizing detection of mesenchymal stem cell exosomes:

[0140] Through experimental design, the test results of the test samples are compared with the positive control samples known to have moisturizing ability. If the column chart of the moisturizing rate of the test samples is higher than that of the positive control, it is determined that the test samples have certain moisturizing ability.

[0141] 1. The experimental group is mesenchymal stem cell exosomes, and the positive controls are glycerol and polyethylene glycol.

[0142] 2. Weigh the glass plate (M0) with a 3-cm adhesive tape attached using an analytical balance. Use a glass rod to evenly apply the sample on the glass plate with a 3-cm adhesive tape attached and accurately record (M1). Place the exosomes and positive control products in a constant temperature and humidity drying device at 25°C and 40% humidity for regular measurement. Take out the glass plate, weigh its mass (M2), record the data, and draw a change graph.

[0143] 3. Please refer to Figure 12 , the column chart of the moisturizing rate of mesenchymal stem cell exosomes is higher than that of the positive control, indicating that mesenchymal stem cell exosomes have certain moisturizing effects.

[0144] Mesenchymal stem cell exosomes repair wounds and promote hair growth:

[0145] Please refer to Figure 13 , a 13-year-old Bichon Frise had a back burn. The exosomes obtained by applying the composite medium were applied once a day. After 2 months, the hair around the wound was significantly longer than that in other shaved areas, indicating that the exosomes promote hair growth.

[0146] Combined with the current actual needs, the above-mentioned implementation methods adopted in this application, the scope of protection is not limited to this. Within the knowledge possessed by those skilled in the art, various changes made without departing from the concept of this application still fall within the protection scope of the present invention.

Claims

1. A method for preparing stem cell exosomes for hair care and regeneration, characterized in that: The following steps are involved: S1. Insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol, and ligustrazine were added to MEMα culture medium containing 10% FBS to culture the 3rd to 5th generations of mesenchymal stem cells. When the cell density reached 80%-90%, the original culture medium was aspirated, and the cells were washed twice with PBS, and the culture medium without FBS was replaced and continued to be cultured in an incubator at a temperature of 37° C. and a carbon dioxide concentration of 5% for 40-50 hours, and then the culture medium was collected; S2, centrifuging the collected culture fluid at a relative centrifugal force of 300 g for 15 min to remove cells and cell debris, then transferring the supernatant to a 0.22 μm filter membrane for filtration, and then centrifuging at a relative centrifugal force of 1500-2500 g for 30 min, and then collecting the concentrate; S3. Centrifuge the collected concentrated solution at 4°C and a relative centrifugal force of 15,000-20,000 g for 30 min-2 h, discard the supernatant, add an appropriate amount of PBS or sterile water for injection, blow and beat the precipitate to obtain an exosome heavy suspension, and then prepare exosome lyophilized powder or conduct subsequent experiments.

2. The method for preparing stem cell exosomes for hair care and regeneration according to claim 1, characterized in that: In the step S1, after adding insulin, L-ascorbic acid 2-phosphate, insulin-like growth factor, epidermal growth factor, resveratrol and ligustrazine to the MEMα culture medium containing 10% FBS, the concentration of insulin in the MEMα culture medium is 50 μM, the concentration of Asc-2-P is 200 μM, the concentration of IGF is 100 ng / ml, the concentration of EGF is 10 nM, the concentration of resveratrol is 1 μM, and the concentration of ligustrazine is 80 μM.

3. The method for preparing stem cell exosomes for hair care and regeneration according to claim 1, characterized in that: The mesenchymal stem cells are separated from fat, umbilical cord, placenta and bone marrow of healthy mammal individuals.

4. The method for preparing stem cell exosomes for hair care and regeneration according to claim 1, characterized in that: The proliferation rate of the mesenchymal stem cells cultured in step S1 is faster than the proliferation rate of the mesenchymal stem cells cultured using only MEMα culture medium containing 10% FBS.

5. The method for preparing stem cell exosomes for hair care and regeneration according to claim 4, characterized in that: The amount of exosomes produced by the mesenchymal stem cells cultured in step S1 is greater than the amount of exosomes produced by the mesenchymal stem cells cultured only in MEMα culture medium containing 10% FBS.

6. The method for preparing stem cell exosomes for hair care and regeneration according to claim 5, characterized in that: The VEGF content in the exosomes produced by the mesenchymal stem cells cultured in the step S1 is higher than the VEGF content in the exosomes produced by the mesenchymal stem cells cultured only in the MEMα culture medium containing 10% FBS.

7. The method for preparing stem cell exosomes for hair care and regeneration according to claim 1, characterized in that: The exosomes are stored in the form of lyophilized powder.

8. The method for preparing stem cell exosomes for hair care and regeneration according to claim 1, characterized in that: The specific method for preparing the lyophilized exosome powder in step S4 is: A1. Prepare an exosome mixed solution with a ratio of 80-90% exosomes, 1-10% mannitol, and 1-10% trehalose. First, weigh an appropriate amount of mannitol in proportion, put it in an appropriate amount of compound electrolyte injection, stir to make the mannitol completely dissolved, then add an appropriate amount of trehalose to the solution in proportion, stir until the trehalose is completely dissolved, leave the solution overnight to fully swell, and then sterilize the swollen solution at high temperature to obtain an exosome auxiliary material. Take an appropriate amount of exosome heavy suspension in proportion, mix it with the prepared exosome auxiliary material to obtain an exosome mixed solution; A2. Transfer the exosome mixture to a freeze dryer with a vacuum degree of <80 mT, and freeze-dry it at -50°C for 36-48 hours to obtain exosome freeze-dried powder.

9. A cosmetic comprising stem cell exosomes obtained by the preparation method according to any one of claims 1 to 8, characterized in that: The cosmetics include shampoo products, scalp care products, and hair care products.

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