Method for preparing exosomes derived from antler periosteal mesenchymal stem cells, exosomes derived from antler periosteal mesenchymal stem cells and applications thereof

Abstract

To provide a method for preparing exosomes derived from antler periosteal mesenchymal stem cells, exosomes derived from antler periosteal mesenchymal stem cells, and methods of use thereof.SOLUTION: The present invention comprises the following steps: a preparation step 110 of obtaining antler periosteal tissue; a collection step 120 of placing the periosteal tissue in a DPBS culture dish containing antibiotics; a degradation step 130 of cutting the periosteal tissue, washing with DPBS, adding digestive enzymes to the cut periosteal tissue, and reacting and decomposing the tissue in an incubator containing carbon dioxide; a collection step 140 of adding the digested and separated periosteal tissue to α-MEM complete culture medium, filtering with a cell strainer, centrifuging, and collecting primary periosteal cells; a culture step 150 of culturing the collected primary periosteal cells in a culture medium to obtain antler periosteal mesenchymal stem cells; and a culture step 160 for mesenchymal stem cell exosomes in which the antler periosteal mesenchymal stem cells are cultured and then filtered to obtain exosomes derived from antler periosteal mesenchymal stem cells; and an exosome filtration and purification step 170.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to velvet antler stem cells, and more specifically to a method for producing exosomes derived from velvet antler periosteum stromal stem cells, exosomes derived from velvet antler periosteum stromal stem cells, and methods for using the same. [Background technology]

[0002] Stem cells have the potential to repair and regenerate tissues and organs. Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in various tissues and organs, and are widely used in stem cell applications due to their excellent immunoregulatory ability and low tumorigenicity.

[0003] Exosomes are vesicular structures secreted by stem cells during physiological activity. They contain physiologically active substances such as RNA and proteins and play an important role in intercellular communication. Exosomes are easy to collect, store, and transport, and their quality can be controlled. Animal experiments have shown that they exhibit therapeutic effects similar to those of interstitial stem cells. However, the composition of exosomes secreted by cells is not constant and varies depending on the cell's condition. Therefore, the search for cells capable of stably secreting large amounts of exosomes is key to achieving major breakthroughs in exosome research and applications.

[0004] Velvet antler has long been used in traditional Chinese medicine, where it is believed to have effects such as replenishing blood and strengthening muscles and bones, and its extracts are also used to treat related diseases.

[0005] Compared with bone marrow stromal stem cells and umbilical cord stromal stem cells, which are currently commonly used in the biotechnology field, antler stem cells (ASCs) are more easily harvestable. Antler stem cells are stromal stem cells derived from the periosteum of antler and possess the characteristics of mesenchymal stem cells.

[0006] Related research has confirmed that velvet antler stem cells have a stronger proliferation ability than general mesenchymal stem cells in an in vitro culture environment. While general mesenchymal stem cells can be proliferated for approximately 15 passages, velvet antler stem cells can be proliferated for up to 55 passages, maintaining stable proliferation ability.

[0007] Therefore, velvet stem cells have the potential to be a stable source of therapeutic exosomes. Currently, exosomes derived from velvet stem cells are primarily used in the fields of bone (joint and cartilage) regeneration and repair, wound healing, cell aging inhibition, and anti-aging. As there are many related patents, detailed descriptions are omitted here.

[0008] In other words, exploring the mechanisms by which exosomes derived from velvet stem cells treat and improve various diseases and developing more effective therapeutic drugs and medical products is extremely important and has great market value.

[0009] In particular, there are currently no similar patents regarding the development of drugs and products that utilize exosomes derived from velvet antler stem cells to improve skin texture and promote hair growth. Summary of the Invention [Problem to be solved by the invention]

[0010] The main objective of the present invention is to provide a method for producing exosomes derived from velvet periosteum stromal stem cells, exosomes derived from velvet periosteum stromal stem cells, and methods for using the same. The exosomes derived from velvet periosteum stromal stem cells produced by the present invention can improve skin texture and promote hair growth. [Means for solving the problem]

[0011] To achieve this objective, the present invention provides a method for producing exosomes derived from velvet antler periosteum stromal stem cells, which comprises at least the following steps: The antler preparation process involves collecting antlers from 1-2 month old deer, washing them with DPBS containing antibiotics, completely scraping off the villi on the surface of the antler, and then washing them again with DPBS containing antibiotics. The periosteal tissue collection process involves peeling off the epidermis of the velvet antler to extract the periosteal tissue, which is then stored in a culture dish containing DPBS containing antibiotics. After completely removing the epidermal tissue and blood remaining on the periosteal tissue, the periosteal tissue was cut into 1 mm cubes and washed with DPBS until the blood was removed. 15 ml of digestive enzyme was added for every 3 g of periosteal tissue, and the mixture was allowed to react in an incubator at 37°C with 20% carbon dioxide for 30 minutes to decompose the periosteal tissue. This was the periosteal tissue decomposition process. The disintegrated periosteal tissue was suspended in 20 ml of α-MEM complete medium, and the cells derived from the periosteal tissue were filtered using a 70 μm cell strainer. The suspension was then centrifuged at 1500 rpm for 10 minutes to collect the precipitated periosteal primary cells. This was the periosteal primary cell collection step. A process for culturing stromal stem cells, which comprises counting the obtained periosteal primary cells and culturing them in a medium containing α-MEM, 10% fetal bovine serum, 10 mg / ml alanine, 9 mg / ml asparagine, 15 mg / ml aspartic acid, 10 mg / ml glycine, 50 mg / ml glutamic acid, 10 mg / ml proline, 10 mg / ml serine, 10 ng / ml basic fibroblast growth factor 2 (bFGF2), and 50 mg / ml gentamicin, changing the medium every 3 days and subculturing after 7 days to obtain and store velvet antler periosteum-derived stromal stem cells (MSCs). This is the exosome culture process for stromal stem cells. Stromal stem cells derived from velvet antler periosteum are cultured until they reach 80% confluence, after which the cell culture medium is removed, the cells are washed twice with DPBS, and α-MEM is added and the cells are cultured continuously for 7 days. This is the exosome filtration and purification process in which α-MEM cultured for 7 days is filtered through a 0.22μm filter and exosomes are separated and concentrated using tangential flow filtration (TFF). The exosome concentration after concentration is 1.53e+10±2.08e+9 particles / ml, and the exosomes are frozen and stored at -80℃.

[0012] The present invention also provides a method for applying exosomes derived from velvet antler periosteum stromal stem cells, which involves applying exosomes derived from velvet antler periosteum stromal stem cells to human fibroblasts to promote cell proliferation and repair.

[0013] The present invention provides a use for improving skin texture by applying exosomes derived from velvet antler periosteum stromal stem cells to human skin.The present invention provides a use for improving skin texture by applying exosomes derived from velvet antler periosteum stromal stem cells to skin care products (cosmetics).

[0014] The present invention provides a use of exosomes derived from velvet antler periosteum stromal stem cells by applying them to human hair to promote hair growth. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a flow chart of the preparation method of the present invention. [Figure 2] FIG. 1 is a polymerase chain reaction (PCR) analysis diagram using marker factors CD9, CD105, Sox2, and CD45 of velvet periosteal stem cells of the present invention. [Figure 3] (a) to (h) are analytical diagrams showing the results of immunofluorescence staining of the velvet periosteal stem cells of the present invention using monoclonal antibodies against the marker factors CD29, CD44, CD73, CD90, CD105, CD34, and CD45, and detecting the marker factors by flow cytometry (FACS). [Figure 4] 1 shows the results of analyzing exosomes from velvet antler periosteum stem cells produced by the present invention using an NTA (nanoparticle tracking analysis) device. [Figure 5] 1(a) to 1(d) are analytical images of immunofluorescence staining using monoclonal antibodies against CD9, CD63, and CD81, which are marker factors of exosomes of velvet periosteal stem cells of the present invention. [Figure 6] (a) and (b) are area analyses and microscopic images of the scratch area when exosomes from velvet antler periosteal stem cells were co-cultured with human fibroblasts at different concentrations and for different times. [Figure 7]1 is a graph showing the analysis of changes in water content over time after applying exosomes from velvet periosteum stem cells of the present invention to the skin. [Figure 8] 1 is a graph showing the analysis of changes in sebum secretion at different times after applying the exosomes of velvet periosteum stem cells of the present invention to the skin. [Figure 9] (a) and (b) are photographs showing the change in sebum secretion before and 4 weeks after the application of exosomes from velvet antler periosteum stem cells of the present invention to the skin of the forehead in a test example (the area marked in yellow is the state of sebum). [Figure 10] 1 is a graph showing the change in glossiness over time after applying the exosomes of velvet periosteum stem cells of the present invention to the skin. [Figure 11] 1 is a graph showing the results of applying the exosomes of velvet periosteum stem cells of the present invention to the skin and analyzing the change in skin roughness over different time periods. [Figure 12] (a) and (b) are photographs showing the change in skin roughness before and 4 weeks after applying the exosomes of velvet periosteum stem cells of the present invention to facial skin. [Figure 13] 1 is a graph showing the results of applying the exosomes of velvet periosteum stem cells of the present invention to the skin and analyzing the change in the number of pores at different times. [Figure 14] (a) and (b) are photographs showing the change in the number of pores before and 4 weeks after applying the exosomes of velvet periosteum stem cells of the present invention to facial skin. [Figure 15] 1 is a graph showing the analysis of changes in dark spots over different time periods after applying the exosomes of velvet periosteum stem cells of the present invention to the skin. [Figure 16] (a) and (b) are photographs showing the change in the lightening of age spots before and 4 weeks after applying the exosomes of velvet periosteum stem cells of the present invention to facial skin. [Figure 17] 1 is a graph showing the analysis of changes in fine wrinkles over different time periods after applying exosomes from velvet periosteum stem cells of the present invention to the skin. [Figure 18] (a) and (b) are photographs showing the change in fine wrinkles before and after 4 weeks of using exosomes from velvet periosteum stem cells of the present invention on facial skin. [Figure 19] 1 is a graph showing the analysis of the change in melanin index at different times after applying the exosomes of velvet periosteum stem cells of the present invention to the skin. [Figure 20] 1 is a graph showing the analysis of changes in hair volume over different time periods after applying the exosomes of velvet periosteum stem cells of the present invention to the scalp. [Figure 21] (a) to (d) are photographs of two test cases showing the change in hair volume before and four weeks after applying the exosomes of velvet periosteum stem cells of the present invention to the scalp. [Figure 22] 1 is a graph showing the analysis of changes in hair diameter over different time periods after applying the exosomes of velvet periosteum stem cells of the present invention to the scalp. [Figure 23] 1 is a graph showing the analysis of the change in melanin index at different times after applying the exosomes of velvet periosteum stem cells of the present invention to the scalp. [Figure 24] 1 is a graph showing the analysis of changes in hair growth rate at different times after applying the exosomes of velvet periosteum stem cells of the present invention to the scalp. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, some preferred embodiments of the present invention will be described in more detail with reference to the drawings.

[0017] First, refer to Figure 1. A preferred embodiment of the method 100 for producing exosomes from venison periosteum stromal stem cells of the present invention is shown, and includes the following steps. The first step is preparation of venison antlers 110. Fresh venison antlers (young antlers) aged 1 to 2 months are harvested. Surface dirt and blood are removed using alcohol and sterile gauze, and then the antlers are washed with HiMedia Dulbecco's Phosphate Buffered Saline (DPBS) containing antibiotics. Next, on a sterile laboratory bench that meets Good Distribution Practice (GDP) standards, a disposable surgical blade is used to completely scrape off the villi from the antler's surface, and the antlers are again washed with DPBS containing antibiotics.

[0018] The second step of the present invention is the collection of periosteum tissue 120. The epidermis of the velvet antler is peeled off using a disposable surgical blade, and the periosteum tissue is collected and stored in a culture dish containing DPBS containing antibiotics.

[0019] The third step of the present invention is the decomposition of the periosteal tissue 130. The epidermal tissue and blood remaining in the periosteal tissue are completely removed, and the periosteal tissue is decomposed to a depth of 1 mm. 2 After washing with DPBS until all blood is removed, 15 ml of digestive enzymes are added for every 3 g of periosteal tissue, and the tissue is digested in an incubator at 37°C with 20% carbon dioxide for 30 minutes.

[0020] The fourth step of the present invention is the collection of periosteal primary cells 140. The periosteal tissue is suspended in 20 ml of α-MEM complete culture medium, and the digested and separated periosteal tissue cells are filtered through a 70 μm cell strainer. The suspension is then centrifuged at 1500 rpm for 10 minutes, and the precipitated periosteal primary cells are collected.

[0021] The fifth step of the present invention is culturing the stromal stem cells 150. The number of cells of the collected periosteal primary cells is counted, and the cells are cultured using a specially prepared culture medium.

[0022] The culture medium contains the following components: α-MEM, 10% fetal bovine serum (FBS), 10mg / ml alanine, 9mg / ml asparagine, 15mg / ml aspartic acid, 10mg / ml glycine, 50mg / ml glutamic acid, 10mg / ml proline, 10mg / ml serine, 10ng / ml basic fibroblast growth factor 2 (bFGF), and 50mg / ml gentamicin. The culture medium is changed every three days, and subculture is performed after seven days to obtain and preserve velvet antler periosteum-derived mesenchymal stem cells (MSCs). The role of each component is as follows:

[0023] Alanine: Involved in cellular glucose metabolism, contributing to energy supply and synthesis of essential organic molecules. Asparagine: Involved in protein synthesis and amino acid metabolic pathways, and also used as a nitrogen source. Aspartic acid: Involved in biochemical processes such as neurotransmission and protein synthesis. Glycine: Used in protein and collagen synthesis, and also functions as a precursor to neurotransmitters. Glutamate: Involved in amino acid metabolic pathways and excitatory neurotransmission in neurotransmission. Proline: Maintains the stability of collagen structure and plays an important role in the formation of skin and connective tissue. Serine: Involved in protein synthesis, maintaining the structure of biological membranes, and important reactions in metabolic pathways. bFGF: A multifunctional cell growth factor that maintains cell proliferation and the self-renewal capacity of stem cells. Gentamicin: An aminoglycoside antibiotic that exhibits high antibacterial activity against gram-negative bacteria, including Escherichia coli, and suppresses bacterial growth by inhibiting bacterial protein synthesis.

[0024] The sixth step of the present invention is the culture of stromal stem cell exosomes 160. Stromal stem cells derived from velvet periosteum are cultured until they reach 80% confluence, the cell culture medium is removed, the cells are washed twice with DPBS, and α-MEM is added and the cells are continuously cultured for 7 days.

[0025] The seventh step of the present invention is exosome filtration and purification. After 7 days of incubation, the α-MEM is filtered through a 0.22 μm filter, and the exosomes are purified and concentrated using tangential flow filtration (TFF). The exosome concentration after concentration is 1.53e+10±2.08e+9 particles / ml and is stored frozen at -80°C.

[0026] Exosomes from velvet antler periosteal stromal stem cells contain growth factors involved in skin repair, such as EGF, bFGF, and TGFb1. Measurement using a human ELISA kit revealed that the growth factor content in exosomes from velvet antler periosteal stromal stem cells was as follows: EGF: 81.1±14.4pg / ml, bFGF: 3.4±2.4pg / ml, TGFb1: 1236.0±67.0pg / ml.

[0027] Furthermore, the present invention provides a method for identifying the species of velvet antler periosteal stromal stem cells.

[0028] Using the stromal stem cells obtained in the 150-step stromal stem cell culture of the present invention, we performed polymerase chain reaction (PCR) for velvet antler stem cell markers CD9, CD105, Sox2, and CD45. The results are shown in Figure 2. Furthermore, we performed immunofluorescence staining using monoclonal antibodies against CD29, CD44, CD73, CD90, CD105, CD34, and CD45, and measured the ratio of labeled factors by flow cytometry (FACS). The results are shown in Figure 3.

[0029] The present invention also provides a method for identifying exosomes from velvet antler periosteum stromal stem cells. The exosomes produced by the present invention were analyzed using nanoparticle tracking analysis (NTA), and the results are shown in Figure 4. The analysis revealed a peak particle size of 103.7 nm. Furthermore, immunofluorescence staining was performed using exosome-specific monoclonal antibodies CD9, CD63, and CD81, and the results are shown in Figure 5.

[0030] In another embodiment, the present invention provides a method for promoting cell proliferation and repair by applying exosomes from velvet periosteal stromal stem cells to human fibroblasts. In this study, exosomes (concentration range: 1%-10%) were co-cultured with human fibroblasts, and the cell behavior was observed after 20 hours. The results confirmed that exosomes from velvet periosteal stromal stem cells significantly promoted the migration of human fibroblasts to the scratch area. Furthermore, after 48 hours, the scratch area in the exosome-added group was almost entirely covered with human fibroblasts (see Figure 6). Human fibroblasts can be used to treat skin defects and other conditions.

[0031] In yet another embodiment, the present invention provides a use of velvet antler periosteum stromal stem cell exosomes by applying them to human skin to improve skin texture. In some embodiments, the present invention provides a use of velvet antler periosteum stromal stem cell exosomes by applying them to skin care products or cosmetics to improve skin texture.

[0032] In one embodiment of the present invention, there is provided a use of exosomes from velvet antler periosteum stromal stem cells by applying them to human hair to promote hair growth. In some embodiments, there is provided a use of exosomes from velvet antler periosteum stromal stem cells in the manufacture of a hair care product that promotes hair growth.

[0033] Below, we will explain the experimental details of the skin test and hair growth test using exosomes from velvet antler periosteal stromal stem cells.

[0034] Skin testing

[0035] subject: The subjects were 30 (25 women and 5 men), with an age range of 22 to 55 years, and a mean age of 32.4 years.

[0036] Measurement conditions: Skin quality measurements were performed in an environment with a relative humidity of 55±5% and a room temperature of 25±1°C.

[0037] Efficacy test method: 1. After washing their face, the subjects waited for the moisture to dry naturally and then underwent skin quality measurements for each item. 2. Skin blemishes, wrinkles, fine lines, roughness, and large pores were measured using a Visia full-face skin quality analyzer (Visia Complexion Analysis, Canfield Scientific, Inc., USA). 3. Skin elasticity was measured using a skin diagnostic system (Aramo TS, Integrated Skin Diagnosis System, Aram HUVIS Co., Ltd. Korea). 4. Skin brightness (L value) was measured using a Minolta Chromameter CM2500d (Japan). 5. Skin moisture content was measured using a C+K Corneometer CM 825 (Courage+Khazaka Electronic, Germany). 6. Skin gloss was measured using a C+K Glossymeter GL 200 (Courage+Khazaka Electronic, Germany). 7. The melanin index of the skin was measured using a Derma-Spectrophotometer (Cortex Technology, Hadsund, Denmark). 8. Exosomes from velvet antler periosteum stromal stem cells were applied to the skin every morning and evening, and measurements were taken again using a skin quality measurement device according to the schedule. 9. We compared the changes in each skin quality item before and after using exosomes from velvet antler periosteal stromal stem cells.

[0038] Evaluation results (facial effect)

[0039] Measurement method: Skin quality was measured before use (W0), and then velvet antler periosteum stromal stem cell exosomes were applied to the entire face every morning and evening. Changes in skin quality were evaluated using a skin quality measurement device every week (W1, W2, W3, W4). Measurement indicators included moisture content, sebum secretion, glossiness, roughness, number of pores, age spots, fine wrinkles, melanin content, and other skin condition indicators.

[0040] Moisture content: See Figure 7. Before use (W0): 45.6 ± 5.8, 1 week later (W1): 58.4 ± 8.6, 2 weeks later (W2): 65.7 ± 10.2, 3 weeks later (W3): 68.2 ± 6.7, 4 weeks later (W4): 68.8 ± 8.8. After 4 weeks (W4), the total average moisture content increased by 23.2%, with an increase of 50.9%.

[0041] Sebum secretion: See Figure 8. Before use (W0): 128.3 ± 9.8, 1 week later (W1): 106.5 ± 12.5, 2 weeks later (W2): 96.2 ± 8.7, 3 weeks later (W3): 94.5 ± 9.5, 4 weeks later (W4): 92.6 ± 11.2. After 4 weeks (W4), the total average sebum secretion amount had decreased by 35.7, confirming a 27.8% reduction. See Figure 9. Comparing the sebum condition on the test subjects' foreheads, the sebum marked in yellow before use had decreased after four weeks, confirming an improvement in sebum secretion.

[0042] Glossiness: See Figure 10. Before use (W0): 4.5±2.6, after 1 week (W1): 8.4±1.4, after 2 weeks (W2): 10.6±1.6, after 3 weeks (W3): 11.2±2.5, after 4 weeks (W4): 12.5±2.8. After 4 weeks, the total average glossiness increased by 8.0%, confirming an improvement of 177.8%.

[0043] Roughness: See Figure 11. Before use (W0): 452.6 ± 31.8, after 1 week (W1): 382.2 ± 45.2, after 2 weeks (W2): 362.5 ± 36.8, after 3 weeks (W3): 358.8 ± 32.7, after 4 weeks (W4): 354.7 ± 28.8. After 4 weeks, the total average value had decreased by 97.9, confirming an improvement of 21.6%. See Figure 12. It was confirmed that the roughness of the test subject's cheeks and nose became smoother than before use, and the skin texture became smoother and brighter.

[0044] Number of pores: See Figure 13. Before use (W0): 512.6 ± 58.6, after 1 week (W1): 462.2 ± 52.5, after 2 weeks (W2): 432.1 ± 48.9, after 3 weeks (W3): 428.3 ± 55.2, after 4 weeks (W4): 421.3 ± 46.6. After 4 weeks, the total average number had decreased by 91.3, confirming an improvement of 17.8%. See Figure 14. The number of pores on the test subject was clearly reduced after 4 weeks, demonstrating improvement.

[0045] Age spots (spots): See Figure 15. Before use (W0): 67.5±22.4, 1 week later (W1): 67.1±20.6, 2 weeks later (W2): 66.2±18.8, 3 weeks later (W3): 64.5±16.7, 4 weeks later (W4): 64.2±15.4. After 4 weeks, the total average age spots had decreased by 3.3%, and an improvement of 4.9% was confirmed. See Figure 16. It was confirmed that the test subject's spots had lightened slightly.

[0046] Fine wrinkles: See Figure 17. Before use (W0): 66.5±11.5, after 1 week (W1): 52.2±12.4, after 2 weeks (W2): 45.8±12.7, after 3 weeks (W3): 41.6±10.5, after 4 weeks (W4): 38.2±10.2. After 4 weeks, the total average number of fine wrinkles had decreased by 28.3, confirming an improvement effect of 42.6%. See Figure 18. It was confirmed that the test subjects' fine lines were clearly reduced.

[0047] Melanin index: See Figure 19. Before use (W0): 38.6 ± 6.5, 1 week later (W1): 37.2 ± 4.2, 2 weeks later (W2): 36.5 ± 2.8, 3 weeks later (W3): 35.4 ± 4.8, 4 weeks later (W4): 33.2 ± 3.4. After 4 weeks, the melanin index decreased by 14.0, and an improvement effect of 5.4% was confirmed.

[0048] Hair growth test

[0049] subject: The subjects were 30 (25 women and 5 men), with an age range of 22 to 55 years, and a mean age of 32.4 years.

[0050] Measurement conditions: Hair and scalp measurements were performed in an environment with a relative humidity of 55±5% and a room temperature of 25±1°C.

[0051] Efficacy test method: 1. Basic measurements of hair and scalp were performed before the start of the study. 2. Hair volume, hair diameter, and other hair / scalp related data were measured using a hair / scalp diagnostic system (Aramo TS, Hair Diagnosis System, Aram HUVIS Co., Ltd. Korea). 3. Exosomes from velvet antler periosteal stromal stem cells were used daily and continuously applied for 4 to 5 months. 4. We compared the changes in various hair / scalp data before and after using exosomes from velvet antler periosteum stromal stem cells.

[0052] Evaluation results (effectiveness on hair)

[0053] Measurement method: The subjects measured their hair volume (using a hair diagnostic system) before using the test sample, and then measured their hair volume in the same way one month, two months, three months, four months (or five months) later. The measurement results were expressed in square centimeters (cm 2 ) and hair efficacy includes hair volume, hair follicle count, hair diameter, melanin index, growth rate and other hair-related indicators.

[0054] Hair volume: See Figure 20. Before use (M0): 112.6±25.7, after 1 month (M1): 116.8±27.2, after 2 months (M2): 118.6±27.5, after 3 months (M3): 120.8±24.1, after 4 months (M4): 122.4±18.5. After 4 months of use, the total average hair volume increased by 9.8 hairs, confirming an improvement of 8.7%. See Figure 21. The test subject's hair volume showed a tendency to increase after four months of continuous use.

[0055] Hair diameter: See Figure 22. Before use (M0): 62.2±2.5 μm, after 1 month (M1): 62.0±1.7 μm, after 2 months (M2): 61.8±1.2 μm, after 3 months (M3): 62.4±1.5 μm, after 4 months (M4): 62.5±1.1 μm. No significant change in hair diameter was observed after 4 months of use.

[0056] Hair melanin index: See Figure 23. Before use (M0): 128.5±21.6, after 1 month (M1): 132.5±15.8, after 2 months (M2): 133.4±18.5, after 3 months (M3): 136.2±25.1, after 4 months (M4): 138.4±27.2. After 4 months of use, the hair melanin index increased by 7.7%.

[0057] Hair growth rate: See Figure 24. 1 month (M0-M1): 1.22±0.02cm, 2 months (M1-M2): 1.26±0.03cm, 3 months (M2-M3): 1.25±0.03cm, 4 months (M3-M4): 1.28±0.02cm, 5 months (M4-M5): 1.32±0.02cm. Hair growth rate increased slightly over the test period.

[0058] Based on the above results, when test subjects used the velvet antler periosteum stromal stem cell exosomes produced by this invention, it was confirmed that skin moisture and luster were significantly increased, sebum secretion, skin roughness, pore count, and fine wrinkles were significantly reduced, and age spots and melanin were also improved, resulting in a significant improvement in skin texture. Furthermore, use of this product tended to increase hair volume, and the melanin index and hair growth rate were also improved, confirming its hair growth promotion effect. In other words, it was demonstrated that velvet antler periosteum stromal stem cell exosomes are effective in improving skin texture and promoting hair growth.

[0059] The above-mentioned are only some of the better embodiments of the present invention, and are not intended to limit the present invention, and those with ordinary knowledge in the technical field can make some modifications, changes and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope of the patent application.

Claims

1. A preparation step of collecting antlers from 1-2 month old deer, washing them with DPBS containing antibiotics, completely scraping off the villi on the surface of the antlers, and washing them again with DPBS containing antibiotics; A periosteum tissue collection step of peeling off the epidermis of the antler to extract the periosteum tissue and storing it in a culture dish containing DPBS containing an antibiotic; a periosteal tissue decomposition step in which the epidermal tissue and blood remaining on the periosteal tissue are completely removed, the periosteal tissue is cut into 1 mm cubes, and washed with DPBS until the blood is removed. 15 ml of digestive enzyme is added for every 3 g of the periosteal tissue, and the mixture is reacted in an incubator at 37°C in a 20% carbon dioxide environment for 30 minutes to decompose the periosteal tissue; a periosteal primary cell collection step of suspending the decomposed periosteal tissue in 20 ml of α-MEM complete culture medium, filtering cells derived from the periosteal tissue using a 70 μm cell strainer, and then centrifuging the filtered cells at 1500 rpm for 10 minutes to collect the precipitated periosteal primary cells; a step of counting the obtained periosteal primary cells and culturing them in a medium containing α-MEM, 10% fetal bovine serum, 10 mg / ml alanine, 9 mg / ml asparagine, 15 mg / ml aspartic acid, 10 mg / ml glycine, 50 mg / ml glutamic acid, 10 mg / ml proline, 10 mg / ml serine, 10 ng / ml basic fibroblast growth factor 2 (bFGF2), and 50 mg / ml gentamicin, wherein the medium is changed every 3 days and subcultured after 7 days to obtain and store velvet antler periosteum-derived stromal stem cells (MSCs); The venison periosteum-derived stromal stem cells are cultured until they reach 80% confluence, and then the cell culture medium is removed, washed twice with DPBS, and α-MEM is added and cultured continuously for 7 days. A process of culturing stromal stem cell exosomes; an exosome filtration and purification step in which the α-MEM cultured for 7 days is filtered through a 0.22 μm filter and exosomes are separated and concentrated using tangential flow filtration (TFF), with the exosome concentration after concentration being 1.53e+10±2.08e+9 particles / ml, and the exosomes are frozen and stored at −80°C; A method for producing exosomes derived from velvet antler periosteum stromal stem cells, comprising:

2. Exosomes derived from velvet antler periosteum stromal stem cells, characterized by a particle size in the range of 50 to 400 nm and growth factor contents of EGF 81.1 ± 14.4 pg / ml, bFGF 3.4 ± 2.4 pg / ml, and TGF-β1 1236.0 ± 67.0 pg / ml.

3. The exosome derived from velvet antler periosteum stromal stem cells according to claim 2, characterized in that the peak particle size is 103.7 nm as a result of analysis using an NTA particle size analyzer.

4. A method for using exosomes derived from velvet antler periosteum stromal stem cells according to claim 2, A method for using exosomes derived from velvet antler periosteum stromal stem cells, characterized in that the exosomes are applied to human fibroblasts to promote the proliferation and repair of the human fibroblasts.

5. A method for using exosomes derived from velvet antler periosteum stromal stem cells according to claim 3, A method for using exosomes derived from velvet antler periosteum stromal stem cells, characterized in that the exosomes are applied to human fibroblasts to promote the proliferation and repair of the human fibroblasts.

6. A method for using exosomes derived from velvet antler periosteum stromal stem cells according to claim 2, applied to human skin to improve skin texture, A method for using exosomes derived from velvet antler periosteum stromal stem cells, characterized in that the effectiveness indicators for improving skin texture include moisture content, sebum secretion, gloss, roughness, number of pores, spots, fine wrinkles, and melanin content.

7. A method for using exosomes derived from velvet antler periosteum stromal stem cells according to claim 3, applied to human skin to improve skin texture, A method for using exosomes derived from velvet antler periosteum stromal stem cells, characterized in that the effectiveness indicators for improving skin texture include moisture content, sebum secretion, gloss, roughness, number of pores, spots, fine wrinkles, and melanin content.

8. A method for using exosomes derived from velvet antler periosteum stromal stem cells according to claim 2, applied to human hair to promote hair growth, A method for using exosomes derived from velvet antler periosteum stromal stem cells, characterized in that the effect of promoting hair growth is determined by indicators including hair volume, hair follicle test results, hair diameter, melanin index, growth rate, and other related indicators.

9. A method for using exosomes derived from velvet antler periosteum stromal stem cells according to claim 3, applied to human hair to promote hair growth, A method for using exosomes derived from velvet antler periosteum stromal stem cells, characterized in that the effect of promoting hair growth is determined by indicators including hair volume, hair follicle test results, hair diameter, melanin index, growth rate, and other related indicators.

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