Exosome obtained by differentiation of embryonic stem cells, preparation method of exosome and application of exosome in products

Abstract

The invention belongs to the technical field of biology, and particularly discloses an exosome obtained by differentiation of embryonic stem cells, a preparation method of the exosome and application of the exosome in products. Compared with a traditional anti-aging cosmetic, the cosmetic developed by utilizing the exosome containing the oligodendroglia cells and the astroglia cells differentiated from the embryonic stem cells has higher bioavailability and better permeability, bioactive molecules can be more effectively transmitted, regeneration and repair of skin cells can be promoted, the skin aging phenomenon can be remarkably improved, and the cosmetic has a good application prospect. For example, skin moisture content is increased, skin elasticity is improved, and wrinkles are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an exosome obtained by differentiation of embryonic stem cells and a preparation method and application thereof in products. BACKGROUND

[0002] With the increasing attention to skin health and beauty, the anti-aging cosmetic market is developing rapidly. Traditional products rely on plant extracts, vitamins and other ingredients, with limited bioavailability and permeability, making it difficult to achieve the desired anti-aging effect. Exosomes, as a new type of bioactive ingredient, have good cell penetration and biocompatibility, and have become a research hotspot in anti-aging. Neural stem cell exosomes are nanoscale vesicles secreted by neural stem cells, which can carry proteins, mRNA, miRNA and other bioactive molecules, and can transmit signals between cells and regulate cell function. Studies have shown that it has potential in promoting skin cell regeneration, repairing damaged cells and inhibiting skin inflammation. However, the current application of neural stem cell exosomes in the field of cosmetics is less, and there is a lack of mature extraction, purification and application technology, and the market is short of related products, which cannot meet the needs of consumers.

[0003] The cells of the central nervous system are mainly composed of neurons and neuroglial cells. The number of glial cells in the central nervous system accounts for about 90%, and the astrocyte (Ast) is the largest in volume among glial cells, and is called macroglia together with oligodendrocytes. It is traditionally believed that astrocytes do not have action potentials and mainly play a supporting role in structure and function for neurons in the nervous system, and are an important structure of the blood-brain barrier. However, with further research, it has been found that Ast plays an important role in the development and pathophysiological processes of the central nervous system, has the functions of uptake, inactivation and supply of neurotransmitters, antioxidant, nutrition, repair, inhibition of excessive excitation of neurons and help in learning and memory. At present, more and more researchers are focusing on astrocytes in the study of nervous system diseases. Oligodendrocytes (OL) form myelin sheaths around axons, which facilitate the rapid transmission of nerve impulses. There are a large number of oligodendrocyte progenitor (OP) cells in mammals before birth, and during the development of the CNS, OP differentiates into mature OL, and axons can be normally myelinated.

[0004] The research found that oligodendrocyte and astrocyte exosomes contain various skin care active ingredients such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), etc., which can promote the proliferation and differentiation of skin cells, stimulate the synthesis of collagen and elastic fibers, increase skin elasticity, reduce wrinkles; such as cadherin, integrin, etc., which can help maintain the connection between skin cells, enhance the firmness and toughness of the skin, and make the skin smoother and more delicate; such as heat shock protein 70 (HSP70), which has a cell protection effect, can help skin cells resist external environmental stress such as ultraviolet radiation, chemical stimulation, etc., reduce cell damage, and delay skin aging; such as ceramide, which is an important lipid component of the skin barrier, can enhance the moisturizing ability of the skin, reduce water loss, and keep the skin moist. At the same time, it can also regulate the function of skin cells, promote the repair and regeneration of the skin, such as cholesterol, which, together with phospholipids, constitutes the exosome membrane structure, helps maintain the stability and fluidity of the skin cell membrane, and enhances the barrier function of the skin. Therefore, oligodendrocyte and astrocyte exosomes have great application prospects as effective ingredients of cosmetics. SUMMARY

[0005] The present application provides an exosome obtained by differentiation of embryonic stem cells, a preparation method thereof and application in products. The cosmetics developed by the exosome containing oligodendrocytes and astrocytes differentiated from embryonic stem cells have higher bioavailability and better permeability compared with traditional anti-aging cosmetics, can more effectively deliver bioactive molecules, promote skin cell regeneration and repair, and significantly improve skin aging phenomena such as increasing skin moisture content, improving skin elasticity and reducing wrinkles.

[0006] The present application solves the technical problems by adopting the following technical solutions: A preparation method of an exosome obtained by differentiation of embryonic stem cells, comprising the following steps: (1) centrifuging cell liquid containing mature oligodendrocytes and astrocytes, filtering to obtain filtrate; (2) centrifuging the filtrate, resuspending with PBS to obtain stem cell exosomes; (3) mixing the stem cell exosomes and ExoQuick reagent uniformly, centrifuging, resuspending with PBS to obtain exosomes.

[0007] As a preferred embodiment of the present application, the filter membrane used for filtering is 0.22 μm.

[0008] As a preferred embodiment of the present application, the volume ratio of the stem cell exosomes and ExoQuick reagent is (3-5):1.

[0009] As a preferred embodiment of the present application, the method for preparing the cell fluid containing mature oligodendrocytes and astrocytes is: The method for preparing the cell fluid containing mature oligodendrocytes and astrocytes is: (11) On day 0 of culture, the embryonic stem cells are inoculated into the first differentiation medium for culture; (12) On day 8 of culture, the medium is aspirated and the second differentiation medium is added for culture; (13) On day 12 of culture, the medium is aspirated and the cells are washed, followed by cell digestion, and then the cells are inoculated into the third differentiation medium for culture; (14) On day 15 of culture, the medium is aspirated and the cells are inoculated into the fourth differentiation medium for culture, and the culture is continued until day 18-20, to obtain the cell fluid containing mature oligodendrocytes and astrocytes.

[0010] As a preferred embodiment of the present application, the first medium comprises the following components: a base medium, 100-300 ng / mL ascorbic acid, 100-300 ng / mL IL-15, 100-300 ng / mL IL-4, 50-200 ng / mL human transferrin, 50-200 ng / mL Nestin and 200-500 ng / mL CD11b. The second medium comprises the following components: a base medium, 100-300 ng / mL CNTF, 200-500 ng / mL β-mercaptoethanol, 50-200 ng / mL Nestin, 100-300 ng / mL IL-4, 100-300 ng / mL IL-2, 200-500 ng / mL CD11b, 100-300 ng / mL NG2 and 400-800 ng / mL GD3.

[0011] As a preferred embodiment of the present application, the third medium comprises the following components: a base medium, 100-300 ng / mL CNTF, 10-40 μM β-mercaptoethanol, 80-100 μg / mL glutathione, 0.01-0.04 mM aspartic acid, 50-200 ng / mL Nestin, 200-500 ng / mL CD11b, 100-300 ng / mL NG2, 200-600 ng / mL GD3, 100-300 ng / mL O4, 50-200 ng / mL MAG and 100-400 ng / mL NeuN. The fourth culture medium comprises the following components: a basic culture medium, 100-300 ng / mL CNTF, 100-300 ng / mL IL-4, 100-300 ng / mL IL-2, glutathione 80-100 μg / mL, 50-200 ng / mL Nestin, 200-500 ng / mL CD11b, 100-400 ng / mL NG2, 400-800 ng / mL GD3, 100-400 ng / mL O4, 50-200 ng / mL MAG, 200-500 ng / mL NeuN, 50-200 ng / mL MBP, 100-400 ng / mL PLP, and 200-600 ng / mL GFAP.

[0012] The application also provides an exosome differentiated from embryonic stem cells, wherein the exosome expresses specific markers CD9, CD81 and CD63.

[0013] The application also provides a use of the exosome differentiated from embryonic stem cells in the preparation of an anti-aging product.

[0014] The application also provides an anti-aging product comprising the following components in the following mass percentages: exosome 0.05-0.2%, glycerol 8-10%, hyaluronic acid 2-4%, azone 0.1-2%, asiaticoside 1-5%, menthol 0.5-2%, hydrolyzed collagen 1-2%, carbomer U20 0.1-0.5%, EDTA-2Na 0.2-0.8%, antibacterial agent 0.1-0.3%, triethanolamine 0.1-0.2%, and the balance being water. The exosome is the exosome described above.

[0015] As a preferred embodiment of the application, the anti-aging product comprises the following components in the following mass percentages: exosome differentiated from embryonic stem cells 0.1%, glycerol 10%, hyaluronic acid 4%, azone 0.5%, asiaticoside 1%, menthol 1%, hydrolyzed collagen 1%, carbomer U20 0.3%, EDTA-2Na 0.5%, antibacterial agent 0.1%, triethanolamine 0.1%, and the balance being water.

[0016] The application has the following beneficial effects: (1) the cosmetic developed by the exosome containing the oligodendrocytes and astrocytes differentiated from embryonic stem cells has higher bioavailability and better permeability compared with traditional anti-aging cosmetics, can more effectively deliver bioactive molecules, promote skin cell regeneration and repair, and significantly improve skin aging phenomena, such as increasing skin moisture content, improving skin elasticity, and reducing wrinkles.

[0017] (2) Compared with the existing stem cell exosome cosmetics (such as umbilical cord mesenchymal stem cell exosome cosmetics), the neural stem cell exosome has a special source, carries more targeted bioactive molecules, has unique advantages in regulating skin cell nerve-related functions and improving skin microenvironment, and can provide more comprehensive care for the skin. The product is easy to use, does not require professional injection operation, expands the audience group, and the natural source of exosomes and good biocompatibility ensure the safety of the product. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the extracted exosome of the embodiment of the present application, which expresses specific surface markers.

[0019] Figure 2 is the right control diagram after using the anti-aging product of the present application for half a year. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0021] In the present application, the technical features described in an open manner include both the closed technical scheme consisting of the listed features and the open technical scheme containing the listed features.

[0022] In the present application, if no special description is made, the numerical range is regarded as continuous and includes the minimum value and the maximum value of the range and each value between the minimum value and the maximum value. Further, when the range refers to an integer, each integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0023] In the present application, the specific dispersion and stirring treatment method is not particularly limited.

[0024] The reagents or instruments used in the present application are not specified by the manufacturer, and are all conventional products that can be obtained by purchase. The raw materials used in the comparative examples and the raw materials used in the parallel experiments of the examples are the same commercially available products unless otherwise specified.

[0025] Example 1 A preparation method of an exosome obtained by differentiation of embryonic stem cells, comprising the following steps: 1. Resuscitating ES cells (1) Pre-warm the water bath to 37°C.

[0026] (2) Place the VTN-coated 6-well plate in the biosafety cabinet for about 2 hours to recover to room temperature in advance.

[0027] (3) Take 10 mL of KnockOut DMEM / F-12 (ThermoFisher) medium, and add 3 μL of 30 mM Blebbistatin at a ratio of 1:8000, and recover to room temperature.

[0028] (4) Take out one frozen ES cell and place it in a 37°C water bath, gently shake it by hand, and thaw it within 1 minute. When the ice crystals in the cell suspension completely disappear, take it out.

[0029] (5) Move the cell suspension to a 15 mL centrifuge tube, then add 10 mL of DMEM / F12 drop by drop, mix the cells gently during the process, and centrifuge at 160 x g for 5 min.

[0030] (6) Aspirate the supernatant, add 6 mL of pre-warmed Blebbistatin + KnockOut DMEM / F-12 medium from step (3), mix the cells, and try to avoid blowing.

[0031] (7) Aspirate the VTN coating solution in the 6-well plate, and inoculate the mixed cells into the 6-well plate at 3 mL / well.

[0032] (8) Shake horizontally and crosswise three times, place it in the cell culture incubator, shake horizontally and crosswise three times again, and culture.

[0033] (9) Change the new KnockOut DMEM / F-12 medium after 24 hours, and change the medium every day thereafter.

[0034] 2. Passage ES cells (1) Selection of passage time: ES cells reach 80% to 90% confluence, generally passaged every 2 to 3 days, even if the clone group is small and the confluence is insufficient, it is recommended not to continuously culture for more than 5 days.

[0035] (2) Passage ratio: according to the cell growth state and experimental needs, passaging can be carried out at a ratio of 1:10.

[0036] (3) Place the VTN-coated 6-well plate in the biosafety cabinet for about 2 hours to recover to room temperature in advance.

[0037] (4) Prepare 3 mL / well of KnockOut DMEM / F-12 medium according to the number of wells inoculated, and add 30 mM Blebbistatin at a ratio of 1:8000, and restore to room temperature.

[0038] (5) Discard the medium in the wells of the ES cell plate, and add 3 mL / well of DPBS (without calcium and magnesium), shake gently and discard.

[0039] (6) Add 1 mL / well of EDTA to completely cover the bottom of the well.

[0040] (7) Incubate in a 37°C incubator for 5 min.

[0041] (8) After digestion, gently take the cell culture plate back to the biosafety cabinet to avoid shaking the cells, and tilt to discard the EDTA.

[0042] (9) Add 3 mL / well of pre-warmed Blebbistatin + KnockOut DMEM / F-12 medium in time, and shake the 6-well plate horizontally and crosswise to detach the cells from the substrate.

[0043] (10) Inoculation: discard the VTN solution in the 6-well plate, and add 3 mL / well of pre-warmed Blebbistatin + KnockOut DMEM / F-12 medium.

[0044] (11) Shake the 6-well plate horizontally and crosswise three times, place it in the cell culture incubator, shake the 6-well plate horizontally and crosswise three more times, and incubate overnight.

[0045] (12) Replace the new KnockOut DMEM / F-12 medium after 24 hours, and change the medium every day thereafter, and continue to subculture or freeze for 3-5 days.

[0046] 3. Differentiation of human embryonic stem cells into oligodendrocytes and astrocytes: S1, DAY0 (1) When the confluence of ES cells in the culture dish reaches 80%, discard the ES medium, then add 3 mL / well of DPBS (without calcium and magnesium), shake gently and discard.

[0047] (2) Add 0.8 mL / well of pre-warmed Accutase + EDTA (volume ratio of Accutase to EDTA is 3:1) to completely cover the bottom of the well, incubate in the incubator for 8 min, and gently shake the plate to completely detach the cells from the substrate.

[0048] (3) Transfer the cell suspension to a 1.5 mL centrifuge tube, and centrifuge in a palm centrifuge for 20 s.

[0049] (4) Aspirate the supernatant, add 4 mL of the first medium, wherein the first medium comprises Neurobasal medium, 200 ng / mL ascorbic acid, 200 ng / mL IL-15, 200 ng / mL IL-4, 100 ng / mL human transferrin, 100 ng / mL Nestin and 400 ng / mL CD11b, resuspend the cells, gently blow 4-8 times to disperse the cells into single cells as much as possible, and count.

[0050] (5) Seed into a Matrigel coated 6-well plate, add 3 mL of the first medium (containing 10 μM Blebbistatin) per well.

[0051] (6) Place in the incubator, shake horizontally 4-8 times, and culture.

[0052] (7) After 24 h of cell culture, aspirate the cell culture medium, add 3 mL of the first medium per well, and place in the incubator for continuous culture, and change the medium every day.

[0053] S2, DAY 8: (1) Aspirate the medium, add 3 mL of the second medium per well, wherein the second medium comprises Neurobasal medium, 200 ng / mL CNTF, 400 ng / mL β-mercaptoethanol, 100 ng / mL Nestin, 200 ng / mL IL-4, 200 ng / mL IL-2, 400 ng / mL CD11b, 200 ng / mL NG2 and 500 ng / mL GD3, and place in the incubator for continuous culture, and change the medium every day (DAY 8-12).

[0054] S3, DAY 12: (1) Aspirate the medium, add 3 mL of DPBS (without calcium and magnesium) per well, gently shake and aspirate.

[0055] (2) Add pre-warmed Accutase+EDTA (volume ratio of Accutase and EDTA is 3:1) at 0.5 mL per well to completely cover the bottom of the well, place in the incubator for 15 min, and gently shake the plate to completely detach the cells.

[0056] (3) Transfer the cell suspension into a 1.5 mL centrifuge tube, and centrifuge in a handheld centrifuge for 20 s.

[0057] (4) Aspirate the supernatant, and add 1.2 mL of the third medium, wherein the third medium comprises Neurobasal medium, 200 ng / mL CNTF, 400 ng / mL β-mercaptoethanol, 100 ng / mL Nestin, 200 ng / mL IL-4, 200 ng / mL IL-2, 400 ng / mL CD11b, 200 ng / mL NG2 and 500 ng / mL GD3; resuspend the cells, and gently blow 4-8 times to disperse the cells into single cells as much as possible, and count the cells.

[0058] (5) Seed the cells into a Matrigel-coated 12-well plate, and add 2 mL / well of the third medium (add Blebbistatin to the third medium to a final concentration of 10 μM).

[0059] (6) Place in an incubator, and shake horizontally 2-7 times, and culture.

[0060] (7) After 24 h of culture, aspirate the cell culture medium, add 3 mL / well of the third medium, and place in an incubator for culture, and change the medium every day (DAY 12-15).

[0061] S4, DAY 15: (1) Aspirate the cell culture medium, and add 3 mL / well of PM differentiation medium 4, wherein the fourth medium comprises Neurobasal medium, 200 ng / mL CNTF, 20 μM β-mercaptoethanol, 80 μg / mL glutathione, 0.02 mM aspartic acid, 100 ng / mL Nestin, 400 ng / mL CD11b, 200 ng / mL NG2, 500 ng / mL GD3, 200 ng / mL O4, 100 ng / mL MAG and 300 ng / mL NeuN.

[0062] 5. The cell fluid containing mature oligodendrocytes and astrocytes can be obtained when the culture is continued to DAY 18-20.

[0063] 4. The differentiated embryonic stem cell-astrocyte co-culture is identified by immunofluorescence staining, comprising: I. Sample: cell climbing sheet of the differentiated cell co-culture of Example 1.

[0064] II. Antibody (both primary antibody and secondary antibody are from Abeam Company): Primary antibody: GFAP (astrocyte characteristic protein) antibody (rabbit anti-human) and MBP (oligodendrocyte characteristic protein) antibody (rabbit anti-human).

[0065] Secondary antibody: fluorescently labeled secondary antibody (FITC-labeled goat anti-rabbit IgG).

[0066] III. Operation steps: (1) Place the cell climbing sheet on the glass slide. Fix with 4% paraformaldehyde for 30 min. Wash with PBS for 3 times, 5 min each time.

[0067] (2) Treat the sample with 0.1% Triton X-100 for 10 min, and wash with PBS for 3 times, 5 min each time.

[0068] (3) Blocking: Cover the sample with blocking solution (5% BSA), incubate at room temperature for 40 min. Wash with PBS for 3 times, 5 min each time.

[0069] (4) Incubation of primary antibody: Dilute the primary antibody according to the antibody instructions: GFAP (astrocyte characteristic protein) antibody (rabbit anti-human) or MBP (oligodendrocyte characteristic protein) antibody (rabbit anti-human) (dilution ratio 1:500). Add the diluted primary antibody to the sample to cover it completely. Incubate in a wet box at 4°C overnight. Wash with PBS for 3 times, 5 min each time.

[0070] (5) Incubation of secondary antibody: Dilute the fluorescently labeled secondary antibody according to the antibody instructions: FITC-labeled goat anti-rabbit IgG (dilution ratio 1:1000). Add the diluted secondary antibody to the sample to cover it completely. Incubate in a wet box for 1 h, avoiding light. Wash with PBS for 3 times, 5 min each time.

[0071] (6) Nuclei are stained with DAPI: Add DAPI (1:1000 dilution) to the sample, incubate at room temperature for 5 min, avoiding light. Wash with PBS for 3 times, 5 min each time.

[0072] (7) Mounting: Mount with anti-fluorescence quenching agent to avoid quenching of the fluorescent signal. Add the mounting agent to the sample, cover with a cover glass, press gently to avoid air bubbles. Store in the dark.

[0073] 5. Extraction of exosomes from co-culture of embryonic stem cell differentiated oligodendrocytes and astrocytes in vitro: (1) Collect the supernatant of the culture medium at the 4th passage, transfer the supernatant to a centrifuge tube, centrifuge at 4°C, 300xg for 10 min, and then centrifuge at 4°C, 2000xg for 10 min to remove dead cells.

[0074] (2) Filter the centrifuged supernatant with a 0.22μm filter, collect the filtrate.

[0075] (3) The filtrate was centrifuged at 100000xg for 70 min at 4°C in a centrifuge tube to remove the supernatant, then resuspended with PBS, and then centrifuged again at 100000xg for 70 min at 4°C in a centrifuge tube to remove the supernatant. The obtained filtrate was resuspended with 550ul PBS to collect stem cell exosomes.

[0076] (4) The exosomes were further purified using a purification reagent (ExoQuick reagent) (volume ratio 1:4 mixed with stem cell exosomes), centrifuged, and resuspended with 550ul PBS to remove residual proteins and impurities to obtain high-purity exosomes.

[0077] (5) The high-purity exosomes were incubated with 4% Roche reagent, 1% HMFS, and RIPA lysis buffer at 4°C for 30 min, then centrifuged at 14000 rpm for 30 min, the supernatant was taken, and SDS-PAGE gel electrophoresis was performed. After electrophoresis, the bands in the gel were transferred to a PVDF membrane. After blocking the PVDF membrane with Western blot blocking solution, it was incubated with CD63 antibody, CD9 antibody, CD81 antibody and Actin antibody at room temperature for 2h and washed with Western blot washing solution for 3 times. Remove the washing solution, add HRP-conjugated secondary antibody, incubate at room temperature for 1h, wash with Western blot washing solution for 3 times, and develop with developing solution. The extracted exosomes all expressed specific surface markers CD9, CD81 and CD63 Figure 1 ).

[0078] Example 2 Preparation of an anti-aging product: (1) The following raw materials were weighed according to the mass percentage: exosomes of Example 1 0.1%, glycerol 10%, hyaluronic acid 4%, azone 0.5%, asiaticoside 1%, menthol 1%, hydrolyzed collagen 1%, carbomer U20 0.3%, EDTA-2Na 0.5%, antibacterial agent 0.1% (sodium benzoate), triethanolamine 0.1%, and the rest was sterile water.

[0079] (2) Exosome treatment: Mix the exosomes with other ingredients in proportion and stir evenly.

[0080] (3) Emulsification: Use emulsification equipment to emulsify the mixed solution to form a stable emulsion.

[0081] (4) Packaging: The prepared emulsion was filled into a cosmetic packaging bottle and sealed for storage.

[0082] Performance test Select 20 subjects over 45 years old into 2 groups, 10 people in group 1 use the exosome serum embodiment, 10 people in group 2 use the commercially available serum, after 15 days, exchange group 2 to use the commercially available serum, and group 1 to use the exosome serum embodiment. During use, the skin of each subject is detected, and the detection indexes include: skin moisture content, skin elasticity, skin wrinkles, and self-evaluation of the subject. Before use, the subjects do not know the information of the two products, and the use frequency and skin care steps are consistent before and after. The detection site is unified as the left cheek, the detection environment is 20-26℃, and the humidity is 55-65%.

[0083] Table 1: The skin moisture detection index statistics of the subjects during use are as follows:

[0084] From Table 1, it can be seen that the skin moisture content of group 1 increases rapidly in the first 15 days, and the skin moisture content of group 2 increases rapidly after the product is exchanged and used in the latter 15 days.

[0085] Table 2: The skin wrinkle improvement percentage index statistics of VISIA detection are as follows:

[0086] From Table 2, it can be seen that the skin wrinkle improvement percentage of group 1 increases rapidly in the first 15 days, and the skin wrinkle improvement percentage of group 2 increases rapidly after the product is exchanged and used in the latter 15 days.

[0087] Table 3: The skin elasticity characterization value index statistics of the skin elasticity tester detection are as follows:

[0088] From Table 3, it can be seen that the skin elasticity characterization value of group 1 increases rapidly in the first 15 days, and the skin elasticity characterization value of group 2 increases rapidly after the product is exchanged and used in the latter 15 days.

[0089] Table 4: The use feeling description of the subjects is as follows:

[0090] As shown in Figure 2 , one of the subjects uses the right cheek skin for half a year, and the improvement situation is shown in Figure 2 , the left is before use, Figure 2 , and the right is after use.

[0091] In summary, the cosmetic developed by using the exosomes containing the oligodendrocytes and astrocytes differentiated from embryonic stem cells has higher bioavailability and better permeability compared with traditional anti-aging cosmetics, can more effectively deliver bioactive molecules, promote skin cell regeneration and repair, and significantly improve skin aging phenomena, such as increasing skin moisture content, improving skin elasticity, and reducing wrinkles.

[0092] It should be pointed out finally that the above embodiments are used to illustrate the technical solutions of the present application but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for producing an exosome differentiated from an embryonic stem cell, characterized by, The method comprises the following steps: (1) centrifuging, filtering and obtaining filtrate from cell liquid containing mature oligodendrocytes and astrocytes; (2) centrifuging the filtrate, resuspending with PBS and obtaining stem cell exosomes; (3) mixing the stem cell exosomes and ExoQuick reagent, centrifuging, resuspending with PBS and obtaining exosomes.

2. The method for preparing exosomes derived from embryonic stem cell differentiation according to claim 1, characterized in that, The filter membrane used for filtering is 0.22 μm.

3. The method for preparing exosomes derived from embryonic stem cell differentiation according to claim 1, characterized in that, The volume ratio of the stem cell exosomes and the ExoQuick reagent is (3-5):

1.

4. The method of claim 1, wherein the exosomes are prepared from embryonic stem cells. The preparation method of the cell liquid containing mature oligodendrocytes and astrocytes is as follows: (11) on day 0, inoculating embryonic stem cells into a first differentiation culture medium for culture; (12) on day 8, discarding the culture medium and adding a second differentiation culture medium for culture; (13) on day 12, discarding the culture medium, washing the cells and then performing cell digestion, and then inoculating the cells into a third differentiation culture medium for culture; (14) on day 15, discarding the culture medium, inoculating the cells into a fourth differentiation culture medium for culture, and culturing to day 18-20 to obtain the cell liquid containing mature oligodendrocytes and astrocytes.

5. The method of claim 4, wherein the exosomes are prepared from embryonic stem cells. The first culture medium comprises the following components: a basic culture medium, 100-300 ng / mL ascorbic acid, 100-300 ng / mL IL-15, 100-300 ng / mL IL-4, 50-200 ng / mL human transferrin, 50-200 ng / mL Nestin and 200-500 ng / mL CD11b. The second culture medium comprises the following components: a basic culture medium, 100-300 ng / mL CNTF, 200-500 ng / mL β-mercaptoethanol, 50-200 ng / mL Nestin, 100-300 ng / mL IL-4, 100-300 ng / mL IL-2, 200-500 ng / mL CD11b, 100-300 ng / mL NG2 and 400-800 ng / mL GD3.

6. The method of claim 4, wherein the exosomes are prepared from embryonic stem cells. The third culture medium comprises the following components: a basic culture medium, 100-300 ng / mL CNTF, 10-40 μM β-mercaptoethanol, 80-100 μg / mL glutathione, 0.01-0.04 mM aspartic acid, 50-200 ng / mL Nestin, 200-500 ng / mL CD11b, 100-300 ng / mL NG2, 200-600 ng / mL GD3, 100-300 ng / mL O4, 50-200 ng / mL MAG and 100-400 ng / mL NeuN. The fourth culture medium comprises the following components: a basic culture medium, 100-300 ng / mL CNTF, 100-300 ng / mL IL-4, 100-300 ng / mL IL-2, glutathione 80-100 μg / mL, 50-200 ng / mL Nestin, 200-500 ng / mL CD11b, 100-400 ng / mL NG2, 400-800 ng / mL GD3, 100-400 ng / mL O4, 50-200 ng / mL MAG, 200-500 ng / mL NeuN, 50-200 ng / mL MBP, 100-400 ng / mL PLP, and 200-600 ng / mL GFAP.

7. An exosome obtained by differentiating an embryonic stem cell, characterized in that, The exosomes express specific markers CD9, CD81 and CD63.

8. Use of the exosomes differentiated from embryonic stem cells according to claim 7 in the preparation of anti-aging products.

9. An anti-aging product characterized in that, The composition comprises the following components in the following mass percentages: exosomes differentiated from embryonic stem cells 0.05-0.2%, glycerin 8-10%, hyaluronic acid 2-4%, azone 0.1-2%, asiaticoside 1-5%, menthol 0.5-2%, hydrolyzed collagen 1-2%, carbomer U20 0.1-0.5%, EDTA-2Na 0.2-0.8%, antibacterial agent 0.1-0.3%, triethanolamine 0.1-0.2%, and the balance water.

10. The anti-aging product of claim 9, wherein, The composition comprises the following components in the following mass percentages: exosomes differentiated from embryonic stem cells 0.1%, glycerin 10%, hyaluronic acid 4%, azone 0.5%, asiaticoside 1%, menthol 1%, hydrolyzed collagen 1%, carbomer U20 0.3%, EDTA-2Na 0.5%, antibacterial agent 0.1%, triethanolamine 0.1%, and the balance water.

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