Stem cell exosome composition and preparation method and application thereof

Abstract

The present invention discloses an embryonic stem cell-derived mesenchymal-like stem cell exosome composition, a preparation method and an application thereof; the exosomes are mesenchymal-like stem cell exosomes derived from embryonic stem cells. The present invention reveals that mesenchymal-like stem cell exosomes from embryonic stem cells have a significant effect on promoting cell proliferation and migration. Surprisingly, at specific concentrations of 0.18-0.22 g / L and 0.38-0.42 g / L, these exosomes significantly enhanced the collagen expression of skin fibroblasts. In addition, the study also found that at the same exosome concentration, embryonic stem cell-derived mesenchymal-like stem cell exosomes are more effective in promoting the migration of skin fibroblasts than umbilical cord mesenchymal stem cell exosomes. These findings provide an important theoretical basis for the development of new skin repair and regeneration therapies.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a stem cell exosome composition and a preparation method and application thereof. Background Art

[0002] Exosomes are small vesicles secreted by all cell types, with a size of about 30-150 nanometers. They are important tools for intercellular communication and can transfer molecules such as proteins, lipids and RNA. There is evidence that exosomes have multiple biological functions such as regulating intercellular communication, repairing tissues, and inhibiting inflammation. In recent years, exosomes have attracted much attention as a research field with broad application prospects, especially in the fields of cell therapy, tissue repair and regenerative medicine.

[0003] Stem cell exosomes, especially stem cell-derived mesenchymal-like stem cell exosomes, have been shown to have significant biological activity, and they retain some key properties of their source cells, such as the ability to promote cell proliferation, migration, and differentiation. Therefore, stem cell exosomes are considered to be a novel, non-cellular therapeutic strategy with significant efficacy and safety advantages.

[0004] In the field of skin care and repair, there is still insufficient comparative research on the effects of exosomes from stem cells of different origins on promoting the proliferation, migration and collagen production of skin fibroblasts. In addition, there is also a lack of detailed research on the appropriate concentration of exosomes in affecting the function of skin fibroblasts. Therefore, the development of stem cell exosome compositions for specific skin problems, especially the clarification of their source cell types and optimal concentration configuration, will be of great significance to the development of skin repair technology. Summary of the invention

[0005] Based on this, the present invention provides an embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which, in addition to being able to promote the proliferation and migration of skin fibroblasts, also has a good effect of enhancing collagen production.

[0006] The first aspect of the present invention provides a stem cell exosome composition, which includes stem cell exosomes and a compound electrolyte solution, wherein the compound electrolyte solution is composed of the following components: 5.23-5.29 g / L sodium chloride, 5-5.05 g / L sodium gluconate, 3.65-3.8 g / L sodium acetate, 0.34-0.40 g / L potassium chloride, and 0.27-0.33 g / L magnesium chloride; the stem cell exosomes are mesenchymal-like stem cell exosomes derived from embryonic stem cells, and the final concentration of the stem cell exosomes is 0.18-0.42 g / L.

[0007] In some embodiments, the composition of the composite electrolyte solution is: 5.25-5.27 g / L sodium chloride, 50.1-5.03 g / L sodium gluconate, 3.67-3.69 g / L sodium acetate, 0.36-0.38 g / L potassium chloride, and 0.29-0.31 g / L magnesium chloride.

[0008] In some embodiments, the composition of the composite electrolyte solution is: 5.26 g / L sodium chloride, 5.02 g / L sodium gluconate, 3.68 g / L sodium acetate, 0.37 g / L potassium chloride, and 0.3 g / L magnesium chloride.

[0009] In some embodiments, the final concentration of the stem cell exosomes is 0.18-0.22 g / L or 0.38-0.42 g / L, preferably the content of the stem cell exosomes is 0.19-0.21 g / L, or 0.39-0.41 g / L.

[0010] In some more preferred embodiments, the content of the stem cell exosomes is 0.2 g / L, or 0.4 g / L.

[0011] The embryonic stem cell-derived mesenchymal-like stem cell exosomes of the present invention can be extracted according to conventional methods. For example, in some embodiments thereof, the method for extracting stem cell exosomes comprises the following steps:

[0012] (1) inoculating and culturing mesenchymal stem cells derived from embryonic stem cells, collecting the cell culture supernatant after the cell confluence reaches more than 90%, centrifuging at 3-5° C. and collecting the centrifuged supernatant to obtain a stem cell exosome mixture;

[0013] (2) Mixing the exosome mixture obtained in step (1) with a PEG6000-NaCl solution, precipitating overnight at 3-5° C., centrifuging, and extracting the centrifugal precipitate to obtain the stem cell exosomes.

[0014] In some preferred embodiments, the volume ratio of the stem cell exosome mixture to the PEG6000-NaCl solution is 1:(0.8-1.2); and / or, the PEG6000-NaCl solution comprises: 0.9-1.1 M sodium chloride, 22-26% (w / v) PEG6000.

[0015] In some preferred embodiments, in step (1), the inoculation density is 1×10 3 Pieces / cm 2 , centrifuged at a gravity acceleration of (490-510)×g for 4-6 minutes, and then centrifuged at a gravity acceleration of (1800-2200)×g for 25-35 minutes; and / or,

[0016] In step (2), the centrifugation is performed at a gravity acceleration of (2800-3200)×g for 55-65 minutes.

[0017] The second object of the present invention is to provide a method for preparing the above-mentioned stem cell exosome composition, comprising the following steps:

[0018] (1) Mixing sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride and water for injection, stirring and dissolving them sufficiently to obtain a composite electrolyte solution;

[0019] (2) adding the embryonic stem cell-derived mesenchymal-like stem cell exosomes into the composite electrolyte solution and mixing them evenly to obtain the composite electrolyte solution.

[0020] The third object of the present invention is to provide the use of the above-mentioned stem cell exosome composition in the preparation of skin care and repair products, in particular, the skin care and repair includes enhancing the collagen expression of skin fibroblasts.

[0021] The skin care and repair also includes the above-mentioned stem cell exosome composition promoting skin cell proliferation, migration and differentiation.

[0022] The present invention reveals that mesenchymal-like stem cell exosomes from embryonic stem cells have a significant effect on promoting cell proliferation, migration and differentiation. Surprisingly, at specific concentrations of 0.18-0.42 g / L, especially at 0.18-0.22 g / L and 0.38-0.42 g / L, these mesenchymal-like stem cell exosomes from embryonic stem cells significantly enhanced the collagen expression of skin fibroblasts. In addition, the study also found that at the same exosome concentration, embryonic stem cell-derived mesenchymal-like stem cell exosomes promoted the migration of skin fibroblasts more effectively than umbilical cord mesenchymal stem cell exosomes. These findings provide an important theoretical basis for the development of new skin repair and regeneration therapies.

[0023] The embryonic stem cell-derived mesenchymal-like stem cell exosome composition of the present invention is used for non-cellular treatment of the skin, without the need to directly use stem cells, reducing the complexity of the operation, increasing safety, and while providing the same biological effects as stem cells, avoiding possible immune rejection and other problems. The embryonic stem cell-derived mesenchymal-like stem cell exosome composition can be used in the field of preparing skin care and repair products, including injection preparations for the skin or coating preparations for the skin, which can effectively promote the proliferation, migration and collagen production of skin fibroblasts, and achieve the effect of skin care and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is a comparison diagram of different concentrations of embryonic stem cell-derived mesenchymal stem cell exosomes promoting the proliferation of skin fibroblasts.

[0025] Figure 2 This is a comparison diagram of different concentrations of embryonic stem cell-derived mesenchymal stem cell exosomes promoting the migration of skin fibroblasts.

[0026] Figure 3 and Figure 4 This is a comparison diagram of different concentrations of embryonic stem cell-derived mesenchymal stem cell exosomes of the present invention promoting the production of type I collagen in skin fibroblasts.

[0027] Figure 5 and Figure 6 This is a comparison of the cell migration rates of exosomes from umbilical cord mesenchymal stem cells and embryonic stem cell-derived mesenchymal-like stem cells at low and high doses.

[0028] Figure 7 This is a comparison of the effects of exosomes from UC-MSC and T-MSC on the proliferation of fibroblasts at different doses. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further illustrated by specific examples below. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] The term "hESC" as used herein refers to human embryonic stem cells, including pluripotent stem cells derived from embryos, inner cell mass, blastomeres or cell lines.

[0032] The term "h-MSC" as used herein refers to mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC) through an intermediate stage in which the cells express Trop-2 and have a trophoblast morphology.

[0033] The term "UC-MSC" as used herein refers to human umbilical cord-derived mesenchymal stem cells.

[0034] The embryonic stem cell-derived mesenchymal stem cell exosomes in the following examples are obtained by the following extraction method:

[0035] (1) The raw materials of embryonic stem cell-derived mesenchymal-like stem cells (T-MSCs) were purchased from the market and induced and cultured according to the method described in patent CN104487568B, which specifically includes the following steps:

[0036] hESCs with a confluence of about 80% on a matrigel-coated dish were digested with 1 mg / ml dispase for 5-10 minutes. The cells were then washed once with mTESR1 medium and seeded in small pieces or single cells on a matrigel-coated dish and cultured in mTESR1 medium for 12 hours. The culture medium was then changed to trophoblast formation medium, which contained BMP4 (2-100 ng / ml), or optionally A83-01 (0.1-1 μM). After 48-72 hours of culture, the cells changed from an hESC-like morphology to a trophoblast morphology, which is characterized by flattening, increased cell size, a small nuclear / cytosol ratio, and diffuse cell borders. The cells were digested with Tryp-LE and washed with MSC growth medium, which was α-MEM containing 20% ​​FBS and non-essential amino acids. The cells were then grown at 5000 cells / cm 2 The cells were seeded at a density of 1.50 μg / cm2 on a culture dish coated with Matrigel. The culture medium was changed after 24 hours and then every 3-4 days. After another 6 days, the cells differentiated into spindle-like cells, similar to the typical morphology of MSCs.

[0037] (2) The above-mentioned mesenchymal stem cells T-MSC were cultured at a rate of 1×10 3 Pieces / cm 2 The cells were inoculated at a density of 100 μg / mL. When the cell confluence reached more than 90%, the cell culture supernatant was collected and centrifuged at 500 × g for 5 min and 2000 × g for 30 min at 4 °C. The supernatant was collected again for exosome extraction. A solution containing 1 M sodium chloride and 24% (w / v) PEG6000 was mixed with the cell supernatant at a ratio of 1:1. The final concentration of PEG6000 in the mixed solution was 12%. The above solution was placed in a 4 °C refrigerator for precipitation overnight. The T-MSC exosomes were harvested by centrifugation at 3000 × g for 60 min the next day. The exosome precipitate was dissolved with the prepared solution and then aliquoted and frozen at -80 °C.

[0038] The following are specific examples.

[0039] Example 1

[0040] A stem cell exosome composition, composed of the above-mentioned embryonic stem cell-derived mesenchymal-like exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 0.001 mg / ml; wherein each 500 ml of the solution contains 0.5 mg of exosomes, 2.63 g of sodium chloride, 2.51 g of sodium gluconate, 1.84 g of sodium acetate, 0.185 g of potassium chloride, and 0.15 g of magnesium chloride.

[0041] The method for preparing the embryonic stem cell-derived mesenchymal-like stem cell exosome composition comprises the following steps:

[0042] (1) Mix the formulated amounts of sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride and an appropriate amount of water for injection, and stir thoroughly until all are dissolved to obtain a composite electrolyte solution;

[0043] (2) adding the mesenchymal stem cell exosomes derived from embryonic stem cells to the composite electrolyte solution and mixing them evenly, then filtering the solution through a microporous filter membrane for sterilization, and finally filling the solution and sealing the cap to obtain the product.

[0044] Example 2

[0045] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, consisting of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 0.01 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0046] The preparation method of this embodiment is the same as that of embodiment 1.

[0047] Example 3

[0048] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which consists of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 0.1 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0049] The preparation method of this embodiment is the same as that of embodiment 1.

[0050] Example 4

[0051] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which consists of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 0.2 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0052] The preparation method of this embodiment is the same as that of embodiment 1.

[0053] Example 5

[0054] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which consists of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 0.3 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0055] The preparation method of this embodiment is the same as that of embodiment 1.

[0056] Example 6

[0057] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which consists of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 0.4 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0058] The preparation method of this embodiment is the same as that of embodiment 1.

[0059] Example 7

[0060] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which consists of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 0.5 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0061] The preparation method of this embodiment is the same as that of embodiment 1.

[0062] Example 8

[0063] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which consists of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 1 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0064] The preparation method of this embodiment is the same as that of embodiment 1.

[0065] Example 9

[0066] An embryonic stem cell-derived mesenchymal-like stem cell exosome composition, which consists of the embryonic stem cell-derived mesenchymal-like stem cell exosomes and a compound electrolyte solution, wherein the final concentration of the embryonic stem cell-derived mesenchymal-like stem cell exosomes is 10 mg / ml; the compound electrolyte solution is the same as that in Example 1.

[0067] The preparation method of this embodiment is the same as that of embodiment 1.

[0068] Comparative Example 1

[0069] A compound electrolyte solution, each 500 ml of the solution contains 2.63 g of sodium chloride, 2.51 g of sodium gluconate, 1.84 g of sodium acetate, 0.185 g of potassium chloride, and 0.15 g of magnesium chloride.

[0070] The method for preparing the composite electrolyte solution comprises the following steps:

[0071] Mix the formulated amounts of sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride and water for injection, and stir thoroughly until all are dissolved to obtain a compound electrolyte solution.

[0072] Comparative Example 2

[0073] A stem cell exosome composition comprises exosomes and the composite electrolyte solution as described in Example 1, wherein the final concentration of the stem cell exosomes is 0.1 mg / ml.

[0074] The stem cell exosomes are exosomes derived from human umbilical cord-derived mesenchymal stem cells (UC-MSCs).

[0075] The method for preparing the stem cell exosome composition comprises the following steps:

[0076] (1) Mixing the formulated amounts of sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride and water for injection, and stirring thoroughly until all are dissolved to obtain a composite electrolyte solution;

[0077] (2) adding human umbilical cord-derived mesenchymal stem cell exosomes to the composite electrolyte solution and mixing them evenly, then filtering the solution through a microporous filter membrane for sterilization, and finally filling the solution and sealing the cap to obtain the product.

[0078] Comparative Example 3

[0079] A stem cell exosome composition comprises exosomes and the composite electrolyte solution as described in Example 1, wherein the final concentration of the stem cell exosomes is 0.2 mg / ml.

[0080] The stem cell exosomes are exosomes derived from human umbilical cord-derived mesenchymal stem cells (UC-MSCs).

[0081] The preparation method of this comparative example is the same as that of comparative example 2.

[0082] Comparative Example 4

[0083] The stem cell exosome composition of this example has the same composition and preparation method as those of Comparative Example 2, except that the final concentration of exosomes in the stem cell exosome composition of this example is 0.4 mg / ml.

[0084] The stem cell exosomes are stem cell exosomes derived from human umbilical cord-derived mesenchymal stem cells (UC-MSCs).

[0085] Comparative Example 5

[0086] The stem cell exosome composition of this embodiment has the same composition and preparation method as those of Comparative Example 2, except that the final concentration of the stem cell exosomes in the stem cell exosome composition of this embodiment is 1 mg / ml. The exosomes are stem cell exosomes derived from human umbilical cord-derived mesenchymal stem cells (UC-MSC).

[0087] Performance Testing

[0088] 1. Experiment on promoting proliferation of skin fibroblasts by exosome composition derived from embryonic stem cells and mesenchymal stem cells

[0089] Human skin fibroblasts were plated in a 96-well cell culture plate at 5000 / well and cultured overnight in DMEM complete medium supplemented with 10% FBS. The next day, the complete medium was discarded and replaced with serum-free medium, and the culture was continued for 24 hours. After the time point was reached, the samples of Examples 1-3, 8-9 and Comparative Example 1 were added for 24 hours. After the action time was reached, 10 μl of CCK-8 detection reagent was added to every 100 μl of culture solution, and the culture was continued in the cell culture incubator for 3 hours. The absorbance value of each well at a wavelength of 450 nm was read using an ELISA reader.

[0090] The results are as follows Figure 1 As shown, the embryonic stem cell-derived mesenchymal stem cell exosomes of the present invention can promote the proliferation of skin fibroblasts. When different concentrations of exosomes act on skin fibroblasts, as the concentration of exosomes increases, the number of skin fibroblasts also increases. Figure 1 The best effect was achieved when the concentration of the embryonic stem cell-derived mesenchymal exosomes was 0.1-1 mg / ml.

[0091] 2. Experiment on promoting migration of skin fibroblasts by exosome composition of embryonic stem cells-derived mesenchymal stem cells

[0092] Human skin fibroblasts were cultured at a rate of 2.5×105 Cells / well were plated in a 12-well plate and cultured overnight in DMEM complete medium supplemented with 10% FBS. The next day, the complete medium was discarded and replaced with serum-free medium for 24 hours. After reaching the action time point, the cells were scratched with a 10 μl gun tip, the medium was discarded, the floating cells were washed with DPBS, and serum-free medium was added for culture, and the samples of Examples 1-3, 8, 9 and Comparative Example 1 were added for action. Photos were taken to record the initial scratch size, and photos were taken after 24 hours to record the confluence of the scratches.

[0093] The results are as follows Figure 2 As shown, compared with comparative example 1 without exosomes, adding exosomes to the solution of the present invention can promote the migration of skin fibroblasts, thereby accelerating the repair of skin wounds. Figure 2 It can be found that as the concentration of embryonic stem cell-derived mesenchymal-like exosomes increases, the migration rate of skin fibroblasts also increases.

[0094] 3. Experiment on the effect of embryonic stem cell-derived mesenchymal stem cell exosome composition on promoting the production of type I collagen in skin fibroblasts

[0095] Human skin fibroblasts were cultured at a rate of 2×10 5 The cells / well were plated in a 6-well plate and cultured overnight using DMEM complete medium supplemented with 10% FBS. The next day, the complete medium was discarded and replaced with serum-free medium for further culturing for 24 hours. After reaching the action time point, samples of Examples 3-7 and Comparative Example 1 were added and treated for 48 hours. After reaching the cell treatment time point, the total RNA of the cells was extracted using an RNA extraction kit, and fluorescent quantitative PCR of CollagenⅠ was performed to obtain data and statistically analyze the experimental results. RNA extraction kit: manufacturer GOODNIE, item number 400-103, qPCR kit: manufacturer ESscience, item number QP002.

[0096] The results are as follows Figure 3 As shown, compared with Comparative Example 1 without exosomes, the addition of the embryonic stem cell-derived mesenchymal stem cell exosome composition of the present invention can promote the production of collagen in skin fibroblasts, thereby accelerating the repair of skin wounds; among them, the embryonic stem cell-derived mesenchymal stem cell exosome composition in Example 4 with an exosome concentration of 0.2 mg / ml achieved the best promoting effect, and the effect of promoting fibroblast collagen production at a high concentration was also more obvious, followed by Example 6 (the exosomes were 0.4 mg / ml), see Figure 3 Moreover, surprisingly, the higher the concentration of exosomes, the stronger the function of enhancing collagen secretion of fibroblasts. Figure 4As a result, in another set of experiments, when the exosomes were at a concentration of 0.2 mg / ml, their function of enhancing the secretion of collagen by fibroblasts was greater than that of the stem cell exosome composition at concentrations of 1 mg / ml and 10 mg / ml.

[0097] 4. Cell migration rate experiment of exosomes of UC-MSC and T-MSC at different doses

[0098] Human skin fibroblasts were cultured at a rate of 2.5×10 5 Cells / well were plated in a 12-well plate and cultured overnight using DMEM complete medium supplemented with 10% FBS. The next day, the complete medium was discarded and replaced with serum-free medium for further culturing for 24 hours. After reaching the action time point, the cells were scratched using a 10 μl gun tip, the medium was discarded, the floating cells were washed with DPBS, and serum-free medium was added for culturing. The samples of Example 3, Example 8, Comparative Example 2, and Comparative Example 5 were added for treatment. Photos were taken to record the initial scratch size, and photos were taken after 24 hours to record the confluence of the scratches.

[0099] The results are as follows Figure 5 As shown, Example 3 and Comparative Example 2 are comparisons of cell mobility of exosomes of T-MSC and UC-MSC at low doses, and Example 8 and Comparative Example 5 are comparisons of cell mobility of exosomes of T-MSC and UC-MSC at high doses. The results show that the cell mobility of the T-MSC exosomes of the present invention is higher than that of the UC-MSC exosomes, whether at low or high doses.

[0100] According to the above method, the samples of Example 4, Example 6, Comparative Example 3 and Comparative Example 4 were added for treatment. Photos were taken to record the initial scratch size, and photos were taken to record the confluence of the scratches after 24 hours. Figure 6 By comparison Figure 6 From the results, we can observe that under the same exosome concentration condition, the cell migration rate of Example 4 is significantly higher than that of Comparative Example 3, and similarly, the cell migration rate of Example 6 is also higher than that of Comparative Example 4. These results clearly show that under the action of the same amount of exosomes, the T-MSC exosomes used in the present invention exhibit higher cell migration efficiency than the UC-MSC exosomes.

[0101] 5. Experiment on the promotion of fibroblast proliferation by exosomes of UC-MSC and T-MSC at different doses

[0102] Human skin fibroblasts were plated in a 96-well cell culture plate at 5000 / well and cultured overnight using DMEM complete medium supplemented with 10% FBS. The next day, the complete medium was discarded and replaced with serum-free medium, and the culture was continued for 24 hours. After the time point was reached, the exosome samples of Example 4, Example 6, and Comparative Examples 3 and 4 were added for 24 hours. After the action time was reached, 10 μl of CCK-8 detection reagent was added to every 100 μl of culture solution, and the culture was continued in the cell culture incubator for 3 hours. The absorbance value of each well at a wavelength of 450 nm was read with an enzyme reader. The experimental results are shown in Figure 7 .

[0103] By contrast Figure 7 From the results, we can observe that under the same exosome concentration condition, the cell proliferation ability of Example 4 is higher than that of Comparative Example 3, and similarly, the cell proliferation ability of Example 6 is also better than that of Comparative Example 4. These results clearly show that under the action of the same amount of exosomes, the T-MSC exosomes used in the present invention are more capable of promoting fibroblast proliferation than the UC-MSC exosomes.

[0104] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A stem cell exosome composition, characterized in that: The stem cell exosome composition includes stem cell exosomes and a compound electrolyte solution, wherein the compound electrolyte solution is composed of the following components: 5.23-5.29 g / L sodium chloride, 5-5.05 g / L sodium gluconate, 3.65-3.8 g / L sodium acetate, 0.34-0.40 g / L potassium chloride, and 0.27-0.33 g / L magnesium chloride; the stem cell exosomes are mesenchymal-like stem cell exosomes derived from embryonic stem cells, and the final concentration of the stem cell exosomes is 0.18-0.22 g / L or 0.38-0.42 g / L.

2. The stem cell exosome composition according to claim 1, characterized in that The composite electrolyte solution is composed of: 5.25-5.27 g / L sodium chloride, 50.1-5.03 g / L sodium gluconate, 3.67-3.69 g / L sodium acetate, 0.36-0.38 g / L potassium chloride, and 0.29-0.31 g / L magnesium chloride.

3. The stem cell exosome composition according to claim 2, characterized in that: The composition of the composite electrolyte solution is: 5.26 g / L sodium chloride, 5.02 g / L sodium gluconate, 3.68 g / L sodium acetate, 0.37 g / L potassium chloride, and 0.3 g / L magnesium chloride.

4. The stem cell exosome composition according to any one of claims 1 to 3, characterized in that: The content of the stem cell exosomes is 0.19-0.21 g / L, or 0.39-0.41 g / L.

5. The stem cell exosome composition according to claim 4, characterized in that The content of the stem cell exosomes is 0.2 g / L, or 0.4 g / L.

6. The stem cell exosome composition according to any one of claims 1 to 3, characterized in that: The method for extracting stem cell exosomes comprises the following steps: (1) inoculating and culturing mesenchymal stem cells derived from embryonic stem cells, collecting the cell culture supernatant after the cell confluence reaches more than 90%, centrifuging at 3-5° C. and collecting the centrifuged supernatant to obtain an exosome mixed solution; (2) Mixing the exosome mixture obtained in step (1) with a PEG6000-NaCl solution, precipitating overnight at 3-5° C., centrifuging, and extracting the centrifugal precipitate to obtain the stem cell exosomes.

7. The stem cell exosome composition according to claim 6, characterized in that: The volume ratio of the stem cell exosome mixture to the PEG6000-NaCl solution is 1:(0.8-1.2); And / or, the PEG6000-NaCl solution comprises: 0.9-1.1 M sodium chloride, 22%-26% w / v PEG6000.

8. The stem cell exosome composition according to claim 6, characterized in that In step (1), the inoculation density is 1×10 3 Pieces / cm 2 , centrifuged at a gravity acceleration of (490-510)×g for 4-6 minutes, and then centrifuged at a gravity acceleration of (1800-2200)×g for 25-35 minutes; and / or, In step (2), the centrifugation is performed at a gravity acceleration of (2800-3200)×g for 55-65 minutes.

9. A method for preparing the stem cell exosome composition according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) Mixing sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride and water for injection, stirring and dissolving them sufficiently to obtain a composite electrolyte solution; (2) adding the embryonic stem cell-derived mesenchymal-like stem cell exosomes into the composite electrolyte solution and mixing them evenly to obtain the composite electrolyte solution.

10. Use of the stem cell exosome composition according to any one of claims 1 to 8 in the preparation of skin care and repair products, wherein the skin care and repair comprises enhancing collagen expression of skin fibroblasts.

Images