A preparation for increasing the exosome secretion capacity of multi-functional stem cells and application thereof

Abstract

The present application relates to a kind of preparation for increasing the excretion capacity of multifunctional stem cell exosome and its application, belong to cell culture technical field.The preparation is composed of culture medium component I and culture medium component II, component I contains transforming growth factor-beta, hepatocyte growth factor etc., for activating stem cell;Component II contains N-acetyl cysteine, vitamin C etc., promote exosome secretion under low oxygen condition.By using two components in a specific order, the exosome secretion amount and biological activity of umbilical cord mesenchymal stem cells can be significantly improved.Compared with the traditional method, the exosome secretion amount treated by the method of the present application is increased by tens of times, and its ability to promote skin fibroblast proliferation and migration is significantly enhanced, so it has important application value in the field of skin repair, tissue regeneration etc..

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and particularly relates to a preparation for increasing the exosome secretion capacity of multi-functional stem cells and application thereof. BACKGROUND

[0002] With the continuous development of stem cell therapy technology, exosomes of stem cells as an important biological material are widely used in disease treatment, tissue repair, and immune regulation fields. Exosomes are small vesicles secreted by cells, which can carry proteins, RNA, lipids and other molecules, and have good biocompatibility and immune regulation effect. Due to its unique biological characteristics, exosomes have shown great potential in regenerative medicine, cell therapy, and anti-inflammatory, anti-tumor and other aspects.

[0003] However, although exosomes have broad prospects in clinical applications, the preparation of exosomes still faces some challenges. Especially in the improvement of exosome secretion, the existing technology still has certain bottlenecks. Traditional methods mainly rely on increasing cell number or changing culture conditions to improve exosome secretion, but these methods are often limited by cell type, culture medium composition, secretion pathway and other factors. Therefore, how to design a multi-functional stem cell exosome secretion promoting preparation to effectively promote the exosome secretion capacity of stem cells has become an important research topic in the field of biological medicine. SUMMARY

[0004] The present application aims to solve the problems of low exosome secretion, insufficient biological activity and low preparation efficiency in the prior art, and provides a preparation for efficiently and reliably promoting the exosome secretion of multi-functional stem cells, as well as related preparation methods and applications.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] Firstly, the present application provides a preparation for increasing the exosome secretion capacity of multi-functional stem cells, which is composed of culture medium component I and culture medium component II;

[0007] The culture medium component I is composed of the following components: 0.25 ng / mL to 1 ng / mL of transforming growth factor-β, 30 ng / mL to 80 ng / mL of hepatocyte growth factor, 2.5 μM to 7.5 μM of Y-27632, 50 μM to 100 μM of thioctic acid, 2 mM to 6 mM of L-glutamine and DMEM / F12 culture medium;

[0008] Said medium component II is composed of the following components: 50-150 μΜ N-acetyl cysteine, 50-150 μΜ vitamin C, 2.5-7.5 μg / L thrombospondin-1, 3-8 mg / L dipalmitoyl phosphatidylcholine, 5-15 ng / mL epidermal growth factor, 25-75 ng / mL insulin-like growth factor-1 and DMEM / F12 medium.

[0009] Preferably, said multi-functional stem cells are umbilical cord mesenchymal stem cells.

[0010] Preferably, said medium component I is composed of the following components: 0.5 ng / mL transforming growth factor-β, 50 ng / mL hepatocyte growth factor, 5 μΜ Y-27632, 50 μΜ thioctic acid, 4 mM L-glutamine and DMEM / F12 medium;

[0011] Said medium component II is composed of the following components: 100 μΜ N-acetyl cysteine, 100 μΜ vitamin C, 5 μg / L thrombospondin-1, 5 mg / L dipalmitoyl phosphatidylcholine, 10 ng / mL epidermal growth factor, 50 ng / mL insulin-like growth factor-1 and DMEM / F12 medium.

[0012] Secondly, the present application provides a culture method for improving the exosome secretion capacity and biological function of multi-functional stem cell exosomes, said method comprising the following steps:

[0013] (1) seeding multi-functional stem cells into culture vessels and culturing to 3-6 passages using medium containing exosome-free FBS;

[0014] (2) replacing the medium with the above-mentioned medium component I and culturing for 24 hours under conventional cell culture conditions;

[0015] (3) replacing the medium with the above-mentioned medium component II and culturing for 24 hours under low-oxygen cell culture conditions;

[0016] (4) collecting the medium for exosome extraction.

[0017] Preferably, said multi-functional stem cells are umbilical cord mesenchymal stem cells;

[0018] Said exosome biological function is the function of exosomes in treating skin damage;

[0019] Said conventional cell culture conditions are 37℃, 5% CO2, 21% O2;

[0020] Said low-oxygen cell culture conditions are 37℃, 5% CO2, 3% O2.

[0021] Preferably, the culture medium component I consists of the following components: 0.5 ng / mL transforming growth factor-β, 50 ng / mL hepatocyte growth factor, 5 μM Y-27632, 50 μM lipoic acid, 4 mM L-glutamine and DMEM / F12 medium;

[0022] The culture medium component II consists of the following components: 100 μM N-acetyl cysteine, 100 μM vitamin C, 5 μg / L thrombospondin-1, 5 mg / L dipalmitoyl phosphatidylcholine, 10 ng / mL epidermal growth factor, 50 ng / mL insulin-like growth factor-1 and DMEM / F12 medium.

[0023] Then, the present application provides a method for efficiently obtaining umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:

[0024] (1) Seed the multi-functional stem cells into a culture vessel, and culture to 3-6 passages using a culture medium containing exosome-free FBS;

[0025] (2) Replace the culture medium with the culture medium component I as claimed in claim 1, and culture at 37°C, 5% CO2 and 21% O2 for 24 hours;

[0026] (3) Replace the culture medium with the culture medium component II as claimed in claim 1, and culture at 37°C, 5% CO2 and 3% O2 for 24 hours;

[0027] (4) Collect the culture medium and extract the exosomes by differential centrifugation.

[0028] Preferably, the differential centrifugation method comprises the following steps:

[0029] (1) Place the collected culture medium into a centrifuge, and centrifuge at 300g for 10 minutes at 4°C;

[0030] (2) Adjust the centrifuge to 2000g and centrifuge for 20 minutes;

[0031] (3) Adjust the centrifuge to 10000g and centrifuge for 30 minutes, discard the precipitate and collect the supernatant;

[0032] (4) Place the supernatant into a centrifuge, and centrifuge at 4000g for 15 minutes;

[0033] (5) Adjust the centrifuge to 100000g and ultracentrifuge for 70 minutes, discard the supernatant and resuspend the precipitate with PBS;

[0034] (6) Centrifuge again at 10000g, discard the supernatant, resuspend the precipitate with PBS and obtain the exosomes.

[0035] Finally, the application provides a use of a medium combination in the preparation of a preparation for improving the skin repair ability of umbilical cord mesenchymal stem cell exosomes, wherein the medium combination is composed of the above-mentioned medium component I and the above-mentioned medium component II.

[0036] Preferably, the improvement of the skin repair ability of umbilical cord mesenchymal stem cell exosomes is reflected in the improvement of the ability of umbilical cord mesenchymal stem cell exosomes to promote the proliferation and migration of skin fibroblasts.

[0037] The application has the following beneficial effects:

[0038] The application uses two specifically designed medium components (medium component I and medium component II) and achieves the synergistic effect of multiple signal pathways through specific sequence use (activation first and stimulation later), thereby significantly improving the exosome secretion capacity of multi-functional stem cells (such as umbilical cord mesenchymal stem cells). Experimental results show that, compared with the traditional method, the use of the preparation of the application can increase the exosome secretion amount by tens of times. This effect breaks through the technical bottleneck of insufficient exosome secretion in the prior art and provides a feasible solution for large-scale preparation of high-concentration exosomes.

[0039] The exosomes prepared by the application not only significantly increase in quantity, but also exhibit stronger biological activity. Experimental verification shows that these exosomes can significantly promote the proliferation and migration ability of skin fibroblasts, thereby exhibiting higher application value in the fields of tissue repair and regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure for detecting the morphology and marker protein expression of the exosomes prepared in Example 1;

[0041] (a) TEM image: showing that the exosomes prepared by the application exhibit a classic cup-shaped vesicle structure, verifying the morphology of the exosomes;

[0042] (b) Western blot image: showing that the CD63 and CD9 protein markers of the exosomes prepared by the application are both positive, confirming the effectiveness of the exosomes;

[0043] Figure 2 Promotion effect of the exosomes prepared in Example 1 on the proliferation ability of skin fibroblasts;

[0044] The absorbance value of the skin fibroblasts treated by the experimental group II (exosomes of Example 1 of the application) is significantly higher than that of the experimental group I (exosomes of Comparative Example 1), indicating that the exosomes prepared in Example 1 have a stronger promotion effect on the proliferation ability of cells;

[0045] Figure 3Promotion effect of the exosomes prepared in Example 1 on the migration ability of skin fibroblasts;

[0046] The number of skin fibroblasts after treatment of the experimental group II (exosomes of Example 1 of the present application) was significantly higher than that of the experimental group I (exosomes of Comparative Example 1), indicating that the exosomes prepared in Example 1 had a stronger promotion effect on the migration ability of cells. DETAILED DESCRIPTION

[0047] The present application will be further described in detail below in conjunction with examples.

[0048] Example 1: Medium and method for increasing the secretion ability of multifunctional stem cell exosomes

[0049] The medium of this Example 1 is composed of medium component I and medium component II;

[0050] The composition of the medium component I is as follows:

[0051] 0.5 ng / mL transforming growth factor-β (TGF-β), 50 ng / mL hepatocyte growth factor (HGF), 5 μM Y-27632, 50 μM thioctic acid, 4 mM L-glutamine and DMEM / F12 medium;

[0052] The composition of the medium component II is as follows:

[0053] 100 μM N-acetylcysteine, 100 μM vitamin C, 5 μg / L thrombospondin-1 (TSP-1), 5 mg / L dipalmitoyl phosphatidylcholine, 10 ng / mL epidermal growth factor (EGF), 50 ng / mL insulin-like growth factor-1 (IGF-1) and DMEM / F12 medium.

[0054] The culture method of this Example 1 is as follows:

[0055] (1) The obtained umbilical cord mesenchymal stem cells were inoculated into a culture dish and cultured to P3 generation using DMEM / F12 medium containing 10% exosome-free FBS;

[0056] (2) The medium was replaced with medium component I and cultured in a cell incubator at 37°C, 5% CO2 and 21% O2 for 24 h;

[0057] (3) The medium was replaced with medium component II and cultured in a cell incubator at 37°C, 5% CO2 and 3% O2 for 24 h;

[0058] (4) The medium was collected for exosome extraction.

[0059] The exosome extraction method of this Example 1 is as follows:

[0060] (1) Put the collected culture medium into the centrifuge, 4°C, 300g, centrifuge for 10 minutes;

[0061] (2) Adjust the centrifuge to 2000g and centrifuge for 20 minutes;

[0062] (3) Adjust the centrifuge to 10000g and centrifuge for 30 minutes, discard the precipitate and collect the supernatant;

[0063] (4) Put the supernatant into the centrifuge, 4000g, centrifuge for 15 minutes;

[0064] (5) Adjust the centrifuge to 100000g and ultracentrifuge for 70 minutes, discard the supernatant and resuspend the precipitate with PBS;

[0065] (6) Purify again by centrifugation at 10000g, discard the supernatant, resuspend the precipitate with PBS, and obtain the exosomes.

[0066] Example 2: Medium and method 2 for increasing the exosome secretion capacity of multi-functional stem cells

[0067] The medium of this example 2 is composed of medium component I and medium component II;

[0068] The composition of the medium component I is as follows:

[0069] 0.25 ng / mL transforming growth factor-β (TGF-β), 80 ng / mL hepatocyte growth factor (HGF), 2.5 μM Y-27632, 100 μM thioctic acid, 6 mM L-glutamine and DMEM / F12 medium;

[0070] The composition of the medium component II is as follows:

[0071] 50 μM N-acetylcysteine, 150 μM vitamin C, 7.5 μg / L thrombospondin-1 (TSP-1), 3 mg / L dipalmitoyl phosphatidylcholine, 5 ng / mL epidermal growth factor (EGF), 75 ng / mL insulin-like growth factor-1 (IGF-1) and DMEM / F12 medium.

[0072] The culture method and exosome extraction method are the same as in example 1.

[0073] Example 3: Medium and method 3 for increasing the exosome secretion capacity of multi-functional stem cells

[0074] The medium of this example 3 is composed of medium component I and medium component II;

[0075] The composition of the medium component I is as follows:

[0076] 1 ng / mL transforming growth factor-β (TGF-β), 30 ng / mL hepatocyte growth factor (HGF), 7.5 μM Y-27632, 25 μM lipoic acid, 2 mM L-glutamine and DMEM / F12 medium;

[0077] The composition of medium component II is as follows:

[0078] 150 μM N-acetyl cysteine, 50 μM vitamin C, 2.5 μg / L thrombospondin-1 (TSP-1), 8 mg / L dipalmitoyl phosphatidylcholine, 15 ng / mL epidermal growth factor (EGF), 25 ng / mL insulin-like growth factor-1 (IGF-1) and DMEM / F12 medium.

[0079] The culture method and the exosome extraction method are the same as in Example 1.

[0080] Example 4

[0081] A. TEM detection of exosome morphology

[0082] (1) Take 10 μl of the exosomes prepared in Example 1 and drop them onto a copper mesh to precipitate for 1 min, and use filter paper to absorb the supernatant;

[0083] (2) Drop 10 μl of uranyl acetate onto a copper mesh to precipitate for 1 min, and use filter paper to absorb the supernatant;

[0084] (3) After drying at room temperature, perform electron microscope imaging at 100 kv.

[0085] B. Detection of exosome marker protein expression

[0086] (1) Mix the exosomes prepared in Example 1 with RIPA buffer at a ratio of 1:10, shake gently, and incubate on ice for 30 min;

[0087] (2) Centrifuge at 12000 g for 15 min, collect the supernatant, and use a BCA protein quantification kit to determine the protein concentration;

[0088] (3) Add the exosome protein sample to the loading buffer and boil for 10 min to obtain the protein sample, and use the cell protein sample of the umbilical cord mesenchymal stem cells as a control;

[0089] (4) Configure a 12% SDS-PAGE gel, and after loading, perform electrophoretic separation;

[0090] (5) Use a transmembrane device to transfer the proteins in the gel to a PVDF membrane at 4°C and 250 mA;

[0091] (6) Put the PVDF membrane into the PBS solution of 5% skim milk, and seal at room temperature for 1 h, so as to reduce non-specific binding;

[0092] (7) Incubate the membrane with diluted CD63 and CD9 primary antibodies at 4°C overnight;

[0093] (8) Wash the membrane with PBS-T for 3 times, each for 5 minutes, to remove unbound antibodies;

[0094] (9) Dilute and add HRP-labeled secondary antibodies to the membrane, and incubate at room temperature for 1 h;

[0095] (10) After washing the membrane with PBS-T for 3 times, put the membrane into ECL luminescent substrate for development, and then detect using a chemiluminescence imaging system.

[0096] The results of the experimental detection are shown in Figure 1 As can be seen from Figure 1 (a), the exosomes prepared by the present application present a classic cup-shaped vesicular structure, and as can be seen from Figure 1 (b), the CD63 and CD9 of the exosome sample are both positive. The above results show that the method of the present application successfully extracts umbilical cord mesenchymal stem cell exosomes.

[0097] Comparative Example 1

[0098] The culture method of this comparative example 1 is as follows:

[0099] (1) The obtained umbilical cord mesenchymal stem cells were inoculated into culture dishes, and cultured to P3 generation using DMEM / F12 medium containing 10% exosome-free FBS;

[0100] (2) The culture medium was replaced with serum-free DMEM medium, and cultured in a cell incubator at 37°C, 5% CO2, and 21% O2 for 48 h;

[0101] (3) The culture medium was collected for exosome extraction.

[0102] The exosome extraction method is the same as that of Example 1.

[0103] Comparative Example 2

[0104] The culture method of this comparative example 2 is as follows:

[0105] (1) The obtained umbilical cord mesenchymal stem cells were inoculated into culture dishes, and cultured to P3 generation using DMEM / F12 medium containing 10% exosome-free FBS;

[0106] (2) The culture medium was replaced with medium component I, and cultured in a cell incubator at 37°C, 5% CO2, and 21% O2 for 24 h;

[0107] (3) replace the medium with medium component I, and place in a cell culture incubator at 37°C, 5% CO2, 3% O2 for 24 h;

[0108] (4) collect the medium for exosome extraction.

[0109] The exosome extraction method is the same as that of Example 1.

[0110] The culture method of Comparative Example 3 is as follows:

[0111] (1) the obtained umbilical cord mesenchymal stem cells are inoculated into a culture dish, and cultured to P3 generation using DMEM / F12 medium containing 10% exosome-free FBS;

[0112] (2) replace the medium with medium component II, and place in a cell culture incubator at 37°C, 5% CO2, 21% O2 for 24 h;

[0113] (3) replace the medium with medium component II, and place in a cell culture incubator at 37°C, 5% CO2, 3% O2 for 24 h;

[0114] (4) collect the medium for exosome extraction.

[0115] The exosome extraction method is the same as that of Example 1.

[0116] Comparative Example 4

[0117] (1) the obtained umbilical cord mesenchymal stem cells are inoculated into a culture dish, and cultured to P3 generation using DMEM / F12 medium containing 10% exosome-free FBS;

[0118] (2) replace the medium with medium component II, and place in a cell culture incubator at 37°C, 5% CO2, 21% O2 for 24 h;

[0119] (3) replace the medium with medium component I, and place in a cell culture incubator at 37°C, 5% CO2, 3% O2 for 24 h;

[0120] (4) collect the medium for exosome extraction.

[0121] The exosome extraction method is the same as that of Example 1.

[0122] Example 5

[0123] (1) take out 1 μl of the exosomes obtained in Example 1, Example 2 and Example 3, and Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4, and dilute 1000 times;

[0124] (2) Diluted exosomes were used to measure the particle size distribution and concentration of exosomes obtained by different methods using a nanoparticle tracking analyzer.

[0125] The results obtained are shown in Table 1.

[0126] Group Average particle size (nm) Particle concentration (particles / mL) Example 1 122.61±7.91 1.48×10*10 Example 2 115.49±6.98 1.02×10*10 Example 3 117.76±7.79 1.13×10*10 Comparative Example 1 105.8±6.60 2.31×10*8 Comparative Example 2 111.56±8.31 1.56×10*9 Comparative Example 3 108.57±8.06 2.12×10*9 Comparative Example 4 112.31±6.79 3.36×10*9

[0127] First, from the perspective of average particle size, although the average particle size of Examples 1-3 is higher than that of the comparative examples without medium stimulation and hypoxic stimulation, the difference with the remaining comparative examples is not large. Therefore, we know that the treatment method of the present application has only a small effect on the change in the particle size of stem cell exosomes.

[0128] From the perspective of exosome concentration, compared with Comparative Example 1 without medium stimulation and hypoxic stimulation, Comparative Example 2 using medium component I alone and hypoxic stimulation increased by 6.75 times, Comparative Example 3 using medium component II alone and hypoxic stimulation increased by 9.17 times, and Comparative Example 4 using medium component II first and then medium component I and hypoxic stimulation increased by 14.54 times.

[0129] And Example 1 using medium component I first and then medium component II and hypoxic stimulation increased by 64.07 times, Example 2 increased by 44.17 times, and Example 3 increased by 48.94 times.

[0130] From the above results, it can be seen that the medium component alone and the wrong stimulation sequence cannot fully play the effect of stimulating umbilical cord mesenchymal stem cell exosomes. And using medium component I to activate stem cells first and then using medium component II and hypoxic stimulation to secrete umbilical cord stem cells (using component I first to activate and then using component II to stimulate), can produce a synergistic promotion effect through the interaction of multiple signals, thereby greatly promoting the secretion of exosomes.

[0131] Example 6

[0132] The application of exosomes in skin regeneration and repair is one of the main uses. Therefore, the effect of the exosomes prepared by the present application on promoting the proliferation and migration ability of skin fibroblasts was detected.

[0133] (A) Ability to promote skin fibroblast proliferation

[0134] (1) First, the exosomes prepared by Example 1 and Comparative Example 1 were diluted with DMEM / F12 medium to a concentration of 1 x 10*9 particles / mL for standby;

[0135] (2) The human skin fibroblasts were prepared into a cell suspension, inoculated into a 96-well plate, and placed in a cell incubator for overnight culture to allow the cells to adhere completely;

[0136] (3) The control group was added with new DMEM / F12 culture medium, the experimental group I was added with the exosome solution prepared in step (1) of the comparative example 1, and the experimental group II was added with the exosome solution prepared in example 1, and 5 repeated holes were set for each experimental group;

[0137] (4) The 96-well plate was placed in a cell incubator for continued culture for 48 hours, and then the absorbance values of different experimental groups were detected by using CCC-8.

[0138] (B) Ability to promote skin fibroblast migration

[0139] (1) First, the exosomes prepared in example 1 and comparative example 1 were diluted with DMEM / F12 culture medium to a concentration of 1×10*9 particles / mL for standby use;

[0140] (2) The comparative example 1 exosome solution and the example 1 exosome solution were used for 24h pretreatment, and the control group was pretreated with DMEM / F12 culture medium;

[0141] (3) After the pretreated cells were digested, a cell suspension was prepared using serum-free DMEM / F12 culture medium, 200 μl of the cell suspension was inoculated into the upper chamber of a Transwell chamber, and 600 μl of DMEM / F12 culture medium containing 10% fetal bovine serum was added into the lower chamber of the Transwell chamber;

[0142] (4) The Transwell chamber was placed in a cell incubator for continued culture for 24h;

[0143] (5) After the culture was completed, the Transwell chamber was taken out, washed with PBS to remove the cells that did not migrate, fixed with formaldehyde, and then stained with crystal violet;

[0144] (6) After washing with PBS, 5 fields of view were randomly selected for photography and counting under a microscope.

[0145] The detection results of the cell proliferation experiment are shown in Table 1. Figure 2 The absorbance value of the cells in the control group was 1.172±0.025, the absorbance value of the cells in the experimental group I was 1.304±0.023, and the absorbance value of the cells in the experimental group II was 1.587±0.027. The results show that the exosomes prepared in example 1 of the present application have a significantly better effect on promoting the proliferation of human skin fibroblasts than the exosomes prepared in comparative example 1.

[0146] The detection results of the cell migration experiment are shown in Table 2.Figure 3 As shown, similar to the results of cell proliferation detection, the number of migration of human skin fibroblasts treated with the exosomes of Example 1 was significantly higher than that of human skin fibroblasts treated with the exosomes of Comparative Example 1. The results show that the exosomes prepared in Example 1 of the present application have a stronger promoting effect on the migration ability of human skin fibroblasts.

[0147] The above results show that the exosomes stimulated by the culture method of the present application have stronger biological activity and can more effectively promote the proliferation and migration ability of skin fibroblasts, thereby making the exosomes exhibit significant advantages in skin repair and regeneration.

Claims

1. A formulation for increasing the exosome secretion capacity of pluripotent stem cells, characterized in that, The formulation consists of culture medium component I and culture medium component II; The culture medium component I consists of the following components: 0.25 ng / mL to 1 ng / mL transforming growth factor-β, 30 ng / mL to 80 ng / mL hepatocyte growth factor, 2.5 μM to 7.5 μM Y-27632, 50 μM to 100 μM lipoic acid, 2 mM to 6 mM L-glutamine and DMEM / F12 medium; The culture medium component II consists of the following components: 50 μM to 150 μM N-acetylcysteine, 50 μM to 150 μM vitamin C, 2.5 μg / L to 7.5 μg / L thrombin-sensitive protein-1, 3 mg / L to 8 mg / L dipalmitoylphosphatidylcholine, 5 ng / mL to 15 ng / mL epidermal growth factor, 25 ng / mL to 75 ng / mL insulin-like growth factor-1 and DMEM / F12 medium; The pluripotent stem cells are umbilical cord mesenchymal stem cells.

2. The formulation according to claim 1, characterized in that, The culture medium component I consists of the following components: 0.5 ng / mL transforming growth factor-β, 50 ng / mL hepatocyte growth factor, 5 μM Y-27632, 50 μM lipoic acid, 4 mM L-glutamine and DMEM / F12 medium. The culture medium component II consists of the following components: 100 μM N-acetylcysteine, 100 μM vitamin C, 5 μg / L thrombin-sensitive protein-1, 5 mg / L dipalmitoylphosphatidylcholine, 10 ng / mL epidermal growth factor, 50 ng / mL insulin-like growth factor-1 and DMEM / F12 medium.

3. A method for culturing exosomes to improve their secretory capacity and biological properties, characterized in that, The method includes the following steps: (1) Seed multipotent stem cells into culture dishes and culture them for 3-6 generations using a medium containing exosome-free FBS; (2) Replace the culture medium with culture medium component I as described in claim 1, and culture for 24 hours under conventional cell culture conditions; (3) Replace the culture medium with culture medium component II as described in claim 1, and culture for 24 hours under hypoxic cell culture conditions; (4) Collect the culture medium for exosome extraction; The pluripotent stem cells are umbilical cord mesenchymal stem cells.

4. The cultivation method according to claim 3, characterized in that, The biological function of the exosomes is to treat skin injuries. The standard cell culture conditions are 37°C, 5% CO2, and 21% O2. The hypoxic cell culture conditions were 37°C, 5% CO2, and 3% O2.

5. The cultivation method according to claim 4, characterized in that, The culture medium component I consists of the following components: 0.5 ng / mL transforming growth factor-β, 50 ng / mL hepatocyte growth factor, 5 μM Y-27632, 50 μM lipoic acid, 4 mL M glutamine and DMEM / F12 medium. The culture medium component II consists of the following components: 100 μM N-acetylcysteine, 100 μM vitamin C, 5 μg / L thrombin-sensitive protein-1, 5 mg / L dipalmitoylphosphatidylcholine, 10 ng / mL epidermal growth factor, 50 ng / mL insulin-like growth factor-1 and DMEM / F12 medium.

6. A method for efficiently obtaining umbilical cord mesenchymal stem cell exosomes, characterized in that, The method includes the following steps: (1) Seed multipotent stem cells into culture dishes and culture them for 3-6 generations using a medium containing exosome-free FBS; (2) Replace the culture medium with culture medium component I as described in claim 1, and culture for 24 hours at 37°C, 5% CO2, and 21% O2. (3) Replace the culture medium with culture medium component II as described in claim 1, and culture for 24 hours at 37°C, 5% CO2, and 3% O2. (4) Collect the culture medium and extract exosomes using differential centrifugation.

7. The method according to claim 6, characterized in that, The differential centrifugation method includes the following steps: (1) Place the collected culture medium into a centrifuge, centrifuge at 4°C and 300g for 10 minutes; (2) Adjust the centrifuge to 2000g and centrifuge for 20 minutes; (3) Adjust the centrifuge to 10000g, centrifuge for 30 minutes, discard the precipitate, and collect the supernatant; (4) Place the supernatant in a centrifuge, 4000g, and centrifuge for 15 minutes; (5) Adjust the centrifuge to 100,000g, centrifuge at ultra-high speed for 70 minutes, discard the supernatant, and add PBS to resuspend the precipitate; (6) Centrifuge again at 10000g, discard the supernatant, add PBS to resuspend the precipitate, and obtain exosomes.

8. The application of a culture medium combination in the preparation of a formulation that enhances the skin repair capacity of umbilical cord mesenchymal stem cell exosomes, characterized in that, The culture medium composition consists of culture medium component I as described in claim 1 and culture medium component II as described in claim 1.

9. The application according to claim 8, characterized in that, The improvement in the skin repair capacity of umbilical cord mesenchymal stem cell exosomes is reflected in the ability of umbilical cord mesenchymal stem cell exosomes to promote the proliferation and migration of skin fibroblasts.

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