Preparation for improving exosome secretion capacity of multifunctional stem cells and application of preparation

Abstract

The invention relates to a preparation for improving exosome secretion capacity of multifunctional stem cells and application of the preparation, and belongs to the technical field of cell culture. The preparation is composed of a culture medium component I and a culture medium component II, and the component I contains transforming growth factors-beta, hepatocyte growth factors and the like and is used for activating stem cells; the component II contains N-acetylcysteine, vitamin C and the like, and exosome secretion is promoted in combination with a low-oxygen condition. By using the two components in a specific sequence, the exosome secretion amount and the biological activity of the umbilical cord mesenchymal stem cells can be remarkably improved. Compared with a traditional method, the method has the advantages that the secretion amount of the exosome treated by the method is increased by dozens of times, and the capability of promoting proliferation and migration of skin fibroblasts is remarkably enhanced, so that the method has important application value in the fields of skin repair, tissue regeneration and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a preparation for increasing the exosome secretion ability of pluripotent stem cells and its application. Background Art

[0002] With the continuous development of stem cell therapy technology, exosomes of stem cells, as an important biological material, are widely used in the fields of disease treatment, tissue repair, and immunomodulation. Exosomes are small vesicles secreted by cells, which can carry molecules such as proteins, RNAs, and lipids, and have good biocompatibility and immunomodulatory effects. Due to their unique biological properties, exosomes have shown great potential in regenerative medicine, cell therapy, anti-inflammation, anti-tumor, etc.

[0003] However, although exosomes have broad prospects in clinical applications, the current preparation of exosomes still faces some challenges. Especially in terms of improving the exosome secretion amount, there are still certain bottlenecks in the existing technology. Traditional methods mainly rely on increasing the cell number or changing the culture conditions to improve exosome secretion, but these methods are often limited by factors such as cell type, culture medium components, and secretion pathways. Therefore, how to design a multifunctional exosome secretion-promoting preparation for stem cells to effectively promote the exosome secretion ability of stem cells has become an important research topic in the current biomedical field. Summary of the Invention

[0004] The present invention aims to solve the problems of low exosome secretion amount, insufficient biological activity, and low preparation efficiency in the prior art, and provides a preparation that can efficiently and reliably promote the exosome secretion of pluripotent stem cells, as well as related preparation methods and applications.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] First, the present invention provides a preparation for increasing the exosome secretion ability of pluripotent stem cells, and the preparation is composed of culture medium component I and culture medium component II;

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

[0008] The culture medium component II consists of the following components: 50 μM to 150 μM of N-acetylcysteine, 50 μM to 150 μM of vitamin C, 2.5 μg / L to 7.5 μg / L of thrombospondin-1, 3 mg / L to 8 mg / L of dipalmitoylphosphatidylcholine, 5 ng / mL to 15 ng / mL of epidermal growth factor, 25 ng / mL to 75 ng / mL of insulin-like growth factor-1, and DMEM / F12 culture medium.

[0009] Preferably, the pluripotent stem cells are umbilical cord mesenchymal stem cells.

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

[0011] The culture medium component II consists of the following components: 100 μM of N-acetylcysteine, 100 μM of vitamin C, 5 μg / L of thrombospondin-1, 5 mg / L of dipalmitoylphosphatidylcholine, 10 ng / mL of epidermal growth factor, 50 ng / mL of insulin-like growth factor-1, and DMEM / F12 culture medium.

[0012] Secondly, the present invention provides a culture method for improving the exosome secretion ability and exosome biological performance of pluripotent stem cells. The method includes the following steps:

[0013] (1) Inoculate the pluripotent stem cells into a culture vessel and culture them with a culture medium containing exosome-free FBS for 3 to 6 passages;

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

[0015] (3) Replace the culture medium with the above-mentioned culture medium component II and culture for 24 hours under hypoxic cell culture conditions;

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

[0017] Preferably, the pluripotent stem cells are umbilical cord mesenchymal stem cells;

[0018] The exosome biological function is the function of exosomes in treating skin injuries;

[0019] The conventional cell culture conditions are 37 °C, 5% CO 2 , 21% O 2 ;

[0020] The hypoxic cell culture conditions are 37°C, 5% CO 2 , 3% O 2 .

[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 culture medium;

[0022] The culture medium component II consists of the following components: 100 μM N-acetylcysteine, 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 culture medium.

[0023] Then, the present invention provides a method for efficiently obtaining umbilical cord mesenchymal stem cell exosomes, and the method includes the following steps:

[0024] (1) Inoculate pluripotent stem cells into a culture vessel and culture them with a culture medium containing exosome-free FBS for 3 to 6 passages;

[0025] (2) Replace the culture medium with the culture medium component I as described in claim 1, and culture for 24 hours under the conditions of 37°C, 5% CO 2 , 21% O 2 ;

[0026] (3) Replace the culture medium with the culture medium component II as described in claim 1, and culture for 24 hours under the conditions of 37°C, 5% CO 2 , 3% O 2 ;

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

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

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

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

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

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

[0033] (5) The centrifuge was adjusted to 100,000 g and centrifuged at ultra-high speed for 70 minutes. The supernatant was discarded, and PBS was added to resuspend the precipitate.

[0034] (6) It was centrifuged again at 10,000 g for purification. The supernatant was discarded, and PBS was added to resuspend the precipitate to obtain exosomes.

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

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

[0037] The beneficial effects of the present invention are as follows:

[0038] The present invention uses two specifically designed culture medium components (culture medium component I and culture medium component II), and through their specific sequential use (activation first and then stimulation), the synergistic effect of multiple signal pathways is achieved, significantly improving the exosome secretion ability of pluripotent stem cells (such as umbilical cord mesenchymal stem cells). Experimental results show that compared with traditional methods, the use of the preparation of the present invention can increase the exosome secretion amount by dozens of times. This effect breaks through the technical bottleneck of insufficient exosome secretion amount in the prior art and provides a feasible solution for large-scale preparation of high-concentration exosomes.

[0039] The exosomes prepared by the present invention not only significantly increase in quantity but also show stronger biological activity. Experimental verification shows that these exosomes can significantly promote the proliferation and migration ability of skin fibroblasts, thus showing higher application value in the fields of tissue repair, regenerative medicine, etc. Description of the Drawings

[0040] Figure 1 It is a detection graph of the morphology and marker protein expression of the exosomes prepared in Example 1;

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

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

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

[0044] The absorbance value of the skin fibroblasts treated with Experimental Group II (the exosomes of Example 1 of the present invention) was significantly higher than that of Experimental Group I (the exosomes of Comparative Example 1), indicating that the exosomes prepared in Example 1 had a stronger promoting effect on cell proliferation ability;

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

[0046] The number of migrated skin fibroblasts treated with Experimental Group II (the exosomes of Example 1 of the present invention) was significantly higher than that of Experimental Group I (the exosomes of Comparative Example 1), indicating that the exosomes prepared in Example 1 had a stronger promoting effect on cell migration ability. Detailed implementation manners

[0047] The following further elaborates on this application in conjunction with examples.

[0048] Example 1: Medium and Method 1 for Increasing the Secretion Ability of Multipotent Stem Cell Exosomes

[0049] The medium of this Example 1 consisted of Medium Component I and Medium Component II;

[0050] Among them, the composition of Medium Component I was 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 lipoic acid, 4 mM L-glutamine, and DMEM / F12 medium;

[0052] The composition of Medium Component II was 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 was as follows:

[0055] (1) Inoculate the obtained umbilical cord mesenchymal stem cells into a culture dish and culture them to passage P3 using DMEM / F12 medium containing 10% exosome-free FBS;

[0056] (2) Replace the medium with Medium Component I, place it in a cell culture incubator, and culture it for 24 h under the conditions of 37 °C, 5% CO 2 , 21% O 2 .

[0057] (3) Replace the culture medium with Culture Medium Component II, place it in a cell incubator at 37°C and 5% CO 2 , 3% O 2 and culture for 24 h under these conditions;

[0058] (4) Collect the culture medium for exosome extraction.

[0059] The method for extracting exosomes in Example 1 is as follows:

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

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

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

[0063] (4) Place the supernatant in a centrifuge and centrifuge at 4000 g for 15 minutes;

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

[0065] (6) Centrifuge again at 10000 g for purification, discard the supernatant, and resuspend the precipitate with PBS to obtain exosomes.

[0066] Example 2: Culture Medium and Method 2 for Increasing the Secretion Capacity of Multipotent Stem Cell Exosomes

[0067] The culture medium in this Example 2 is composed of Culture Medium Component I and Culture Medium Component II;

[0068] Among them, the composition of Culture 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 lipoic acid, 6 mM L-glutamine and DMEM / F12 culture medium;

[0070] The composition of Culture 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 culture medium.

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

[0073] Example 3: Medium and Method 3 for Increasing the Secretion Capacity of Multipotent Stem Cell Exosomes

[0074] The medium of this Example 3 is composed of Medium Component I and Medium Component II;

[0075] Among them, the composition of 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-acetylcysteine, 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 exosome extraction method are the same as those in Example 1.

[0080] Example 4

[0081] A. TEM detection of exosome morphology

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

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

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

[0085] B. Detection of the expression of exosome marker proteins

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

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

[0088] (3) Add the exosome protein sample to the loading buffer and boil for 10 minutes to obtain the protein sample. At the same time, use the cellular protein sample of umbilical cord mesenchymal stem cells as a control.

[0089] (4) Prepare a 12% SDS-PAGE gel. After loading the sample, perform electrophoresis separation.

[0090] (5) At 4 °C and 250 mA, use a membrane transfer device to transfer the proteins in the gel to a PVDF membrane.

[0091] (6) Place the PVDF membrane into a PBS solution containing 5% skim milk and block at room temperature for 1 h to reduce non-specific binding.

[0092] (7) Use diluted anti-CD63 and anti-CD9 primary antibodies and incubate the membrane overnight at 4 °C.

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

[0094] (9) Use an HRP-labeled secondary antibody, dilute and add it to the membrane, and incubate at room temperature for 1 h.

[0095] (10) After washing the membrane 3 times with PBS-T, place the membrane into an ECL chemiluminescent substrate for development, and then use a chemiluminescent imaging system for detection.

[0096] The results of the experimental detection are as Figure 1 shown. From Figure 1 (a), it can be seen that the exosomes prepared by the present invention present a classic cup-shaped vesicle structure. From Figure 1 (b), it can be seen that both CD63 and CD9 of the exosome sample are positive. The above results indicate that the method of the present invention 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) Inoculate the obtained umbilical cord mesenchymal stem cells into a culture dish and culture them to passage P3 using DMEM / F12 medium containing 10% exosome-free FBS.

[0100] (2) Replace the medium with serum-free DMEM medium and place it in a cell culture incubator at 37 °C, 5% CO 2 , 21% O 2 and culture for 48 h.

[0101] (3) Collect the medium for exosome extraction.

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

[0103] Comparative Example 2

[0104] The culture method of this Comparative Example 2 is as follows:

[0105] (1) Inoculate the obtained umbilical cord mesenchymal stem cells into a culture dish, and culture them to passage P3 using DMEM / F12 medium containing 10% exosome-free FBS;

[0106] (2) Replace the medium with Medium Component I, place it in a cell culture incubator, and culture it for 24 h under the conditions of 37 °C, 5% CO 2 , 21% O 2 ;

[0107] (3) Replace the medium with Medium Component I, place it in a cell culture incubator, and culture it for 24 h under the conditions of 37 °C, 5% CO 2 , 3% O 2 ;

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

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

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

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

[0112] (2) Replace the medium with Medium Component II, place it in a cell culture incubator, and culture it for 24 h under the conditions of 37 °C, 5% CO 2 , 21% O 2 ;

[0113] (3) Replace the medium with Medium Component II, place it in a cell culture incubator, and culture it for 24 h under the conditions of 37 °C, 5% CO 2 , 3% O 2 ;

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

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

[0116] Comparative Example 4

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

[0118] (2) Replace the medium with Medium Component II, place it in a cell culture incubator, and culture it at 37 °C, 5% CO2 , 21% O 2 Cultivate for 24 h under the condition of

[0119] (3) Replace the culture medium with Culture Medium Component I, place it in a cell culture incubator at 37 °C, 5% CO 2 , 3% O 2 Cultivate for 24 h under the condition of

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

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

[0122] Example 5

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

[0124] (2) Use a nanoparticle tracking analyzer to measure the particle size distribution and concentration of the exosomes obtained by different methods for the diluted exosomes.

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

[0126] Grouping 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 the average particle size, it can be seen that although the average particle sizes of Examples 1 - 3 are higher than those of the comparative examples without medium stimulation and hypoxia stimulation, the gap with the other comparative examples is not large. Therefore, we can know that the treatment method of the present invention has only a small impact 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 hypoxia stimulation, Comparative Example 2 using only Culture Medium Component I and hypoxia stimulation increased by 6.75 times, Comparative Example 3 using only Culture Medium Component II and hypoxia stimulation increased by 9.17 times, and Comparative Example 4 using Culture Medium Component II stimulation first and then Culture Medium Component I and hypoxia stimulation increased by 14.54 times;

[0129] While Example 1 using Culture Medium Component I stimulation first and then Culture Medium Component II and hypoxia stimulation increased by 64.07 times, Example 2 increased by 44.17 times, and Example 3 increased by 48.94 times.

[0130] As can be seen from the above results, the individual culture medium components and the wrong stimulation order cannot fully exert the effect of stimulating umbilical cord mesenchymal stem cell exosomes. However, when stem cells are activated with culture medium component I first and then umbilical cord stem cells are stimulated with culture medium component II and hypoxia (activate with component I first and then stimulate with component II), a synergistic promoting effect can be produced through the interaction of multiple signals, thus greatly promoting the secretion of exosomes.

[0131] Example 6

[0132] One of the main uses of exosomes is their application in skin regeneration and repair. Therefore, in this example, the effects of the exosomes prepared by the present invention on promoting the proliferation and migration of skin fibroblasts were detected.

[0133] (A) Ability to promote the proliferation of skin fibroblasts

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

[0135] (2) Prepare a cell suspension of human skin fibroblasts, inoculate it into a 96-well plate, place it in a cell culture incubator, and culture it overnight to allow the cells to adhere completely;

[0136] (3) Add fresh DMEM / F12 medium to the control group, add the exosome solution prepared in Comparative Example 1 diluted in step (1) to Experimental Group I, and add the exosome solution prepared in Example 1 to Experimental Group II. Set 5 replicate wells for each experimental group;

[0137] (4) Place the 96-well plate in a cell culture incubator and continue to culture for 48 hours. Then, use CCC-8 to detect the absorbance values of different experimental groups.

[0138] (B) Ability to promote the migration of skin fibroblasts

[0139] (1) First, dilute the exosomes prepared in Example 1 and Comparative Example 1 with DMEM / F12 medium to a concentration of 1×10*9 particles / mL for standby;

[0140] (2) Pretreat with the exosome solution of Comparative Example 1 and the exosome solution of Example 1 for 24 hours, and pretreat the control group with DMEM / F12 medium;

[0141] (3) After digesting the pretreated cells, prepare a cell suspension using serum-free DMEM / F12 medium. Inoculate 200 μl of the cell suspension into the upper chamber of a Transwell insert, and add 600 μl of DMEM / F12 medium containing 10% fetal bovine serum to the lower chamber of the Transwell insert.

[0142] (4) Place the Transwell insert in a cell culture incubator and continue culturing for 24 h.

[0143] (5) After the culture is completed, take out the Transwell insert, wash away the non-migrated cells with PBS, fix with formaldehyde, and stain with crystal violet.

[0144] (6) After washing with PBS, randomly select 5 fields of view using a microscope for photographing and counting.

[0145] The detection results of the cell proliferation experiment are as Figure 2 shown. Among them, the absorbance value of the control group cells is 1.172 ± 0.025, the absorbance value of the experimental group I cells is 1.304 ± 0.023, and the absorbance value of the experimental group II cells is 1.587 ± 0.027. This result shows that: the exosomes prepared in Example 1 of the present invention have a significantly better promoting effect on the proliferation ability of human skin fibroblasts than the exosomes prepared in Comparative Example 1.

[0146] The detection results of the cell migration experiment are as Figure 3 shown. Similar to the results of the cell proliferation detection, the migration number of human skin fibroblasts treated with the exosomes of Example 1 is significantly higher than that of human skin fibroblasts treated with the exosomes of Comparative Example 1. This result shows that: the exosomes prepared in Example 1 of the present invention 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 invention have stronger biological activity, can more effectively promote the proliferation and migration ability of skin fibroblasts, and thus show significant advantages in skin repair and regeneration.

Claims

1. A preparation for increasing the exosome secretion capacity of pluripotent stem cells, characterized in that: The preparation consists of culture medium component I and culture medium component II; The medium component I is composed 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 is composed 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-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 culture medium.

2. The preparation according to claim 1, characterized in that The multipotent stem cells are umbilical cord mesenchymal stem cells.

3. The preparation according to claim 2, characterized in that The medium component I is composed 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 medium component II consists of the following components: 100 μM N-acetylcysteine, 100 μM vitamin C, 5 μg / L thrombin-1, 5 mg / L dipalmitoylphosphatidylcholine, 10 ng / mL epidermal growth factor, 50 ng / mL insulin-like growth factor-1 and DMEM / F12 medium.

4. A method for cultivating pluripotent stem cells to improve their exosome secretion capacity and exosome biological properties, characterized in that: The method comprises the following steps: (1) Inoculate pluripotent stem cells into a culture vessel and culture them for 3-6 generations using a culture medium containing exosome-free FBS; (2) replacing the culture medium with the medium component I as described in claim 1, and culturing for 24 hours under conventional cell culture conditions; (3) replacing the culture medium with component II of the culture medium as described in claim 1, and culturing the cells under hypoxic cell culture conditions for 24 hours; (4) Collecting culture medium for exosome extraction.

5. The culture method according to claim 4, characterized in that The multipotent stem cells are umbilical cord mesenchymal stem cells; The biological function of exosomes is the function of exosomes in treating skin damage; The conventional cell culture conditions are 37°C, 5% CO2, 21% O2; The hypoxic cell culture conditions are 37° C., 5% CO 2 , and 3% O 2 .

6. The culture method according to claim 5, characterized in that: The medium component I is composed 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 medium component II consists of the following components: 100 μM N-acetylcysteine, 100 μM vitamin C, 5 μg / L thrombin-1, 5 mg / L dipalmitoylphosphatidylcholine, 10 ng / mL epidermal growth factor, 50 ng / mL insulin-like growth factor-1 and DMEM / F12 medium.

7. A method for efficiently obtaining umbilical cord mesenchymal stem cell exosomes, characterized in that: The method comprises the following steps: (1) Inoculate pluripotent stem cells into a culture vessel and culture them for 3-6 generations using a culture medium containing exosome-free FBS; (2) replacing the culture medium with component I of the culture medium as described in claim 1, and culturing for 24 hours at 37° C., 5% CO 2 , 21% O 2 ; (3) replacing the culture medium with component II of the culture medium as described in claim 1, and culturing for 24 hours at 37° C., 5% CO 2 , 3% O 2 ; (4) Collect the culture medium and extract exosomes by differential centrifugation.

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

9. Use of a culture medium combination in preparing a preparation for improving the skin repair ability of umbilical cord mesenchymal stem cell exosomes, characterized in that: The culture medium combination consists of the culture medium component I according to claim 1 and the culture medium component II according to claim 1.

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

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