Method for large-scale acquisition of mesenchymal stem cell exosome with high expression of IL-10 and application of mesenchymal stem cell exosome

Abstract

The invention discloses a method for large-scale acquisition of mesenchymal stem cell exosomes with high expression of IL-10 and application of the mesenchymal stem cell exosomes, and relates to the technical field of umbilical cord mesenchymal stem cell exosomes. The method comprises the following steps: carrying out cross culture on umbilical cord mesenchymal stem cells by adopting complete culture and hunger culture, and collecting a solution discharged from a cell factory when the hunger culture is finished each time, so as to obtain an exosome stock solution; and filtering the exosome stock solution to remove impurities, so as to obtain the mesenchymal stem cell exosome with high expression of IL-10. According to the method, proliferation of the umbilical cord mesenchymal stem cells can be effectively inhibited through alternate culture of the starvation culture medium and the complete culture medium, exosome secretion is stimulated, and high-yield, high-efficiency and stable exosome production is achieved. The mesenchymal stem cell exosome with high expression of IL-10 is applied to clinical treatment of HFpEF, has an anti-apoptosis protection effect on myocardial cells, and effectively promotes angiogenesis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of umbilical cord mesenchymal stem cell-derived exosomes, in particular to a method for large-scale obtaining of mesenchymal stem cell exosomes with high expression of IL-10 and application thereof. BACKGROUND

[0002] Umbilical cord mesenchymal stem cell-derived exosomes (UC-MSC-Exos) refer to extracellular vesicles (about 30-150nm in diameter) secreted from umbilical cord mesenchymal stem cells (UC-MSCs), containing biological active substances such as proteins, nucleic acids (such as mRNA, miRNA), lipids, etc., which can regulate inflammation and repair, promote angiogenesis and tissue regeneration, and participate in cell communication.

[0003] Heart failure with preserved ejection fraction (HFpEF) is a complex cardiovascular disease, and its pathological mechanism involves chronic low-grade inflammation, microvascular dysfunction, and myocardial fibrosis. At present, the treatment of HFpEF is limited, and new treatment strategies need to be developed. IL-10 is a key anti-inflammatory cytokine, which can inhibit the NF-κB and STAT3 pathways, reduce the release of pro-inflammatory factors (TNF-α, IL-6, IL-1β); it can also promote the polarization of regulatory T cells (Treg) and M2 macrophages, and establish an immune tolerant microenvironment. Therefore, IL-10 can significantly enhance the immune regulation and tissue repair capacity of exosomes. The miRNAs (such as miR-21, miR-146a) carried by exosomes can synergize with IL-10 to reduce tissue damage, promote angiogenesis (up-regulation of VEGF) and collagen orderly deposition (reduction of fibrosis), therefore, umbilical cord mesenchymal stem cell-derived exosomes with high expression of IL-10 (UC-MSC-Exos-IL10) is a new type of therapeutic vesicle that enhances the content of IL-10 in exosomes, and is expected to become an effective treatment strategy for HFpEF in clinic.

[0004] With the deepening of the research on exosomes in the treatment of diseases, the clinical demand for exosomes is increasing. However, the traditional exosome preparation method cannot meet the demand of large-scale application; and the exosomes prepared by the conventional method have functional defects such as insufficient expression of anti-inflammatory factors. Therefore, developing a preparation method that can realize industrial production and ensure high expression of anti-inflammatory factors and functional stability of exosomes is of great significance for promoting the clinical transformation and treatment of diseases such as HFpEF. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a method for large-scale obtaining of mesenchymal stem cell exosomes with high expression of IL-10 and application thereof.

[0006] In a first aspect, the present application provides a method for obtaining high IL-10 expression mesenchymal stem cell exosomes on a large scale, comprising:

[0007] The umbilical cord mesenchymal stem cells are subjected to complete culture and starvation culture for cross culture, wherein the complete culture is adding complete culture medium to the cell factory and culturing for a period T1; the starvation culture is adding starvation culture medium to the cell factory and culturing for a period T2; the cross culture is repeated for multiple times;

[0008] And the solution discharged from the cell factory at the end of each starvation culture is collected to obtain an exosome stock solution;

[0009] The exosome stock solution is filtered to remove impurities, thereby obtaining the high IL-10 expression mesenchymal stem cell exosomes.

[0010] Further, in the above method, the complete culture medium is DMEM base medium added with 5-20% serum substitute; and the starvation culture medium is DMEM base medium.

[0011] It is explained that, by alternating the starvation culture medium and the complete culture medium, the proliferation of umbilical cord mesenchymal stem cells can be effectively inhibited, and the exosome secretion can be stimulated, and the exosomes can have the effects of up-regulating the expression of anti-inflammatory factor IL-10 and blocking the secretion of inflammatory factor TNF-α, thereby realizing the production of exosomes with high yield, high efficiency and stability.

[0012] In the above scheme, it can be understood that, on the one hand, by adding 5-20% serum substitute in the DMEM base medium, the serum substitute can provide nutritional support for the proliferation of umbilical cord mesenchymal stem cells. On the other hand, since the starvation culture medium does not contain FBS, the cell proliferation will be inhibited under this condition, which may be due to the stimulation of the starvation state to the cells, so that more exosomes are released for paracrine supplementation (supplementary note: paracrine supplementation refers to the way that cells secrete some substances to surrounding cells to stimulate and promote the growth of surrounding cells), in which case the collected exosomes up-regulate IL-10 and down-regulate TNF-a (for details, see the data support in the examples). Moreover, after the complete culture, the starvation culture mode is switched, and since the starvation culture medium does not contain serum substitute, the collection of the solution discharged from the cell factory at the end of each starvation culture can effectively avoid the influence of protein components in the serum (or nutritional support) on the purity of the exosomes, thereby obtaining an exosome stock solution with relatively higher purity.

[0013] Further preferably, in the above method, the T1 is 1-2 days, the T2 is 1-2 days, and the cross culture is repeated for 6-8 times.

[0014] It can be understood that, in actual operation, the values of T1 and T2 are not strictly limited, and those skilled in the art can adjust the values of T1 and T2 according to needs, but in the present application, the above values are a relatively preferred value range determined by us through long-term repeated experiments, and T1 and T2 slightly less than 1 day or slightly more than 2 days can also achieve the large-scale and stable acquisition of mesenchymal stem cell exosomes. As for the number of repeated cross-cultures, it is not strictly limited to 6-8 times in actual operation, and those skilled in the art can adjust the number appropriately according to needs, and the adjustment does not exceed the protection scope described in the present application.

[0015] As an alternative to the above first aspect, before the cross-culture, further comprising stable culture, wherein the stable culture is that umbilical cord mesenchymal stem cells are inoculated in a cell factory, and complete culture medium is used for stable recovery culture under normal temperature and normal oxygen condition for 6-8 days.

[0016] Description: The cells in the frozen state can recover cell functions through a period of stable culture.

[0017] Further, the cell factory is a stacked structure, and the number of layers is 1-40 layers.

[0018] Further, the inoculation amount of umbilical cord mesenchymal stem cells in the cell factory is 3-5 x 10 6 cells per layer.

[0019] Secondly, the present application also provides the use of the high-IL-10-expressing mesenchymal stem cell exosomes obtained by the method of the above first aspect in the preparation of a drug for treating HFpEF.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] Firstly, the method of the present application can continuously produce exosomes without increasing the cost of raw materials and culture consumables;

[0022] Secondly, the method of the present application uses a starvation medium to harvest exosomes, which can effectively avoid the influence of protein components in serum (or nutritional support) on the purity of exosomes;

[0023] Thirdly, the method of the present application uses alternating culture of starvation medium and complete medium, which can not only effectively inhibit the proliferation of umbilical cord mesenchymal stem cells and stimulate the secretion of exosomes, but also make the exosomes have the functions of up-regulating the expression of anti-inflammatory factor IL-10 and blocking the secretion of inflammatory factor TNF-α, thereby realizing the production of exosomes with high yield, high efficiency and stability and reliability;

[0024] Fourthly, the mesenchymal stem cell exosome obtained by the method has good performance stability.

[0025] Fifthly, the mesenchymal stem cell exosome obtained by the method can be applied to the treatment of HFpEF in clinic, has an anti-apoptotic protective effect on cardiomyocytes, and effectively promotes angiogenesis. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure for umbilical cord mesenchymal stem cell exosome yield under three different culture conditions of Example 1, Comparative Example 1 and Comparative Example 2;

[0027] Figure 2 Figure for detection of umbilical cord mesenchymal stem cell exosome markers CD63, CD81 and CD9 in the embodiments of the present application;

[0028] Figure 3 Figure for electron microscope morphology detection of umbilical cord mesenchymal stem cell exosome in the embodiments of the present application;

[0029] Figure 4 Figure for detection of the angiogenesis-promoting effect of umbilical cord mesenchymal stem cell exosome in the embodiments of the present application;

[0030] Figure 5 Figure for detection of the immune regulation-IL-10 secretion-promoting effect of umbilical cord mesenchymal stem cell exosome in the embodiments of the present application;

[0031] Figure 6 Figure for detection of the immune regulation-TNF-α inhibition effect of umbilical cord mesenchymal stem cell exosome in the embodiments of the present application. DETAILED DESCRIPTION

[0032] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0033] In the embodiments of the present application, the words such as "exemplary" or "for example" are used to represent as an example, illustration or description. Any embodiment or design scheme described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as more preferred or more advantageous than other embodiments or design schemes. Rather, the words such as "exemplary" or "for example" are intended to present the relevant concept in a specific manner.

[0034] Some professional terms involved in the present application are explained as follows:

[0035] Umbilical cord mesenchymal stem cells: Umbilical cord mesenchymal stem cells (UC-MSCs) are a kind of multipotent stem cells isolated from the umbilical cord tissue of newborns, which have the advantages of strong proliferation ability, low immunogenicity, and few ethical controversies.

[0036] Cell factory: (Cell Factory) refers to a technical platform for efficient and controllable biological manufacturing using engineered cells (such as microorganisms, mammalian cells, plant cells, etc.) to produce drugs, chemicals, fuels or other high-value products. Its core is to transform cells into "production factories" through synthetic biology, metabolic engineering and biological process optimization. The cell factory used in this application belongs to a mammalian cell factory, which relies on adherent growth, preferably a multi-layer cell factory, and the cell factory is a stacked structure with 1-40 layers.

[0037] HFpEF: Heart Failure with preserved Ejection Fraction, i.e. heart failure with preserved ejection fraction.

[0038] Next, the materials used in the examples of the present application are described as follows:

[0039] Source of umbilical cord mesenchymal stem cells: derived from the main library.

[0040] DMEM (Dulbecco's Modified Eagle Medium) basal medium: one of the most commonly used basal media for cell culture, especially suitable for the culture of various adherent cells such as umbilical cord mesenchymal stem cells (UC-MSCs), fibroblasts, epithelial cells, etc. The basic formula is shown in Table 1 below:

[0041] Table 1: DMEM basal medium formula

[0042] Ingredients Concentration (mg / L) Function Glucose 4500 (high sugar version) Main energy source L-glutamine 584 Provide nitrogen source, involved in protein synthesis Sodium pyruvate 110 Replace energy substrate, protect cells Amino acids (e.g. lysine) Customization Essential for protein synthesis (more than EMEM) Inorganic salts (Ca 2+ , Na + , etc.) In appropriate amounts Maintain osmotic pressure and signaling

[0043] Example 1:

[0044] This example describes one method of obtaining high expression of IL-10 mesenchymal stem cell exosomes on a large scale, which includes:

[0045] S101, stable culture: umbilical cord mesenchymal stem cells are inoculated into a cell factory, and complete culture medium is used for stable recovery culture at room temperature and normal oxygen conditions for 7 days.

[0046] Among them, the room temperature and normal oxygen conditions in the above scheme are: 37℃, 5% CO2, 20% O2.

[0047] The cell factory is a stacked structure with 20 layers, and the umbilical cord mesenchymal stem cells are inoculated in the cell factory at a quantity of 4 x 10 6 cells per layer.

[0048] The complete culture medium used is: 10% serum substitute is added to the DMEM basic culture medium.

[0049] S102, the umbilical cord mesenchymal stem cells are subjected to complete culture and starvation culture for cross culture, wherein the complete culture is: adding complete culture medium to the cell factory, and culturing for a period T1 (in this embodiment, T1 is specifically 1.5 days); the starvation culture is: adding starvation culture medium to the cell factory, and culturing for a period T2 (in this embodiment, T2 is specifically 1.5 days); the cross culture is repeated 8 times;

[0050] The complete culture medium used is the same as that in S101, and the starvation culture medium is DMEM basic culture medium, and the cell factory is also the same as that in S101.

[0051] S103, collecting the solution discharged from the cell factory at the end of each starvation culture in S102 to obtain an exosome stock solution;

[0052] S104, filtering and removing impurities from the obtained exosome stock solution, thereby obtaining the mesenchymal stem cell exosomes with high expression of IL-10.

[0053] The filtering and impurity removal of the obtained exosome stock solution is carried out by using the filtering means in the prior art, for example, a commercially available chromatography column or a nanoscale filter, and the present application does not make special limitations thereon, and a person skilled in the art can select a corresponding filtering tool according to the needs. The main macromolecular protein impurities removed by filtering include cell fragment residues, protein aggregates and other vesicle contaminants such as apoptotic bodies (diameter greater than 100 nm).

[0054] Example 2:

[0055] This embodiment is based on the basis of Example 1, and describes another method for obtaining mesenchymal stem cell exosomes with high expression of IL-10 on a large scale under another parameter, which is basically the same as the method of Example 1, and the difference is that:

[0056] In S101, the cell factory has 5 layers, and the umbilical cord mesenchymal stem cells are inoculated in the cell factory at a quantity of 3 x 10 6 cells per layer; the stable recovery culture is 6 days; and the complete culture medium used is: 5% serum substitute is added to the DMEM basic culture medium.

[0057] In S102, T1 is 1 day, T2 is 1 day, and the cross culture is repeated 6 times in total.

[0058] Example 3:

[0059] This embodiment is based on Example 1, and describes a method for large-scale acquisition of mesenchymal stem cell exosomes with high IL-10 expression under different parameters. It is basically the same as the method in Example 1, except that:

[0060] In S101, the cell factory has 40 layers, and the seeding density of umbilical cord mesenchymal stem cells in the cell factory is 5 × 10⁻⁶. 6 Cells / layer; stable recovery culture for 8 days; the complete culture medium used was DMEM basal medium with 20% serum substitute added;

[0061] In S102, T1 was 2 days, T2 was 2 days, and the crossover culture was repeated 7 times.

[0062] It is understandable that Examples 2 and 3 are based on Example 1 with some adjustments to the relevant parameters. All of the above adjustments are within an acceptable range, and the exosome yields obtained within this range are basically similar.

[0063] Investigation 1: Investigating the effects of culture temperature and oxygen content on exosome yield.

[0064] To investigate the effects of inoculation and culture conditions (including culture temperature and oxygen content) on exosome yield from umbilical cord mesenchymal stem cells, comparative example 1 (room temperature and hypoxia) and comparative example 2 (low temperature and normoxic conditions) were set up as follows:

[0065] Comparative Example 1:

[0066] The difference between Comparative Example 1 and Example 1 is that the umbilical cord mesenchymal stem cells were cultured under normal temperature and low oxygen conditions (37°C, 5% CO2, 1% O2).

[0067] Comparative Example 2:

[0068] The difference between Comparative Example 2 and Example 1 is that the umbilical cord mesenchymal stem cells were cultured under low temperature and normoxic conditions (4°C, 5% CO2, 20% O2).

[0069] The exosomes of umbilical cord mesenchymal stem cells obtained in Example 1, Comparative Example 1, and Comparative Example 2 were analyzed, and the number of exosomes extracted per mL of supernatant was measured. The yield obtained in Example 1 was 4.7 × 10⁻⁶. 10 p / mL, the yield obtained in Comparative Example 1 was 3.77 × 10⁻⁶. 10 p / mL, the yield obtained in Comparative Example 2 was 0.376 × 10⁻⁶. 10 p / mL, see appendix for detailed results. Figure 1 .

[0070] Depend on Figure 1It can be seen that, on the one hand, the culture temperature has a greater impact on the yield of exosomes, and the yield is higher at room temperature, and the yield of exosomes is greatly reduced under low temperature conditions, therefore, the room temperature condition is better. On the other hand, the oxygen content also has a certain influence on the yield of exosomes, and too low oxygen content will lead to a decrease in the yield of exosomes. In summary, the effect is better when the oxygen content is about 20%.

[0071] Exploration II, explore the influence of cross-culture and whole-process starvation culture on the yield of exosomes

[0072] Based on the scheme of Example 1, simultaneously set Comparative Example 3, on the basis of the scheme of Example 1, after the first, second and eighth replacement of the starvation medium, the solution (i.e. exosome stock solution) discharged from the cell factory was obtained, and the umbilical cord mesenchymal stem cell exosomes extracted from the exosome stock solution were obtained, respectively, to obtain umbilical cord mesenchymal stem cell exosomes obtained by replacing the starvation medium once, umbilical cord mesenchymal stem cell exosomes obtained by replacing the starvation medium twice, and umbilical cord mesenchymal stem cell exosomes obtained by replacing the starvation medium eight times.

[0073] Comparative Example 3: whole-process starvation culture

[0074] Culture of umbilical cord mesenchymal stem cells: inoculate 4x10 6 cells per layer of umbilical cord mesenchymal stem cells in the cell factory, use complete culture medium to stabilize and recover culture for 2 days under room temperature and normal oxygen (37℃, 5% CO2, 20% O2), replace with starvation medium for culture to 6 days, obtain exosome stock solution, and the umbilical cord mesenchymal stem cell exosomes extracted from the exosome stock solution are whole-process starvation medium obtained umbilical cord mesenchymal stem cell exosomes.

[0075] The exosomes obtained by replacing the starvation medium once, twice, eight times and whole-process starvation culture were counted, and the results are shown in Table 2 as follows:

[0076] Table 2: Exosome situation obtained by different harvesting times and culture methods

[0077] Experimental conditions Exosome harvest data 1x change of starvation medium 4.70 x 10 10 p / mL 2x change of starvation medium 4.53 x 10 10 p / mL 8x change of starvation medium 3.91 x 10 10 p / mL <!-- 5 -->]]> Full course of starvation medium 4.5 x 10 6 p / mL

[0078] As can be seen from the data in Table 2, compared with the single whole-process starvation culture of the prior art, the cross-culture method of the present application significantly improves the yield of exosomes, and during the cross-culture period, the yield of exosomes after each replacement of the starvation medium can still maintain a high level, which also indicates that the cross-culture method of the present application can continuously produce exosomes without increasing the cost of raw materials and culture consumables.

[0079] The above examples and comparative examples effectively prove that the technical scheme of the present application can realize high-yield, high-efficiency, stable and reliable exosome production.

[0080] The surface markers and membrane structures of the exosomes obtained by the scheme of the application will be detected below.

[0081] I. Determining the expression amount of markers CD63, CD81 and CD9 of the umbilical cord mesenchymal stem cell exosomes obtained by the application.

[0082] It can be understood that the exosome surface markers (such as CD63, CD81 and CD9) are key molecules for identifying exosomes, and CD63, CD81 and CD9 are the most typical three markers. Common detection methods include: flow cytometry (Flow Cytometry), enzyme-linked immunosorbent assay (ELISA), Western Blot, Immuno-EM, nanoparticle tracking analysis (NTA) + antibody labeling and other means.

[0083] The enzyme-linked immunosorbent assay is used in this detection, and the specific operation is as follows:

[0084] 1. Coating capture antibody: dilute the capture antibody to 1-5 μg / mL with coating buffer, add 100 μL per well to the ELISA plate, and incubate at 4°C overnight (or 37°C for 2 hours); wash the plate: wash 3 times with washing buffer, soak for 1 minute each time and pat dry.

[0085] 2. Blocking

[0086] Add 200 μL of blocking solution per well, and block at 37°C for 1 hour; wash the plate 3 times (as above).

[0087] 3. Sample and standard addition

[0088] Standard curve: use known concentration of recombinant CD63 / CD81 / CD9 protein or quantitative exosome standard, gradient dilution (such as 0-1000 pg / mL);

[0089] Test sample: dilute the exosome sample with blocking solution (determine the optimal dilution multiple by gradient pre-experiment).

[0090] Add 100 μL of sample or standard per well, and incubate at 37°C for 2 hours; wash the plate 5 times (to avoid cross-reaction).

[0091] 4. Add detection antibody

[0092] Dilute the biotin-labeled detection antibody with blocking solution (according to the concentration in the instructions, usually 0.1-1 μg / mL), add 100 μL per well, and incubate at 37°C for 1 hour; wash the plate 5 times.

[0093] 5. Add streptavidin-HRP

[0094] Dilute streptavidin-HRP (usually 1:1000-1:5000) with blocking solution, add 100 μL per well, and incubate at 37°C for 30 min in the dark; wash the plate 5 times.

[0095] 6. Color development

[0096] Add 100 μL TMB substrate per well, and react at room temperature for 10-30 min in the dark (blue color develops).

[0097] The umbilical cord mesenchymal stem cell exosomes obtained by the scheme of the present application (i.e., complete medium and starvation medium cross-culture), the umbilical cord mesenchymal stem cell exosomes obtained by starvation culture (i.e., whole starvation culture), and the control group starvation medium and PBS were subjected to CD63, CD81, and CD9 detection.

[0098] The detection results are shown in Table 1. Figure 2 As shown in Table 1, the results show that the color of the group of complete medium and starvation medium cross-culture is significantly deeper than that of the whole starvation culture group, indicating that the former significantly expresses typical exosome membrane proteins CD63, CD81, and CD9. In addition, neither the starvation medium nor the PBS expresses CD63, CD81, and CD9, further demonstrating the reliability of the experiment.

[0099] II. Electron microscope detection of the membrane structure of the umbilical cord mesenchymal stem cell exosomes obtained by the scheme of the present application.

[0100] The specific operation is as follows:

[0101] 1. Sample preparation: The umbilical cord mesenchymal stem cell exosomes extracted in Example 1 are suspended in an appropriate amount of physiological saline to a concentration (e.g., 1-5 x 10 10 particles / mL) suitable for electron microscope observation. A small amount (e.g., 3 μL) of the umbilical cord mesenchymal stem cell exosome solution is added dropwise onto a copper grid to spread it evenly.

[0102] 2. Negative staining: Heavy metal salt solutions such as phosphotungstic acid or uranyl acetate are used to negatively stain the umbilical cord mesenchymal stem cell exosomes on the copper grid to enhance the contrast. The copper grid is immersed in the negative staining solution for a certain period of time (e.g., 30-60 s), and then the excess staining solution is absorbed with filter paper.

[0103] 3. Drying and observation: The stained copper grid is naturally dried or dried with nitrogen. Subsequently, the copper grid is placed under a transmission electron microscope (TEM) for observation and photography.

[0104] The TEM results are shown in Table 2. Figure 3 As shown in Table 2, the results show that the exosomes harvested by the complete medium and starvation medium cross-culture are round or cup-shaped vesicle structures under the electron microscope, with an intact membrane structure and a diameter of 30-150 nm.

[0105] Therefore, the umbilical cord mesenchymal stem cell exosomes obtained by the application significantly express typical exosome membrane proteins CD63, CD81 and CD9, and the umbilical cord mesenchymal stem cell exosomes are in circular or cup-shaped vesicular structures under electron microscopy, and the membrane structure is complete.

[0106] On this basis, we further study and confirm that the umbilical cord mesenchymal stem cell exosomes obtained by the application have therapeutic effects on HFpEF, including anti-cardiomyocyte apoptosis, promotion of angiogenesis, immune regulation and the like.

[0107] III. Anti-cardiomyocyte apoptosis detection of umbilical cord mesenchymal stem cell exosomes (in vitro)

[0108] Detection 1: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs, 4 x 10 4 were inoculated into a 12-well plate coated with Matrigel in advance, and cultured at 37°C overnight under the condition of RPMI1640 medium containing B27 supplement, and then washed with DPBS. Then, 0.3 mL of DMEM medium containing or not containing exosomes was added to each well; incubated for 48 hours under serum-free conditions (simulating heart failure modeling) of 5% CO2, 94% N2 and 1% O2, and the CellTiter AQueous One Solution Cell Proliferation Assay kit (MTS) (Promega, USA). The myocardial cell apoptosis was detected, and the detection results are shown in Table 3.

[0109] Table 3: Anti-cardiomyocyte apoptosis detection of umbilical cord mesenchymal stem cell exosomes under serum-free hypoxic conditions (in vitro)

[0110] Experimental conditions Viability ratio DMEM medium with FBS 2.1 DMEM medium with FBS exosomes 1.3 DMEM medium with 1st exosomes 1.7 DMEM medium with 8th exosomes 1.5 DMEM medium without exosomes 1

[0111] Note: Under each experimental condition, the myocardial cell biological activity detected by the MTS kit is calculated by taking the activity of the myocardial cells under the condition of DMEM medium without exosomes as the base value.

[0112] As can be seen from Table 3, the exosomes extracted from the first to the eighth time have anti-apoptosis effect on myocardial cells under early hypoxic-ischemic conditions under hypoxic and serum-free conditions.

[0113] Detection 2: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs, 4 x 10 4(Number of exosomes / well) were seeded into 12-well plates pre-coated with Matrigel and incubated overnight at 37°C with RPMI 1640 medium and B27 supplement, followed by washing with DPBS. After induction with 0.01 μM STAUROSPORINE for 1 h, 0.3 mL of DMEM medium with or without exosomes was added to each well; the plates were then cultured for 48 h under serum-free conditions of 5% CO2, 94% N2, and 1% O2 (simulating heart failure modeling), and the cells were analyzed using the CellTiter. AQueous One Solution Cell Proliferation Assay kit (MTS) (Promega, USA). Cardiomyocyte apoptosis was detected; the results are shown in Table 4.

[0114] Table 4. Detection of anti-apoptotic effects of umbilical cord mesenchymal stem cell exosomes under induced cardiomyocyte apoptosis conditions (in vitro).

[0115] Experimental conditions Viability ratio DMEM medium with FBS 2.3 DMEM medium with FBS exosomes 1.5 DMEM medium with 1st exosomes 1.9 DMEM medium with 8th exosomes 1.6 DMEM medium without exosomes 1

[0116] As shown in Table 4, under the premise of inducing cardiomyocyte apoptosis, the exosomes extracted from the first to the eighth extractions had an anti-apoptotic protective effect on cardiomyocytes under hypoxic and serum-free conditions.

[0117] IV. Detection of the angiogenesis-promoting effect of umbilical cord mesenchymal stem cell exosomes

[0118] The detection method is as follows: 3×10 5 Cardiac microvascular endothelial cells were seeded into 12-well plates and cultured for 24 hours. After the cells formed a complete monolayer, three parallel "scratches" were made in each well using a P200 pipette tip, followed by washing the cells twice with PBS.

[0119] After co-incubating the cells with culture media containing or without exosomes, the scratched areas were photographed and counted using a bright-field microscope, and the image data were analyzed using Prism 5.0 software.

[0120] Test results as follows Figure 4 As shown, the results indicated that the exosomes of umbilical cord mesenchymal stem cells extracted from the 1st to the 8th extractions significantly repaired the scratched area, with the confluence of the scratched area reaching 17.8%–23.0%, all of which had an angiogenesis-promoting effect.

[0121] V. Detection of Immunomodulatory Effects of Umbilical Cord Mesenchymal Stem Cell Exosomes

[0122] To investigate the effect of EV-MSC on the production of IL-10 and the regulation of inflammatory factor TNF-α in LPS-induced THP-1 cells, we performed the following experiments:

[0123] 1. Cell culture and grouping:

[0124] THP-1 cells (human monocyte cell line) were cultured and divided into control group (untreated), LPS stimulation group (inflammatory model), LPS+EV-MSC treatment group (experimental group), and LPS+MV negative control group (exosome solvent group).

[0125] 2. Inflammatory factor determination:

[0126] Cell supernatant was collected, and the secretion levels of TNF-α and IL-10 were detected by ELISA.

[0127] The results of the determination are shown in Figure 5 , Figure 6 The results show that IL-10 is an anti-inflammatory cytokine that can inhibit inflammatory response and promote tissue repair. EV-MSC can induce THP-1 cells to secrete IL-10 by delivering signaling molecules such as miR-21 or TGF-β, which is significantly different from the simple LPS treatment group (P<0.001). TNF-α is a pro-inflammatory cytokine involved in inflammation amplification and tissue damage. EV-MSC inhibits the secretion of TNF-α by THP-1 cells, which is significantly different from the simple LPS treatment group (P<0.01).

[0128] Each embodiment in the specification is described in a progressive manner, and the same or similar parts between each embodiment can be referred to each other. Each embodiment focuses on the differences from other embodiments.

[0129] Finally, it should be noted that the above examples are used to illustrate the technical solutions of the present application, but not to limit them. Although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for some technical features. These modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for large-scale acquisition of mesenchymal stem cell exosomes with high expression of IL-10, characterized in that, The application relates to a method for preparing mesenchymal stem cell exosomes with high IL-10 expression. The method comprises the following steps: Cross-culturing umbilical cord mesenchymal stem cells by complete culture and starvation culture, wherein the complete culture is that complete culture medium is added into a cell factory and cultured for a period T1; the starvation culture is that starvation culture medium is added into the cell factory and cultured for a period T2; the cross-culturing is repeated for multiple times; Solution discharged from the cell factory at the end of each starvation culture is collected to obtain an exosome stock solution; 2. The method of claim 1, wherein the method is for large-scale production of the mesenchymal stem cell exosomes highly expressing IL-10. The exosome stock solution is filtered to remove impurities, and mesenchymal stem cell exosomes with high IL-10 expression are obtained.

3. The method of large-scale obtaining of mesenchymal stem cell exosomes highly expressing IL-10 according to claim 1, characterized in that, The complete culture medium is DMEM basic medium added with 5-20% serum substitute; the starvation culture medium is DMEM basic medium.

4. The method of claim 1, wherein the method is for large-scale production of the mesenchymal stem cell exosomes highly expressing IL-10. The T1 is 1-2 days, the T2 is 1-2 days, and the cross-culturing is repeated for 6-8 times in total.

5. The method of large-scale obtaining of mesenchymal stem cell exosomes highly expressing IL-10 according to claim 1, characterized in that, Before the cross-culturing, stable culture is further included, wherein the stable culture is that umbilical cord mesenchymal stem cells are inoculated into a cell factory, and the cells are stably recovered and cultured for 6-8 days under normal temperature and normal oxygen condition by using complete culture medium.

6. The method of claim 5, wherein the method is performed on a large scale. The umbilical cord mesenchymal stem cells are inoculated in the cell factory at 3-5 x 10 6 cells per layer.

7. Use of mesenchymal stem cell exosomes of IL-10 obtained by the method according to any one of claims 1 to 6, characterized in that, The cell factory is a stacked structure, and the number of layers is 1-40. The mesenchymal stem cell exosomes with high IL-10 expression are used for preparing a drug for treating HFpEF.

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