Energizing method for improving exosome secretion amount and immunosuppression function of mesenchymal stem cells

Abstract

The invention discloses an energizing method for improving the exosome secretion amount and the immunosuppression function of mesenchymal stem cells, and relates to the technical field of biomedicine, the energizing method for improving the exosome secretion amount and the immunosuppression function of the mesenchymal stem cells comprises the following steps: S1, performing multiplication culture on the mesenchymal stem cells; s2, when the fusion degree of the mesenchymal stem cells reaches 60% or above, mitomycin C is added for co-culture pretreatment; s3, performing energizing pretreatment on the mesenchymal stem cells by adopting a pretreatment culture medium added with cell factors; and S4, exosome extraction and purification. MSCs are treated through mitomycin C, so that proliferation of the MSCs is stopped at a proper fusion degree; specific cell factors are added into a culture medium to form a new pretreatment culture medium, and the MSCs are pretreated by using the pretreatment culture medium, so that the yield of the exosome is remarkably increased, and the energizing exosome with a stronger immunosuppression function is obtained.

Description

Technical Field

[0001] The present invention relates to biomedical technology, and in particular to a method for increasing the secretion amount of exosomes of mesenchymal stem cells and the immunosuppressive function. Background Art

[0002] Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells originally isolated from the bone marrow. They are a type of stem cell with self-renewal, self-replication, and multidirectional differentiation capabilities. They participate in the formation of the bone marrow hematopoietic microenvironment, and have a significant supportive effect on the proliferation and differentiation of hematopoietic stem cells. Their low immunogenicity and high immunoregulatory ability have broad application prospects in drugs for a variety of autoimmune diseases. The biological characteristics and functions of MSCs from different tissues are not the same; subsequent confirmation shows that even MSCs from the same tissue source have different cell types, showing the "heterogeneity" characteristics of MSCs. When MSCs exert their immunosuppressive function, they need to receive the stimulation of microenvironmental cytokines in the body before they can exert a specific immunosuppressive effect. However, due to their "heterogeneity" characteristics, the functions they exert after the response are also inconsistent, which will cause instability in clinical effects. Studies have shown that after being stimulated by specific cytokines, MSCs tend to form cell subpopulations with specific functions, which have the characteristics of cell homogeneity. Based on the above, MSCs with specific functional subpopulations can help promote the realization of precision applications, making them dominant in the field of regenerative medicine. However, after MSCs are infused, they remain in the body for a short time, and the number in the body gradually decreases. In order to ensure the application effect, in addition to increasing the number of MSCs infusions, it is crucial to improve the function of MSCs.

[0003] An important way for MSCs to function is paracrine, and a key component of paracrine substances is exosomes. MSCs can play a more long-term function through the exosome pathway. Exosomes are a type of extracellular vesicles, which are tiny vesicles formed by a lipid bilayer encapsulating a variety of contents, with a diameter of generally 30-200nm. Exosomes originate from the endocytosis of the plasma membrane. The plasma membrane invaginates to form early endosomes. During the invagination process, proteins, nucleic acids, amino acids, metabolites and other substances in the cytoplasm are taken in to form intraluminal vesicles. Except for the intraluminal vesicles degraded by lysosomes, the remaining intraluminal vesicles fuse with the plasma membrane and release exosomes to the outside of the cell.

[0004] However, due to the unique way in which exosomes are formed, their size, contents, functions and sources are heterogeneous. Exosomes secreted by different types of cells have different components, and even exosomes secreted by the same type of cells may differ. The exosomes produced by pretreated mesenchymal stem cells not only have the advantages of low immunogenicity, non-tumorigenicity and high clinical safety of ordinary exosomes, but also have the advantages of high efficiency and precision of pretreated stem cells, achieving precise application on exosomes.

[0005] Currently, most exosomes are extracted by collecting the culture medium supernatant when the cell fusion degree reaches more than 90%, and then enriching and extracting the exosomes using ultracentrifugation, density gradient centrifugation, ultrafiltration and other methods. Under this method, exosomes can only be collected once from each generation of cells, which limits the production of exosomes and cannot meet the clinical requirements for exosomes. The existing methods for increasing the secretion yield of exosomes from mesenchymal stem cells are difficult to produce a large number of exosomes in a short period of time, which limits their industrial production and market application.

[0006] In summary, in view of this situation, it is urgent to develop an empowerment method to increase the secretion amount and immunosuppressive function of mesenchymal stem cell exosomes to meet practical needs. Summary of the invention

[0007] In view of this, the present invention aims to address the deficiencies in the prior art, and its main purpose is to provide an empowerment method for increasing the secretion amount of exosomes and the immunosuppressive function of mesenchymal stem cells. MSCs are treated with mitomycin C to stop the proliferation of MSCs at an appropriate degree of confluence; in addition, specific cytokines are added to the culture medium to form a new pretreatment culture medium, and MSCs are pretreated with the pretreatment culture medium, which can significantly increase the exosome production and obtain empowered exosomes with stronger immunosuppressive function.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions: A method for increasing the secretion amount of exosomes of mesenchymal stem cells and the immunosuppressive function thereof comprises the following steps: S1. Proliferation and culture of mesenchymal stem cells; S2, when the confluence of mesenchymal stem cells reached more than 60%, mitomycin C was added for co-culture pretreatment; S3, using a pretreatment medium supplemented with cytokines to perform empowerment pretreatment on mesenchymal stem cells; S4. Continue to culture the mesenchymal stem cells after empowerment pretreatment, collect the mesenchymal stem cell culture fluid, and extract and purify the exosomes.

[0009] As a preferred solution: when the degree of confluence of mesenchymal stem cells in step S2 reaches more than 60%, mitomycin C is added to the culture medium; the concentration of mitomycin C is 5-20 ug / ml, and the co-culture pretreatment time is 2-6 hours.

[0010] As a preferred solution: when the degree of confluence of mesenchymal stem cells reaches 90% in step S2, mitomycin C is added to the culture medium; the concentration of mitomycin C is 10 ug / ml, and the co-culture pretreatment time is 2 hours.

[0011] As a preferred embodiment: in step S3, at least one cytokine selected from the group consisting of IFN-γ, IL-27, TNF-α, IL-4 and IL-17A is added to the pretreatment culture medium.

[0012] As a preferred solution: the concentration of cytokines added in step S3 is 10-300 ng / ml, and the pretreatment time is 6-48 hours.

[0013] As a preferred solution: the cytokine added to the pretreatment culture medium in step S3 is IFN-γ, the concentration of IFN-γ is 20 ng / ml, and the empowerment pretreatment time is 12 hours.

[0014] As a preferred embodiment: the cytokines added to the pretreatment culture medium in step S3 include IFN-γ, IL-27 and TNF-α, with corresponding concentrations of 10 ng / ml, 20 ng / ml and 20 ng / ml, respectively, and the empowerment pretreatment time is 12 hours.

[0015] As a preferred solution: the pretreatment medium in step S3 is serum-free medium; the addition method of mitomycin C in step S2 includes: adding before cytokine pretreatment, adding simultaneously with cytokines, and adding after cytokine pretreatment.

[0016] As a preferred solution: the method of adding mitomycin C in step S2 is: adding it before cytokine pretreatment.

[0017] As a preferred embodiment, the mesenchymal stem cells in step S1 are derived from at least one of human tissue, induced pluripotent stem cells and embryonic stem cells.

[0018] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that by treating MSCs with mitomycin C, MSCs stop proliferating when the degree of confluence reaches more than 60%; in addition, specific cytokines are added to the culture medium to form a new pretreatment culture medium, and MSCs are pretreated with the pretreatment culture medium. MSCs can be activated by the cytokines in the culture medium, and the activated empowered MSCs can effectively promote the secretion of exosomes during the subsequent culture for 1-3 days. MSCs treated with mitomycin C can continuously secrete a large amount of exosomes, and the ability to secrete exosomes does not decrease due to excessive cell confluence or cell aging. After collecting the exosomes once, the serum-free culture medium is replaced to continue culturing the cells, and the exosomes can still be secreted. By isolating a higher content of exosomes from the supernatant of the cell culture medium, it is possible to repeatedly collect exosomes at the same generation of cells, which can significantly increase the exosome yield; in addition, the exosomes secreted by the pretreated MSCs have better immunosuppressive function against T cells, and can be better applied to immune-related diseases in the future; a pretreatment culture medium containing cytokines is provided for pretreatment and culture of mesenchymal stem cells; human mesenchymal stem cells pretreated with a pretreatment culture medium supplemented with cytokines can secrete 3-5 times more empowered exosomes with stronger immunosuppressive function; the obtained empowered exosomes can highly express IDO1, PD-L1, TGF-β and PGE-2 immune regulatory molecules, and have a stronger inhibitory effect on the proliferation of activated T cells.

[0019] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A flow chart of a method for increasing the secretion amount and immunosuppressive function of exosomes of mesenchymal stem cells according to the present invention; Figure 2 A is a schematic diagram of the NTA particle size measurement results of human umbilical cord mesenchymal stem cell exosomes after treatment with mitomycin C in Example 2 of the present invention; A is the addition of IFN-γ to the culture medium; ABC is the simultaneous addition of IFN-γ, IL-27 and TNF-α to the culture medium, and the results of the pretreatment with different cytokines; Figure 3 This is a schematic diagram of the number of exosome particles (number of particles / ml) contained in each ml of the cell culture supernatant collected on the first, second and third days after being treated with mitomycin C in Example 2 of the present invention under different cytokine pretreatments; Figure 4This is a schematic diagram of the number of exosome particles (number of particles / ml) contained in each ml of the cell culture supernatant of the first collection (72h) and the second collection (72h) of human umbilical cord mesenchymal stem cells of the same generation after being treated with mitomycin C in Example 2 of the present invention under different cytokine pretreatments; Figure 5 This is a schematic diagram of the average number of exosome particles secreted by each MSCs (number of particles / cell) in the cell culture supernatant collected on the first, second and third days after being treated with mitomycin C and pretreated with different cytokines in Example 2 of the present invention; Figure 6 This is a schematic diagram of the average number of exosome particles (number of particles / cell) secreted by each MSCs in the cell culture supernatant of the first collection (72h) and the second collection (72h) of the same generation of human umbilical cord mesenchymal stem cells after being treated with mitomycin C in Example 2 of the present invention under different cytokine pretreatments; Figure 7 This is a schematic diagram of the Western Blot detection results of the characteristic expressed proteins of the exosomes of human umbilical cord mesenchymal stem cells in each group in Example 1 of the present invention; Figure 8 This is a schematic diagram of the "tray-like" morphology of each group of exosomes in Example 1 of the present invention under a transmission electron microscope; Fig. 9 This is a schematic diagram of the gene expression of various immune regulatory factors in the exosomes of human umbilical cord mesenchymal stem cells in each group after being treated with mitomycin C and pretreated with different cytokines in Example 1 of the present invention; Fig.10 This is a schematic diagram of the immunosuppressive function of exosomes of human umbilical cord mesenchymal stem cells in vitro on T cell proliferation after being treated with mitomycin C and pretreated with different cytokines in Example 2 of the present invention; Fig.11 Schematic diagram of the flow cytometry results in Example 2 of the present invention, wherein a is the flow cytometry detection of each group of exosomes on CD3 + Schematic diagram of the effect of exosomes on T cell proliferation, quantified by T cell marker CFSE; b is the effect of exosomes on CD3 + Schematic diagram of T cell proliferation inhibition rate results. DETAILED DESCRIPTION

[0021] The present invention Figure 1 As shown in FIG11 , a method for increasing the secretion amount of exosomes of mesenchymal stem cells and the immunosuppressive function thereof comprises the following steps: S1. Proliferation and culture of mesenchymal stem cells; S2, when the confluence of mesenchymal stem cells reached more than 60%, mitomycin C was added for co-culture pretreatment; S3, using a pretreatment medium supplemented with cytokines to perform empowerment pretreatment on mesenchymal stem cells; S4. Continue to culture the mesenchymal stem cells after empowerment pretreatment, collect the mesenchymal stem cell culture fluid, and extract and purify the exosomes.

[0022] The supernatant of the pretreated MSCs was collected after 1-3 days of in vitro culture, and the exosomes were extracted by differential ultracentrifugation.

[0023] Specifically, the collected stem cell culture medium is first centrifuged at a relative centrifugal force of 3000 g to remove cell debris and dead cells in the culture medium.

[0024] The culture medium was then centrifuged at a relative centrifugal force of 100,000 g to extract and purify exosomes.

[0025] The exosomes obtained by ultracentrifugation were sterilized and the larger diameter vesicles were removed using a 0.22 μm filter to obtain the final purified exosomes. The subsequent in vitro experimental verification was performed using this exosome product.

[0026] In step S2, when the confluence of the mesenchymal stem cells reaches more than 60%, mitomycin C is added to the culture medium; the concentration of mitomycin C is 5-20 ug / ml, and the co-culture pretreatment time is 2-6 hours.

[0027] In step S2, when the confluence of the mesenchymal stem cells reaches 90%, mitomycin C is added to the culture medium; the concentration of mitomycin C is 10 ug / ml, and the co-culture pretreatment time is 2 hours.

[0028] In step S3, at least one cytokine selected from the group consisting of IFN-γ, IL-27, TNF-α, IL-4 and IL-17A is added to the pretreatment culture medium.

[0029] The concentration of cytokines added in step S3 is 10-300ng / ml; the concentration of IFN-γ is 10ng / ml-200ng / ml; the concentration of IL-27 is 10ng / ml-200ng / ml; the concentration of TNF-α is 20-300ng / ml; the concentration of IL-4 is 10ng / ml-100ng / ml; the concentration of IL-17A is 30ng / ml-200ng / ml, and the pretreatment time is 6-48 hours.

[0030] The cytokine added to the pretreatment medium in step S3 is IFN-γ, the concentration of IFN-γ is 20 ng / ml, the empowerment pretreatment time is 12 hours, and the pretreatment medium has an empowerment effect.

[0031] The cytokines added to the pretreatment culture medium in step S3 include IFN-γ, IL-27 and TNF-α, with corresponding concentrations of 10 ng / ml, 20 ng / ml and 20 ng / ml, respectively, and the empowerment pretreatment time is 12 hours.

[0032] The pretreatment medium in step S3 is a serum-free medium; the serum-free medium is any medium commonly used for culturing mammalian cells, such as Dulbecco's modified Eagle's medium (DMEM), a mixture of Dulbecco's modified Eagle's medium and F12 (DMEM / F12), and mesenchymal stem cell serum-free medium (MSCM-cf).

[0033] The addition method of mitomycin C in step S2 includes: adding before cytokine pretreatment, adding simultaneously with cytokine, and adding after cytokine pretreatment.

[0034] The method of adding mitomycin C in step S2 is: adding it before cytokine pretreatment.

[0035] In step S1, the mesenchymal stem cells are derived from at least one of human tissue, induced pluripotent stem cells and embryonic stem cells.

[0036] The human tissues include bone marrow, fat, placenta, umbilical cord, umbilical cord blood and dental pulp with good cell growth status; human umbilical cord MSCs are selected for use.

[0037] In the present invention, the reagents and test materials used can be obtained from commercial channels, and the sources of biological materials are as follows: IFN-γ, IL-27, TNF-α, IL-4 and IL-17A cytokines were purchased from R&D system.

[0038] Example 1: A method for increasing the secretion of exosomes from mesenchymal stem cells and the immunosuppressive function S1. Proliferation and culture of primary umbilical cord mesenchymal stem cells; Primary umbilical cord mesenchymal stem cells (P0) with normal cell morphology and good growth rate were isolated from Wharton's jelly of fresh umbilical cord, and cultured in well plates and / or culture bottles and / or cell factories using serum-free medium and the adherence method. When the cell confluence reached more than 90%, the original medium was discarded, the cells were washed twice with PBS, and trypsin was added for digestion. The cells were re-seeded in new well plates and / or culture bottles and / or cell factories. After 4 passages of primary umbilical cord mesenchymal stem cells, P4 umbilical cord mesenchymal stem cells were obtained for subsequent experiments.

[0039] S2, when the degree of confluence of mesenchymal stem cells reaches more than 60%, add mitomycin C and co-culture with P4 umbilical cord mesenchymal stem cells; P4 umbilical cord mesenchymal stem cells are 4000-13000 cells / cm 2 Seed in well plates and / or culture flasks and / or cell factories at a density of 4000 cells / cm 2 The inoculation density of the culture flask is 8000 cells / cm 2 The cell factory seeding density is 13,000 cells / cm 2 .

[0040] When the cell confluence reached 90%, the cells were treated with mitomycin C.

[0041] The original serum-free medium was replaced with a serum-free medium supplemented with mitomycin C, and the cells were co-cultured for 2 h; the added concentration of mitomycin C was 10 μg / ml.

[0042] S3, using a pretreatment medium supplemented with cytokines to perform empowerment pretreatment on P4 generation umbilical cord mesenchymal stem cells; The original serum-free medium supplemented with mitomycin C was replaced with a pretreatment medium supplemented with at least one cytokine among IFN-γ, IL-27, TNF-α, IL-4 and IL-17A, and the cells were co-cultured for 12-36 hours.

[0043] The types of pretreatment culture medium were A: culture medium with IFN-γ added; ABC: culture medium with IFN-γ, IL-27, and TNF-α added; and the culture time was 12 hours.

[0044] The concentration of IFN-γ factor in the basic culture medium is 20 ng / ml; the concentrations of the cytokine composition of IFN-γ, IL-27, and TNF-α in the basic culture medium are 10 ng / ml, 20 ng / ml, and 20 ng / ml, respectively.

[0045] S4, continuing to culture the human umbilical cord mesenchymal stem cells after empowerment pretreatment, collecting the mesenchymal stem cell culture fluid, and extracting and purifying exosomes; The serum-free medium used can be any medium commonly used for culturing mammalian cells, such as Dulbecco's modified Eagle's medium (DMEM), a mixture of Dulbecco's modified Eagle's medium and F12 (DMEM / F12), and mesenchymal stem cell serum-free medium (MSCM-cf). In this embodiment, Nutristem MSC XF medium (Sartorius) is used.

[0046] After 12 hours of pretreatment, the culture medium in the cells was replaced with a new serum-free culture medium without added cytokines, and the cells were starved for 24-72 hours, and the cell culture supernatant was collected.

[0047] After collecting the cell culture supernatant for 72 hours, the culture medium in the cells was replaced with a new serum-free culture medium without added cytokines, and the culture was continued for 72 hours. After 72 hours, the cell culture supernatant was collected for the second time. The collected cell culture supernatant can be temporarily stored in a -80°C ultra-low temperature refrigerator for subsequent extraction and purification of exosomes.

[0048] The method for extracting exosomes is differential ultracentrifugation, and the specific implementation steps are as follows: 01) Perform a preliminary centrifugation on the collected serum-free culture medium. The centrifugation conditions and parameters are 4°C, 3000g, and time for 30 minutes. The purpose is to remove cell debris and dead cells in the culture medium. After the preliminary centrifugation, retain the supernatant and transfer it to a clean new container.

[0049] 02) The supernatant retained after centrifugation in 01) was centrifuged again, and the centrifugation conditions and parameters were 4°C, 100,000 g, and time for 100 min, and the centrifugal precipitate was retained.

[0050] 03) Add 30 ml of D-PBS to the centrifugal precipitate of 02) for washing and centrifugation. The centrifugation conditions and parameters are 4°C, 100,000 g, and time for 100 min. The centrifugal precipitate is retained, which is the extracted exosomes.

[0051] 04) Add 1 ml of D-PBS to the final centrifugal precipitate to dissolve and mix. In order to facilitate subsequent experiments, use a 0.22 μm filter to sterilize and remove vesicles with larger diameters to obtain the final purified exosomes for subsequent experiments.

[0052] Effect Example 1: Detection of Gene Expression of Multiple Immunomodulatory Factors in Empowered Exosomes Extracted from Pretreated Human Umbilical Cord Mesenchymal Stem Cell Culture Medium: The method for detecting the gene expression of multiple immune regulatory factors in exosomes is real-time fluorescence quantitative PCR.

[0053] Specifically: Total RNA was extracted from exosomes of different cytokine pretreatment groups (UCMSC-EXO, A-EXO, ABC-EXO) using an RNA extraction kit (QIAGEN), and the expression of immune regulatory factor genes in exosomes (IDO, PD-L1, TGF-β1, PGE-2 and IL-6) was detected using fluorescent quantitative PCR.

[0054] According to the results of fluorescence quantitative PCR, compared with the enabled exosomes extracted from the culture medium of untreated human umbilical cord mesenchymal stem cells, the expression of immune regulatory factors IDO, PD-L1, TGF-β1, PGE-2 and IL-6 in the enabled exosomes extracted from the culture medium of pretreated human umbilical cord mesenchymal stem cells was significantly increased, with significant statistical differences between the groups, among which the expression level of immune regulatory factors in the exosomes of the ABC group was the highest.

[0055] Effect Example 2: In vitro immunosuppressive function detection of empowered exosomes extracted from pretreated human umbilical cord mesenchymal stem cell culture medium.

[0056] The in vitro method for detecting the immune function of pretreated human umbilical cord mesenchymal stem cell-empowered exosomes is to co-culture the exosomes with human peripheral blood mononuclear cells (PBMCs) in vitro.

[0057] Specifically: human peripheral blood mononuclear cells were first labeled with CFSE, and then stimulated and activated with CD3 and CD28 monoclonal antibodies, and then co-cultured with exosomes (UCMSC-EXO, A-EXO, ABC-EXO) from different cytokine pretreatment groups. Flow cytometry was performed after 3 days. The amount of exosomes added in the implementation plan was determined by the number of PBMC cells. Taking a 24-well plate as an example, two experimental schemes with two ratios were set (PBMC:EXO = 1:20000 and 1:30000). The specific implementation steps were as follows: 01) Resuspend PBMC in DPBS to 1×10 7 / ml density, add CFSE staining at a ratio of 1ul CFSE / ml to the PBMC suspension, incubate at 37°C in the dark for 10 minutes, shake intermittently within 10 minutes, add 2 volumes of ice DPBS after 10 minutes, wash and centrifuge to remove the supernatant, then add 10ml 1640 culture medium to wash and centrifuge, remove the supernatant, and finally add 1640 culture medium containing 10% FBS to resuspend PBMC.

[0058] 02) Prepare PBMC stimulation solution with 1640 culture medium containing 10% FBS, add CD3 and CD28 to the culture medium, mix well, and add to the PBMC activation wells in the 24-well plate.

[0059] 03) The PBMC suspension in 01) was diluted to 5×10 5 PBMC / well cells were added into 24-well plates.

[0060] 04) According to two different ratios of PBMC:EXO = 1: 20000 and 1:30000 (cell mass: particle number), different groups of exosomes were added and co-cultured in a 5% CO2 cell culture incubator at 37°C in the dark for 3 days. After 3 days, CD3 was detected by flow cytometry. +T cell proliferation.

[0061] According to the flow cytometry results, the UCMSC-EXO, A-EXO, and ABC-EXO groups all decreased CD3 + The proliferation ratio of T cells was different among the groups. The CD3 + The proliferation rate of T cells decreased significantly, which was higher than that of CD3 T cells co-cultured with exosomes produced by human umbilical cord mesenchymal stem cells (UCMSC-EXO) without cytokine pretreatment. + There were significant statistical differences in T cells among the groups.

[0062] From the perspective of the proliferation inhibition rate of exosomes, the inhibition rate ratio of A-EXO and ABC-EXO was significantly higher than that of UCMSC-EXO; in addition, as the amount of exosomes added increased, the exosomes had a greater effect on CD3 + The higher the T cell proliferation inhibition rate, the more obvious the inhibition effect of the ABC-EXO (30000) group was.

[0063] In summary, the MSCs obtained by double pretreatment with the combination of mitomycin C and cytokines of the present invention can produce more exosomes than ordinary human umbilical cord MSCs; after empowerment, the secretion of exosomes increased by about 3-5 times; and after treatment with mitomycin C, it is possible to repeatedly collect the supernatant of the cell culture medium under the same generation of cells, and still separate a high content of exosomes from the supernatant of the cell culture medium, significantly improving the exosome yield. In addition, cytokine pretreatment also improves the immunosuppressive function of exosomes. Compared with ordinary exosomes, empowered exosomes can exert stronger immunosuppressive ability, and the three-factor group of three cytokine combinations has better effects and is more suitable for precise application in immune diseases.

[0064] The design focus of the present invention is that MSCs are treated with mitomycin C to stop the proliferation of MSCs when the degree of confluence reaches more than 60%; in addition, specific cytokines are added to the culture medium to form a new pretreatment culture medium, and the MSCs are pretreated with the pretreatment culture medium, so that the MSCs can be activated by the cytokines in the culture medium, and the activated empowered MSCs can effectively promote the secretion of exosomes during the subsequent culture for 1-3 days. The MSCs treated with mitomycin C can continuously secrete a large amount of exosomes, and the ability to secrete exosomes does not decrease due to excessive cell confluence or cell aging. After collecting exosomes once, the serum-free culture medium is replaced to continue culturing the cells, and the exosomes can still be separated from the supernatant of the cell culture medium. A higher content of exosomes can achieve repeated collection of exosomes under the same generation of cells, which can significantly increase the exosome yield; in addition, the exosomes secreted by pretreated MSCs have better immunosuppressive function against T cells, and can be better applied to immune-related diseases in the future; a pretreatment medium containing cytokines is provided for pretreatment and culture of mesenchymal stem cells; human mesenchymal stem cells pretreated with a pretreatment medium supplemented with cytokines can secrete 3-5 times more empowered exosomes with stronger immunosuppressive function; the obtained empowered exosomes can highly express IDO1, PD-L1, TGF-β and PGE-2 immune regulatory molecules, and have a stronger inhibitory effect on the proliferation of activated T cells.

[0065] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for increasing the secretion of exosomes from mesenchymal stem cells and the immunosuppressive function, characterized in that: The steps include: S1. Proliferation and culture of mesenchymal stem cells; S2, when the confluence of mesenchymal stem cells reached more than 60%, mitomycin C was added for co-culture pretreatment; S3, using a pretreatment medium supplemented with cytokines to perform empowerment pretreatment on mesenchymal stem cells; S4. Continue to culture the mesenchymal stem cells after empowerment pretreatment, collect the mesenchymal stem cell culture fluid, and extract and purify the exosomes.

2. A method for increasing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 1, characterized in that: When the degree of confluence of mesenchymal stem cells reaches more than 60% in step S2, mitomycin C is added to the culture medium; the concentration of mitomycin C is 5-20 ug / ml, and the co-culture pretreatment time is 2-6 hours.

3. A method for increasing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 2, characterized in that: When the degree of confluence of mesenchymal stem cells reaches 90% in step S2, mitomycin C is added to the culture medium; the concentration of mitomycin C is 10 ug / ml, and the co-culture pretreatment time is 2 hours.

4. The method for enhancing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 1, characterized in that: In step S3, at least one cytokine selected from the group consisting of IFN-γ, IL-27, TNF-α, IL-4 and IL-17A is added to the pretreatment culture medium.

5. A method for increasing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 4, characterized in that: The concentration of cytokines added in step S3 is 10-300 ng / ml, and the pretreatment time is 6-48 hours.

6. A method for increasing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 5, characterized in that: The cytokine added to the pretreatment culture medium in step S3 is IFN-γ, the concentration of IFN-γ is 20 ng / ml, and the empowerment pretreatment time is 12 hours.

7. The method for enhancing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 5, characterized in that: The cytokines added to the pretreatment culture medium in step S3 include IFN-γ, IL-27 and TNF-α, with corresponding concentrations of 10 ng / ml, 20 ng / ml and 20 ng / ml, respectively, and the empowerment pretreatment time is 12 hours.

8. The method for enhancing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 1, characterized in that: The pretreatment medium in step S3 is a serum-free medium; The addition method of mitomycin C in step S2 includes: adding before cytokine pretreatment, adding simultaneously with cytokine, and adding after cytokine pretreatment.

9. The method for enhancing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 8, characterized in that: The method of adding mitomycin C in step S2 is: adding it before cytokine pretreatment.

10. The method for enhancing the secretion of exosomes of mesenchymal stem cells and the immunosuppressive function according to claim 1, characterized in that: In step S1, the mesenchymal stem cells are derived from at least one of human tissue, induced pluripotent stem cells and embryonic stem cells.

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