Preparation method of extracellular vesicles with immune regulation function and tolerant dendritic cells induced by extracellular vesicles and application of extracellular vesicles in treatment of allergic diseases

Abstract

The invention discloses an extracellular vesicle with an immunoregulation function and a preparation method of tolerance dendritic cells induced by the extracellular vesicle, and the extracellular vesicle is prepared from a conditioned medium collected by culturing stem cells on a large scale by a serum-free stem cell medium with definite components under a low oxygen partial pressure condition. Inducing the immature dendritic cells to obtain tolerant dendritic cells; the serum-free culture medium with determined components is provided for large-scale culture of stem cells, so that a large number of extracellular vesicles prepared by separation and purification of the conditioned culture medium can be collected and can be used for inducing to obtain tolerant dendritic cells, and the extracellular vesicles have the effects of resisting anaphylaxis, inhibiting inflammation, regulating immunity and improving tissue remodeling. Under the condition of low oxygen partial pressure, the tolerance dendritic cells induced by the stem cell-derived extracellular vesicles cultured by a serum-free stem cell culture medium with definite components are prepared into a biological agent, and the biological agent is applied to drugs for treating allergic diseases.

Description

Technical Field

[0001] The present invention relates to extracellular vesicles (EVs) with immunomodulatory functions and the tolerant dendritic cells induced therefrom. The conditioned medium collected from stem cells cultured on a large scale under hypoxic atmospheric conditions is separated and purified to obtain the extracellular vesicles, which are used to induce immature dendritic cells to obtain tolerant dendritic cells. The present invention provides a serum-free medium with defined components for large-scale stem cell culture, from which a large amount of conditioned medium can be collected, and the extracellular vesicles obtained can be used to induce tolerant dendritic cells, which have the effects of preventing allergic reactions, suppressing inflammation, regulating immunity, and improving tissue remodeling. The stem cell-derived extracellular vesicles cultured under hypoxic atmospheric conditions and the tolerant dendritic cells induced therefrom are prepared into biological preparations, which can be prepared into pharmaceutical compositions for treating allergic diseases. Background Art

[0002] Allergic diseases are immune responses triggered by allergens, shifting naive CD4 helper T cells (Th) toward Th2 and Th17 polarization and downregulating Th1 and regulatory T cells (Treg). This promotes the proliferation of eosinophils and neutrophils, triggering inflammation and airway hyperresponsiveness. These diseases include asthma, rhinitis, bronchitis, Henoch-Schönlein purpura, contact dermatitis, eczema, and urticaria. Modernization has led to an annual increase in the incidence of these diseases, driven by excessive cleanliness and hygiene practices, and the disease is increasingly affecting younger people.

[0003] Currently, allergic diseases are primarily treated with antihistamines and steroids, which are clinically effective, but have significant side effects with long-term use. Developing drugs with fewer side effects and longer-lasting efficacy is a research hotspot. Furthermore, allergen-specific therapy, a treatment that targets tolerance to allergens and allows for causal treatment that naturally induces tolerance, may offer a cure for these diseases. However, due to its time-consuming nature, high costs, and currently low efficacy, less than 5% of patients receive this treatment. Improving this treatment approach is urgent, and the purpose of AIT in inducing tolerance is closely related to dendritic cells (DCs). Dendritic cells (DCs) play a key role in adaptive immunity and are particularly important in developing tolerance in allergic diseases.

[0004] Dendritic cells are the largest antigen-presenting cells in the body, making the generation of tolerogenic dendritic cells a hot topic in the study of allergic and autoimmune diseases. Their generation induces tolerance mechanisms, including the proliferation of regulatory T cells, regulatory B cells, M2 macrophages, and regulatory NK cells, as well as the secretion of anti-inflammatory factors, thereby generating tolerance to allergens and regulating immune inflammation. Currently, chemical agents such as interleukin-10, transforming growth factor-β, vitamin D3, lipopolysaccharide, dexamethasone, and rapamycin are commonly used to induce tolerogenic dendritic cells. The present invention has discovered a new substance that induces tolerogenic dendritic cells: stem cell-derived extracellular vesicles, which are 40-1000 nm in size and secreted by stem cells. They have an intact membrane structure and contain RNA, proteins, carbohydrates, and lipids. They can penetrate the cell membrane and are primarily involved in the transport of substances and information between cells.

[0005] As extracellular vesicles derived from human stem cells, they have good compatibility with dendritic cells and naturally induce tolerance, without causing various adverse reactions in disease treatment. At the same time, the present invention selects a microenvironment for cell growth in the body that includes not only a reticular scaffold of the extracellular matrix, but also various proteins and cytokines adhered to the reticular scaffold, as well as factors such as the growth environment, to jointly create optimal survival conditions for stem cells and obtain better stem cells and extracellular vesicles derived from them. That is, stem cells are cultured under three-dimensional and hypoxic atmospheric conditions, and their extracellular vesicles are prepared, thereby inducing tolerant dendritic cells. Our research found that the tolerant dendritic cells of the present invention can not only stimulate the proliferation of regulatory T cells and the expression of anti-inflammatory factors, but also activate the production of new immune regulatory cells, that is, the proliferation of anergic T cells, indicating that they truly activate the autoimmune tolerance mechanism in the body and can produce a better tolerance response to allergens. Summary of the Invention

[0006] The present invention is based on our discovery that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) cultured under long-term hypoxia can better produce immune regulation function than those cultured under normoxia, inhibiting CD3 + CD8 +It can induce the production of tolerant dendritic cells, activate anergic T cells (Tan) and promote the proliferation of regulatory T lymphocytes (Treg) in animals with allergic airway inflammation; inhibit the expression of pro-inflammatory molecules (interleukin 4, IL-4, interleukin 17, IL-17 and interleukin 5, IL-5), promote the expression of anti-inflammatory molecules (interleukin 10, IL-10 and transforming growth factor beta-1, TGF-β1), inhibit the expression of follicular helper T cells (T follicular helper cell, Tfh), effectively controlling the polarization of Th0 to Th2 / Th17; producing a good immune regulatory response in vivo and in vitro; at the same time, extracellular vesicles can induce the acquisition of tolerant dendritic cells, which can not only stimulate the proliferation of regulatory T cells and the expression of anti-inflammatory factors, but also activate the production of new immune regulatory cells, that is, the proliferation of anergic T cells, which can induce a good immune regulatory effect in the body, thereby producing tolerance to allergens.

[0007] The immunomodulatory extracellular vesicles of the present invention are derived from mesenchymal stem cells cultured in a serum-free culture system that simulates the growth environment of stem cells in the body. This includes factors such as the extracellular matrix, nutrients, and an oxygen atmosphere. Studies using small molecule metabolites on stem cell proliferation, differentiation, survival, and immunomodulation have revealed that these cells exhibit excellent immunomodulatory properties and induce tolerance to dendritic cell induction. This induction process, which utilizes extracellular vesicles directly to facilitate cell-to-cell communication without the aid of chemical inducers, is more natural and has better compatibility.

[0008] The immunomodulatory extracellular vesicles of the present invention are 40-1000nm double-membrane vesicles secreted from stem cells. They carry a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, and other proteins, participating in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. Due to their small size, excellent stability, excellent biological functions, and convenient source, they have become a new drug for disease treatment.

[0009] The large-scale stem cell culture described in the present invention is to simulate the body's low-oxygen atmospheric environment, that is, 1-10% oxygen atmospheric concentration, and to culture the stem cells in a serum-free conditioned medium with a defined composition, from the primary culture to the P30 generation, while still maintaining good cell stemness and immunomodulatory effects; and the conditioned medium from each passage is collected.

[0010] Preferably, the stem cells are cultured in a three-dimensional system such as a hydrogel or microcarrier.

[0011] The method for large-scale stem cell culture described in the present invention is to directly use discarded tissue after multiple washing and mincing for primary culture, or to digest human tissue with digestive enzymes to form a single-cell suspension for primary culture. That is, the cells are added to a cell culture vessel and continue to grow in a three-gas incubator at 37°C, 5% CO2 and a low oxygen atmosphere of 1-10%, and fresh cell culture medium containing 1x serum-free supplement is replaced every 3 days; when the stem cells grow and become more than 80% confluent, they are collected by trypsin digestion to obtain primary stem cells and stored in a refrigerator at 4°C until needed; the primary cells are added to a matrix hydrogel-coated cell culture vessel for subculture, and are continuously amplified for large-scale subculture. Each time the stem cells grow and become more confluent for about 2-3 days, their conditioned medium is collected and used to prepare extracellular vesicles. The subcultured cells can be prepared into stem cell preparations or frozen.

[0012] The hypoxic culture condition is a hypoxic atmosphere environment of 1-10%; preferably, a hypoxic atmosphere environment of 1-5% is selected.

[0013] The stem cells prepared after 30 subcultures have a stronger cell proliferation rate than stem cells cultured under normal oxygen atmospheric conditions, maintain good cell morphology and cell phenotype of the stem cells, and can differentiate into corresponding adult cells; preferably, the prepared stem cells survive longer in the animal body and have good stem cell characteristics.

[0014] The human tissues include discarded umbilical cord, umbilical cord blood, placenta, amnion, urine, fat, bone marrow, skin, menstrual blood, dental pulp, synovial fluid, and synovial membrane, etc., and the corresponding stem cells are prepared, namely umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental pluripotent stem cells, urine stem cells, amniotic epithelial progenitor cells, adipose-derived stem cells, bone marrow mesenchymal stem cells, bone marrow endothelial progenitor cells, menstrual blood endometrial progenitor cells, dental pulp stem cells, synovial fluid mesenchymal stem cells, and synovial membrane mesenchymal stem cells. The stem cells can be resuspended in serum-free freezing medium (commercial product) and stored in a -196°C liquid nitrogen tank for future use after thawing.

[0015] Extracellular vesicles with immunomodulatory functions were isolated and purified from conditioned medium obtained from stem cell cultures using ultracentrifugation at 100,000 centrifugal force, ultrafiltration, size exclusion chromatography, and immunoaffinity methods. Particle size analysis revealed a range of 40-150 μm. Western blot analysis revealed the expression of extracellular vesicle markers including CD9, CD63, and TSG101.

[0016] Preferably, extracellular vesicles are added to lymphocytes for co-culture to inhibit CD3 + CD4 + 、CD3+ CD8 + and CD3 - CD56 + / 16 + Lymphocyte proliferation, promotes the proliferation of regulatory T lymphocytes.

[0017] The conditioned medium of the present invention is a serum-free stem cell culture medium with defined components, namely, containing recombinant human serum albumin, serotonin, recombinant human transferrin, recombinant human insulin, ethanolamine, sodium selenite, β-mercaptoethanol, non-essential amino acids, alanyl-glutamine, a lipid concentrate with defined components, L-ascorbic acid-2-phosphate, progesterone, taurine, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human insulin-like growth factor, recombinant human platelet-derived growth factor, Iscove's modified Dulbecco's medium or alpha low-limit Eagle's medium. e culture medium or keratinocyte basal culture medium; at the same time, the present invention adds key small molecules, namely lipostatin-1, which inhibits stem cell ferroptosis, 2-hydroxy-D-glutamate disodium salt, which prevents the increase of reactive oxygen species and causes oxidative stress in stem cells, and activator polypeptide 740Y-P, which has cell-permeable PI3K activators and activates stem cell proliferation activity. The entire stem cell culture process does not involve animal and human serum or plasma, and therefore does not involve extracellular vesicles from other sources. The conditioned medium obtained from stem cell culture can be directly collected and the extracellular vesicles therein can be extracted. We found that MSCs prepared with key molecules have better proliferation activity, telomerase activity, and stemness maintenance than MSCs prepared without key molecules and with currently commonly used additives.

[0018] Preferably, the serum-free culture additive solution does not contain other components of the basal culture medium. It can be placed in a freeze dryer for freeze drying to obtain a serum-free culture additive freeze-dried powder. After sealing with a cap, it can be stored in a refrigerator at 2-8°C, which is convenient for storage and transportation and has a longer shelf life.

[0019] The extracellular vesicles with immunomodulatory function provided by the present invention were transplanted to treat a mouse model of allergic airway inflammation, producing significant therapeutic effects. The specific indicators are as follows:

[0020] Through Dif rapid staining and small animal blood routine tests, it was found that extracellular vesicles and the tolerogenic dendritic cells they induced reduced the number of inflammatory cells in the bronchoalveolar lavage fluid of mice with allergic airway inflammation, especially neutrophils and eosinophils.

[0021] Pulmonary function testers showed that extracellular vesicles improved airway hyperresponsiveness, that is, they suppressed the lung airway resistance and lung elastic resistance of mice with allergic airway inflammation and increased lung compliance.

[0022] Enzyme-linked immunosorbent assay (ELISA) showed that extracellular vesicles and the tolerant dendritic cells they induced reduced the expression of pro-inflammatory molecules (IL-4, IL-17, and IL-5) and promoted the expression of anti-inflammatory molecules (IL-10 and TGF-β1).

[0023] Pathological staining of lung tissue using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining kit showed that the infiltration of inflammatory cells in the lungs was inhibited and the number of inflammatory cells in the lungs was significantly reduced; Masson's trichrome staining significantly reduced collagen fiber deposition in the pulmonary airways.

[0024] The present invention also provides a method for preparing tolerant dendritic cells induced by extracellular vesicles with immunomodulatory function, comprising the following steps:

[0025] In the first step, mononuclear cells are obtained from human peripheral blood by density gradient centrifugation or apheresis using a blood cell separator. Adherent cells are induced with the cytokines GM-CSF and IL-4 or Flt-3L for 4-7 days to obtain immature dendritic cells.

[0026] In step 2, extracellular vesicles are added to immature dendritic cells and the induction continues for 12-96 hours to obtain tolerogenic dendritic cells.

[0027] Extracellular vesicles with immunomodulatory functions induce tolerant dendritic cells, which downregulate CD11c molecules and co-stimulatory molecules (CD83, CD86, CD80, CD40) and express the immunosuppressive molecule programmed cell death protein 1 (PD-1), thereby promoting the proliferation of regulatory T lymphocytes.

[0028] Co-culture with lymphocytes inhibited CD3 + CD4 + cells, and the expression of IL-4 and IL-17.

[0029] Preferably, the tolerogenic dendritic cells suppress CD4 + CXCR5 + Follicular helper T cells effectively control the polarization of Th0 to Th2;

[0030] More preferably, the tolerogenic dendritic cells promote the expression of PD-1 and CD44 + CD73 + TIGIT + The proliferation of anergic T cells promotes the proliferation of regulatory T lymphocytes.

[0031] The extracellular vesicle-induced tolerogenic dendritic cells with immunomodulatory function provided by the present invention produce significant therapeutic effects in the immunotherapy of a mouse model of allergic airway inflammation. The specific indicators are as follows:

[0032] Through Dif rapid staining and small animal blood routine tests, it was found that extracellular vesicles and the tolerogenic dendritic cells they induced reduced the number of inflammatory cells in the bronchoalveolar lavage fluid of mice with allergic airway inflammation, especially neutrophils and eosinophils.

[0033] Pulmonary function tests revealed that the extracellular vesicles and the tolerant dendritic cells they induced reduced airway hyperresponsiveness, suppressing the lung airway resistance and lung elastic resistance of mice with allergic airway inflammation and improving lung compliance.

[0034] ELISA detection showed that extracellular vesicles and the tolerant dendritic cells they induced reduced the expression of pro-inflammatory molecules (IL-4, IL-17 and IL-5) and promoted the expression of anti-inflammatory molecules (IL-10 and TGF-β1).

[0035] Furthermore, it was surprising to find that in the mouse model of allergic airway inflammation treated with transplantation, a tolerance response was generated in the body, that is, immune tolerance CD11c was enhanced. + PD-1 + cells and CD44 + CD73 + TIGIT + The proliferation of anergic T cells, thereby activating the proliferation of regulatory T lymphocytes.

[0036] The above data indicate that extracellular vesicles derived from mesenchymal stem cells cultured in long-term hypoxia are more effective than those cultured in normoxia in treating chronic allergic airway inflammation and can better prevent airway remodeling in chronic airway inflammation.

[0037] The present invention provides a kind of stem cells prepared by culturing in a serum-free culture system under low-oxygen atmospheric environment conditions. The obtained extracellular vesicles with immunomodulatory function and the tolerant dendritic cells induced by them can be prepared into biological products and pharmaceutical compositions for the treatment of allergic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figure 3 shows the flow cytometry results of mesenchymal stem cells cultured to the 30th generation.

[0039] Figure 2 Comparison of the proliferation and telomerase activity of MSCs prepared in Example 2 and those prepared without key molecules

[0040] Figure 3Flow cytometry results of MSC-EVs-induced lymphocyte proliferation prepared in Example 4

[0041] Figure 4 Statistical analysis of the AHR results of the AAI mouse model treated with MSC-EVs prepared in Example 4 measured by a small animal respiratory function detector

[0042] Figure 5 Figure 4 shows the Difference in the immunoglobulin staining of immune cells in the alveolar lavage fluid after MSC-EVs treatment.

[0043] Figure 6 Results of ELISA testing of total serum IgE and IgG levels against HDM after MSC-EVs treatment

[0044] Figure 7 HE and PAS staining of lung tissue after MSC-EVs treatment prepared in Example 4

[0045] Figure 8 Figure 2 shows the flow cytometry results of the specific phenotypes of dendritic cells induced by No-EVs and Hy-EVs.

[0046] Figure 9 Flow cytometry of regulatory T cells after co-culture of No-EV-DCs and Hy-EV-DCs with PBLs

[0047] Figure 10 Figure 2 shows the flow cytometry results of IL-4 and IL-17 after co-culture of Tol-DCs prepared in Example 7 with lymphocytes.

[0048] Figure 11 Figure 7 shows the flow cytometry results of Tfh cells after co-culture of Tol-DCs and lymphocytes prepared in Example 7

[0049] Figure 12 Figure 2 is the flow cytometry result of PD-1 after co-culture of Tol-DCs and lymphocytes prepared in Example 7

[0050] Figure 13 Figure 7 shows the flow cytometry results of Tan cells after co-culture of Tol-DCs and lymphocytes prepared in Example 7

[0051] Figure 14 Statistical analysis of the results of AHR after treatment with Tol-DCs prepared in Example 7 measured by a small animal respiratory function detector

[0052] Figure 15 Figure 7 shows the immune cell count analysis of the alveolar lavage fluid of small animals after treatment with Tol-DCs prepared in Example 7

[0053] Figure 16Figure 1 shows the ELISA test for the secretion level of cytokines in the serum of mice after treatment with Tol-DCs prepared in Example 7. DETAILED DESCRIPTION

[0054] As used herein, the term "dendritic cells" is the largest antigen-presenting cell in the body and plays an important role in adaptive immunity. Not only can it phagocytize, process and present antigens and activate immune cells to fight tumors and viruses, but it can also produce tolerance under the action of anti-inflammatory molecules, and participate in immune regulation by producing tolerance to antigens, including allergens. Dendritic cells are mainly found in the thymus, lymph nodes and spleen in the body, and there are also a small amount of about 0.1%-4% in the blood. Therefore, they participate in the adaptive immunity caused by the main antigens of the body and are clinically used to treat tumors and viral infections. Its tolerance role has been paid attention to in recent years, and it has gradually been found that it plays a key role in autoimmune diseases and allergic diseases.

[0055] As used herein, the term "extracellular vesicles" refers to small, 40-1000 nm, double-membrane vesicles secreted by many cells. These vesicles carry a variety of proteins, lipids, DNA, mRNA, miRNA, and other molecules involved in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. The immunomodulatory extracellular vesicles of the present invention are derived from stem cells, namely mesenchymal stem cells, epithelial progenitor cells, subtotipotent stem cells, and endothelial progenitor cells. Due to their small size, excellent stability, and involvement in tissue and organ repair, particularly immune regulation, they represent a novel therapeutic agent.

[0056] The present invention provides a serum-free culture system, meaning that no animal or human serum is used during the cell culture process, nor are any other components containing extracellular vesicles. The serum-free culture system has a clear composition, and the stem cell culture supernatant can be collected after primary and continuous subculture and directly used for isolation and purification to produce extracellular vesicles. Furthermore, continuous culture is preferably performed in a three-dimensional culture environment under a hypoxic atmosphere, which better preserves stem cell activity, proliferation capacity, survival time, differentiation potential, and immunomodulatory function, closely resembling the internal environment of the human body. Both the stem cells themselves and the extracellular vesicles they secrete have improved tissue compatibility, making them more suitable for human application.

[0057] The stem cells described in the present invention include mesenchymal stem cells, epithelial progenitor cells, subtotipotent stem cells and endothelial progenitor cells, etc., which can be derived from discarded umbilical cord, umbilical cord blood, placenta, amniotic membrane, fat, urine, bone marrow, skin, menstrual blood, teeth, synovial fluid and synovial membrane, etc. Preferably, they are mesenchymal stem cells derived from umbilical cord and epithelial progenitor cells derived from amniotic membrane.

[0058] The method for culturing stem cells on a large scale of the present invention is to wash human tissue with physiological saline three times and cut it into 1 mm pieces with ophthalmic scissors. 3The tissue was minced for culture; or the single cell suspension isolated and purified from human tissue was filtered through a 40 μm cell sieve for culture, that is, cell culture medium containing 1 times serum-free culture supplement was added to resuspend, and the cells were counted after moss blue staining and counted according to 0.1-1×10 5 Cells / mL were added to the matrix hydrogel-coated cell culture chamber and grown in a three-gas incubator at 37°C, 5% CO2 and 1-10% hypoxic atmosphere. When the stem cells grew to more than 80% confluence in the cell culture chamber, they were digested and counted, and the concentration was calculated as 0.1-1×10 4 Cells / mL were added to a new matrix hydrogel-coated cell factory culture flask for subculture, i.e., grown in a three-gas incubator at 37°C, 5% CO2, and a hypoxic atmosphere of 1-10%, with fresh cell culture medium containing 1x serum-free supplement replaced every 3 days. When the stem cells reached more than 80% confluence, the stem cells were harvested by trypsin digestion as described above and cultured for 30 consecutive generations. The stem cells obtained after each passage were resuspended in physiological saline, stained with moss blue, counted, and refrigerated at 4°C until further use. The conditioned medium from the stem cell culture was collected after each passage for use in preparing extracellular vesicles.

[0059] The human tissues include discarded umbilical cord, umbilical cord blood, placenta, amnion, urine, fat, bone marrow, skin, menstrual blood, dental pulp, synovial fluid, and synovial membrane, etc., and the corresponding stem cells are prepared, namely umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental sub-totipotent stem cells, urine stem cells, amniotic epithelial progenitor stem cells, adipose stem cells, bone marrow mesenchymal stem cells, bone marrow endothelial progenitor cells, menstrual blood endometrial progenitor cells, dental pulp stem cells, synovial fluid mesenchymal stem cells, and synovial membrane mesenchymal stem cells. The stem cells are stored in a serum-free cryopreservation solution (commercial product) at -196°C in a liquid nitrogen tank for future use after thawing.

[0060] The hypoxic culture condition is a hypoxic atmosphere environment of 1-10%; preferably, a hypoxic atmosphere environment of 1-5% is selected.

[0061] The stem cells prepared after 30 passages of culture have a stronger cell proliferation rate than stem cells cultured under normal oxygen atmospheric conditions, and maintain good cell morphology and cell phenotype of the stem cells, and can differentiate into corresponding adult cells; preferably, the prepared stem cells survive longer in the animal body and have good stem cell characteristics.

[0062] Extracellular vesicles with immunomodulatory functions were isolated and purified from conditioned medium obtained from stem cell cultures using ultracentrifugation at 100,000 centrifugal force, ultrafiltration, size exclusion chromatography, and immunoaffinity methods. Particle size analysis revealed a range of 40-150 μm. Western blot analysis revealed the expression of extracellular vesicle markers including CD9, CD63, and TSG101.

[0063] Preferably, extracellular vesicles are added to lymphocytes for co-culture to inhibit CD3 + CD4 + 、CD3 + CD8 + and CD3 - CD56 / 16 + Lymphocyte proliferation, promotes the proliferation of regulatory T lymphocytes.

[0064] The conditioned medium of the present invention is a serum-free stem cell culture medium with defined components, namely, recombinant human albumin, serotonin, recombinant human transferrin, recombinant human insulin, ethanolamine, sodium selenite, β-mercaptoethanol, non-essential amino acids, alanyl-glutamine, a lipid concentrate with defined components, L-ascorbic acid-2-phosphate, progesterone, taurine, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human insulin-like growth factor, recombinant human platelet-derived growth factor, Iscove's Modified Dulbecco's Medium (IMDM) or Alpha Minimum Eagle's Medium (Alpha Minimum Eagle's Medium). Essential Medium (aMEM) or keratinocyte basal medium is included; at the same time, the present invention adds key small molecules, namely lipostatin-1, 2-hydroxy-D-glutamate disodium salt and activator polypeptide 740Y-P. The entire stem cell culture process does not involve animal and human serum or plasma, and therefore does not involve extracellular vesicles from other sources. The conditioned medium obtained from stem cell culture can be directly collected and the extracellular vesicles therein can be extracted.

[0065] Preferably, the serum-free culture supplement solution is placed in a freeze dryer for freeze drying to obtain a serum-free culture supplement freeze-dried powder, which is then sealed with a cap and stored in a refrigerator at 2-8° C. for ease of storage and transportation and a longer shelf life.

[0066] The immunomodulatory extracellular vesicles provided by the present invention were transplanted to treat a mouse model of allergic airway inflammation, producing significant therapeutic effects. The specific indicators are as follows:

[0067] Through Dif rapid staining and small animal blood routine tests, it was found that extracellular vesicles and the tolerogenic dendritic cells they induced reduced the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) of mice with allergic airway inflammation, especially neutrophils and eosinophils.

[0068] Pulmonary function testers showed that extracellular vesicles improved airway hyperresponsiveness, that is, they suppressed the lung airway resistance and lung elastic resistance of mice with allergic airway inflammation and increased lung compliance.

[0069] Enzyme-linked immunosorbent assay (ELISA) showed that extracellular vesicles and the tolerant dendritic cells they induced reduced the expression of pro-inflammatory molecules (IL-4, IL-17, and IL-5) and promoted the expression of anti-inflammatory molecules (IL-10 and TGF-β1).

[0070] Pathological staining of lung tissue using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining kit showed that the infiltration of inflammatory cells in the lungs was inhibited and the number of inflammatory cells in the lungs was significantly reduced; Masson's trichrome staining significantly reduced collagen fiber deposition in the pulmonary airways.

[0071] The method for preparing tolerant dendritic cells induced by extracellular vesicles with immunomodulatory function provided by the present invention comprises the following steps:

[0072] In the first step, mononuclear cells are obtained from human peripheral blood by density gradient centrifugation or apheresis using a blood cell separator. Adherent cells are induced with the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) (100-2000 IU / mL) and IL-4 (50-1000 IU / mL) or FMS-related tyrosine kinase 3 ligand (Flt-3L) (50-1000 IU / mL) for 4-7 days to obtain immature dendritic cells.

[0073] In step 2, extracellular vesicles (1 μg-200 μg / mL) are added to immature dendritic cells and the induction continues for 12-96 hours to obtain tolerogenic dendritic cells.

[0074] Extracellular vesicles with immunomodulatory functions induce tolerant dendritic cells, which downregulate CD11c molecules and co-stimulatory molecules (CD83, CD86, CD80, CD40) and express the immunosuppressive molecule PD-1, thereby promoting the proliferation of regulatory T lymphocytes.

[0075] Co-culture with lymphocytes inhibited CD3 + CD4 + cells, and the expression of IL-4 and IL-17.

[0076] Preferably, the tolerogenic dendritic cells suppress CD4 + CXCR5 +Follicular helper T cells effectively control the polarization of Th0 to Th2 / Th17;

[0077] More preferably, the tolerogenic dendritic cells promote the expression of PD-1 and CD44 + CD73 + TIGIT + The proliferation of anergic T cells enhances the differentiation of regulatory T lymphocytes.

[0078] The extracellular vesicle-induced tolerogenic dendritic cells with immunomodulatory function provided by the present invention were transplanted to treat a mouse model of allergic airway inflammation, producing significant therapeutic effects. The specific indicators are as follows:

[0079] Dif rapid staining and small animal blood routine tests showed that extracellular vesicle-induced tolerogenic dendritic cells reduced the number of inflammatory cells in bronchoalveolar lavage fluid, especially the number of neutrophils and eosinophils;

[0080] Pulmonary function tests showed that the tolerant dendritic cells induced by extracellular vesicles improved airway hyperresponsiveness, that is, they suppressed the lung airway resistance and lung elastic resistance of mice and increased lung compliance.

[0081] ELISA and qRT-PCR detection showed that extracellular vesicle-induced tolerogenic dendritic cells reduced the expression of pro-inflammatory molecules (IL-4, IL-17 and IL-5) and promoted the expression of anti-inflammatory molecules (IL-10 and TGF-β1).

[0082] In particular, in the mouse model of allergic airway inflammation treated with transplantation, a tolerance response was generated in the body, which activated the proliferation of regulatory T lymphocytes and increased the immune tolerance CD11c + PD-1 + cells and CD44 + CD73 + TIGIT + Proliferation of anergic T cells.

[0083] The extracellular vesicles with immunomodulatory function provided by the present invention and the tolerant dendritic cells induced therein can be prepared into biological products, pharmaceutical compositions for the treatment of allergic diseases, and cell therapy products for clinical treatment.

[0084] Hereinafter, specific embodiments of the present invention will be described, but the technical scope of the present invention is not limited to these examples.

[0085] Example 1 Preparation of serum-free supplement for stem cells

[0086] The purchased biological reagents were prepared into serum-free supplements at a 50-fold dilution. The specific preparation method was as follows: the components listed in Table 1 were weighed separately and dissolved in sterile deionized water. Recombinant human insulin was dissolved in 1 mM hydrochloric acid and then added to the solution. The solution was dissolved in a chromatography cabinet at 4°C under magnetic stirring for 30 minutes. The pH was adjusted to 7.2-7.6. The prepared solution was continuously filtered through 0.22 μm and 0.1 μm vacuum filters to remove bacteria and pathogens. The solution was then aseptically filled into vials or plastic bottles, each containing 10 mL. The vials were sealed with a cap and stored in a -86°C ultra-low temperature freezer.

[0087] The filled 10 mL vials or plastic bottles were placed in a freeze dryer for freeze drying to obtain serum-free additive freeze-dried powder, which was sealed with a cap and stored in a refrigerator at 2-8°C.

[0088] Table 1 The concentrations of the components of the 50x serum-free supplement are as follows:

[0089]

[0090]

[0091] The vial or plastic bottle is labeled and placed in a packaging box, and an instruction manual is placed in it, and then sealed to produce a product.

[0092] Example 2 Three-dimensional scale culture of mesenchymal stem cells under hypoxic atmospheric conditions

[0093] Discarded human umbilical cord, amniotic membrane or placenta (with informed consent signed by the donor) was soaked in 75% alcohol for 3-5 minutes and then placed in 2 mL of 1× PBS (pH 7.4) containing 2x double antibody, washed three times, and minced into 1 mm pieces with ophthalmic scissors. 3 The cells were transferred to a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded and the cell pellet was washed once with 1× PBS (pH 7.4). The cell pellet was resuspended in αMEM and IMDM (1:2, Gibco, USA) containing the 1× serum-free supplement prepared in Example 1. The pellet was stained with moss blue and counted according to the 1-10×10 6 cells / mL were added to a 10 cm cell culture dish coated with 3D cell culture hydrogel (Guangzhou Chuangsai Biotechnology) and grown in a three-gas incubator at 37°C, 5% CO2 and 1% hypoxic atmosphere for 48 h.

[0094] After 48 hours of culture, the mesenchymal stem cells were cultured with a 5 mL pipette, and the culture medium was removed. The serum-free medium containing αMEM and IMDM (1:2) was added. The cells were cultured in the 10 cm cell culture dish in a 37°C, 5% CO2, and 1-5% hypoxic atmosphere in a three-gas incubator. The culture medium was replaced with fresh culture medium every two days.

[0095] Mesenchymal stem cells grew in a cell culture vessel and reached more than 80% fusion. The culture medium was discarded with a 5 mL pipette, 5 mL of 1×PBS was taken to wash the bottom of the culture flask once, 0.5 mL of 0.25% trypsin (containing 0.05% EDTA) (Shanghai Yisheng Biological) was added to the culture flask, and the flask was placed in a 37°C, 5% CO2 incubator for digestion for 2-3 minutes; after digestion, 200 μL of fetal bovine serum was added to terminate the digestion, and 5 mL of normal saline was added, pipetted with a 5 mL pipette, and aspirated into a 15 mL centrifuge tube, and then washed once with 5 mL of normal saline, added to the same 15 mL centrifuge tube, and centrifuged at 1000 rpm for 5 minutes to collect the mesenchymal stem cells; after centrifugation, the cells were resuspended with 1 mL of fresh culture medium, counted, and 10 mL of fresh cell culture medium was added to a new cell factory culture bottle coated with matrix hydrogel and continued to grow in a three-gas incubator at 37°C, 5% CO2 and 1% hypoxic atmosphere. Fresh cell culture medium containing 1x serum-free additive was replaced every 3 days and the cell culture medium was collected. When the stem cell growth and fusion reached more than 80%, the mesenchymal stem cells were collected by trypsin digestion as described above, and the cells were scaled up in the cell factory culture bottle and subcultured to the 10th, 20th and 30th generations. The obtained mesenchymal stem cells were resuspended in physiological saline, stained with moss blue and counted to obtain mesenchymal stem cells. They were placed in a refrigerator at 4°C for storage. The 10th, 20th and 30th generations of mesenchymal stem cells were cultured in a cell culture vessel with matrix hydrogel under hypoxic atmosphere conditions and observed under an inverted microscope. The cells were observed under a low-power microscope (4x microscope) and showed small protrusions, round or spindle-shaped, growing in a spiral shape (see Figure 1 ), A and B show the morphology of MSCs at passages 20 and 30, respectively. The mesenchymal stem cells prepared by the present invention achieved a proliferation rate exceeding 3200-fold at passage P10; this demonstrates that culturing mesenchymal stem cells in a matrix hydrogel-coated cell culture chamber under hypoxic atmospheric conditions exhibits excellent proliferation capacity, significantly increasing the expansion rate.

[0096] Example 3 Mesenchymal stem cell phenotype, proliferation activity and telomerase activity

[0097] Take 3×10 6MSCs at passage 30 were divided into three groups. Group 1 was the isotype control and was added with 20 μL FITC-labeled mouse IgG1, 20 μL PE-labeled mouse IgG1 and 20 μL PerCP-labeled mouse IgG1; Group 2 was added with 20 μL FITC-labeled mouse anti-human CD34 monoclonal antibody, 20 μL PE-labeled mouse anti-human CD90 monoclonal antibody and 20 μL PerCP-labeled mouse anti-human HLA-DR monoclonal antibody; Group 3 was added with 20 μL FITC-labeled mouse anti-human CD44 monoclonal antibody and 20 μL PE-labeled mouse anti-human CD73 monoclonal antibody; Group 4 was added with 20 μL FITC-labeled mouse anti-human CD45 monoclonal antibody and 20 μL PE-labeled mouse anti-human CD105 monoclonal antibody (all flow cytometry antibodies were purchased from Biolegend, USA).

[0098] The samples were placed in a 4°C refrigerator for staining for 30 minutes, then washed three times with 1 mL of 1× PBS (pH 7.4), and finally resuspended in 0.5 mL of 1× PBS. The washed cells were detected using a FACS Calibur flow cytometer (BD Biosciences, USA). Figure 1 This is a flow cytometry test result of mesenchymal stem cells cultured to the 30th passage. The results show that the human MSCs prepared by the present invention highly express CD90, CD73, CD105 and CD44, and lowly express CD34, CD45 and HLA-DR, indicating that MSCs can still maintain the requirements of mesenchymal stem cell phenotypic markers after continuous culture to the 30th passage.

[0099] MSCs were cultured without key molecules (lipostatin-1, 2-hydroxy-D-glutamate disodium salt and activator polypeptide 740Y-P) and replaced with currently commonly used human platelet lysate. The proliferation activity and telomerase activity of the prepared MSCs were tested together with the MSCs prepared in Example 2. Figure 2 The results of CCK-8 kit test in A show that the proliferation ability of human MSCs prepared by the present invention is much higher than that of MSCs without adding key molecules (**P<0.01, ***P<0.001 and ****P<0.0001), and it has better telomerase activity (see Figure 2 B), indicating that the MSCs prepared by the present invention have better proliferation activity and cell stemness.

[0100] Example 4 Preparation of Extracellular Vesicles from Mesenchymal Stem Cell Conditioned Medium

[0101] Example 2 During the culture of mesenchymal stem cells in a hypoxic environment, the conditioned medium was collected before each liquid exchange and passage digestion, and the ultra-high-speed refrigerated centrifugation method was as follows: the conditioned medium was centrifuged once at 1000 × g for 10 min, once at 3000 × g for 15 min, and once at 10,000 × g for 30 min. The supernatant was then transferred to an ultra-transparent tube, balanced, and centrifuged at 100,000 × g for 1.5 h in a SW32Ti rotor at 4 ° C (Beckman Coulter, USA), and then the pellet after centrifugation was carefully suspended in 1 mL of PBS and used immediately or stored in a -80 ° C ultra-low temperature freezer, i.e., extracellular vesicles derived from MSCs cultured in a hypoxic atmosphere (Hypoxic cultured MSC derived extracellular vesicles, Hy-EVs). The experimental control was normoxic cultured MSC-derived extracellular vesicles (No-EVs), that is, cultured in an incubator at 37°C, 5% CO2 and 21% normal oxygen, using the currently commonly used αMEM and IMDM (1:2, Gibco, USA) culture medium without the key molecules lipostatin-1, 2-hydroxy-D-glutamate disodium salt and activator peptide 740Y-P, and supplemented with human platelet lysate.

[0102] The extracted extracellular vesicles all expressed CD9, CD63, and TSG101 exosome-specific markers, and their surface structure was observed using a scanning electron microscope. Scanning electron microscopy and ultrasensitive flow cytometry detected the morphology of the mesenchymal stem cell-derived extracellular vesicles prepared in Example 2, and their particle size was 75.83 nm, ranging from 30 nm to 150 nm, which meets the exosome particle size standard.

[0103] The extracted extracellular vesicles were quantified using a BCA protein detection kit (Thermo Fisher Scientific, USA). According to the kit instructions, a microplate reader was used to prepare a standard curve to calculate the protein concentration and quantify it.

[0104] Table 2 Content of extracellular vesicles derived from mesenchymal stem cells in conditioned medium

[0105]

[0106] Take 25 ml of conditioned medium from MSCs of different passages, and the corresponding MSC cell number is 1×10 7, and then the EVs were extracted. The results in Table 2 show that the human MSCs prepared by the present invention can secrete more EVs, which is much higher than that of the normoxic control (**P < 0.01), indicating that more extracellular vesicles can be extracted by the preparation method of the present invention, which can become an important process indicator for the large-scale preparation of extracellular vesicles.

[0107] Example 5 Analysis of the immunomodulatory function of extracellular vesicles derived from mesenchymal stem cells

[0108] 20 mL of peripheral blood was collected from five healthy donors (who signed informed consent forms) and peripheral blood lymphocytes (PBLs) were obtained after density gradient centrifugation with lymphocyte separation medium. PBLs were divided into 1×10 6 Cells were added into a 6-well plate containing RPMI1640 containing 10% fetal bovine serum (FBS, Gibco, USA);

[0109] Hy-MSC-EVs and No-MSC-EVs (20 μg / mL) prepared in Example 4 were added to PBLs in a 6-well plate in RPMI 1640 containing 10% FBS, and then cultured continuously in a 5% CO2, 37°C incubator for 3 days.

[0110] On day 3, suspension cells were collected and stained with FITC-CD3, PE-CD56 / 16, and PerCP-CD8 (all from BioLegend) for 30 minutes at 4°C in the dark, and their surface markers were detected according to the above protocol. Other suspension cells were stained with FITC-Foxp3, PE-CD25, and PerCP-CD4 to detect the intracellular expression of Foxp3 using fixation buffer and intracellular staining permeabilization and washing buffer (BioLegend) according to the manufacturer's instructions. The intracellular control was PE mouse IgG1, kappa isotype.

[0111] After EVs were added to PBL for culture, MSC-EVs showed good immunomodulatory ability, especially in a hypoxic environment. Figure 3 The flow cytometry results of MSC-EVs-induced lymphocyte proliferation, including T cells, NK cells and Treg cells. The percentage of MSC-EVs-inhibited lymphocytes, including CD3 + CD8 + T cells and CD3 - CD56 / 16 +NK cells, which was significantly different between Hy-EVs and No-EVs (4.27-fold and 2.07-fold, 9.47-fold and 2.35-fold, respectively). Compared with PBL, both Hy-EVs and No-EVs stimulated CD4 + CD25 + Foxp3 + The production of Treg cells was significantly increased (increased by 2.18 and 3.50 times, respectively), but Hy-EVs had a stronger promoting effect compared with No-EVs. This suggests that MSC-EVs have good immunomodulatory function and can be used to treat allergic diseases, especially Hy-EVs.

[0112] Example 6 In vivo experiment on the treatment of asthma model with extracellular vesicles derived from mesenchymal stem cells

[0113] Six-week-old female Balb / c mice were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (Changsha, China). The mice were randomly divided into the following five groups (n = 8 mice per group): normal control group, AAI model (PBS treatment), HDM treatment group, No-EVs prepared in Example 4 combined with HDM treatment (No-EVs treatment), and Hy-EVs prepared in Example 4 combined with HDM treatment (Hy-EVs treatment). According to previous reports, chronic AAI was induced. Briefly, from day 0, mice were sensitized with 50 μg HDM (Greerlabs, USA) and 5 mg aluminum hydroxide (Sigma) in 100 μL PBS by intraperitoneal injection, once every 2 weeks for a total of two times, except for the control group. No-EVs and Hy-EVs (200 μg / 200 μL PBS) were injected via the tail vein every other day, and HDM (250 μg / 100 μL PBS) was injected subcutaneously every other day from day 35 to day 40, for a total of three times. Starting from day 47, HDM (25 μg / 25 μL PBS) was challenged intranasally every other day for a total of six times. The mice were sacrificed on day 58.

[0114] Experimental mice airway hyperresponsiveness (Airway hyperresponsiveness, AHR) analysis: the AHR of experimental mice was measured by FlexiVent system (U.S. SCIREQ company). The ventilator was set to produce a tidal volume of 10mL / kg at a frequency of 150 breaths per minute. Continuous doses of 3.125, 6.25, 12.5, 25, 50 and 100mg / mL of methacholine chloride (MethacholineChloride, Mch, U.S. MCE company) were atomized to challenge the airway until the dose caused continuous airway resistance, which was approximately four to five times the baseline. Respiratory resistance was measured by the disturbance of SnapShot, including respiratory resistance (resistance of respiratory system, Rrs), elastic resistance (elastance resistance, Ers) and static compliance (compliance of respiratory systern, Crs).

[0115] Figure 4 The statistical analysis of the AHR results of MSC-EVs-treated AAI mouse model measured by a small animal respiratory function tester showed that Rrs and Ers continued to increase with increasing Mch concentrations, with both Rrs and Ers increasing sharply in the AAI model and HDM treatment groups. Comparing the results of two concentrations (31.25 and 62.5 mg / mL Mch) compared to HDM treatment, both No-EVs and Hy-EVs treatments miraculously reduced Rrs (5.02- and 3.74-fold in No-EVs, and 9.77- and 13.54-fold in Hy-EVs) and Ers (9.25- and 35.51-fold in No-EVs, and 3.55- and 5.11-fold in Hy-EVs). At the same time, both No-EVs and Hy-EVs treatment significantly promoted AHR Crs (No-EVs increased by 8.29-fold and 1.46-fold, respectively, and Hy-EVs increased by 8.75-fold and 3.21-fold, respectively), with significant differences compared to the AAI model and HDM treatment. Furthermore, Hy-EVs treatment also achieved a superior Crs to No-EVs treatment. This confirms that MSC-EVs can modulate inflammatory responses and improve AHR, with the effect of Hy-EVs being more pronounced.

[0116] Bronchoalveolar lavage fluid (BALF) was collected after the last HDM intranasal challenge. After euthanasia, the trachea was ligated at the upper layer, and 1 mL of cold PBS was gently instilled into the nasopharynx via a 21-gauge catheter. The collected BALF was centrifuged at 3000 rpm for 5 minutes at 4°C. The cells in the BALF were resuspended in 50 μL of PBS and then stained for immune cells using Diffusion's fast staining. Different immune cells were stained with different colors, namely monocytes, basophils, eosinophils, and neutrophils.

[0117] Figure 5 Figure 2 shows a Diffusion stain of immune cells in bronchoalveolar lavage fluid (BALF) after MSC-EV treatment. Counting these stained immune cells revealed a decrease in both total and viable cell counts across all treatment groups, with the Hy-EVs-treated group showing the lowest number, significantly different from the No-EVs-treated group. Compared to the AAI model, both the HDM and MSC-EVs-treated groups significantly reduced monocyte levels in the BALF, similar to those in the normal control group. However, the HDM-treated group increased basophil and neutrophil numbers, significantly exceeding those in the MSC-EVs-treated group. Compared to the AAI model and HDM-treated groups, both the No-EVs and Hy-EVs-treated groups significantly suppressed basophil and neutrophil proliferation in the BALF. Furthermore, while the AAI model dramatically increases eosinophil numbers in the BALF, the inhibitory effect of each treatment group was far less than that observed in the AAI model (reductions of 77.08%, 57.67%, and 82.62% in the HDM, No-EVs, and Hy-EVs-treated groups, respectively). The number of eosinophils in the Hy-EVs treatment group was also lower than that in the No-EV treatment group (decreased by 58.95%).

[0118] Anti-HDM-specific immunoglobulin detection: After the experimental mice were sacrificed, the total serum IgE and IgG against HDM were measured using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Jingmei Biotechnology).

[0119] Figure 6 Figure 3 shows the results of ELISA testing for total serum IgE and IgG against HDM after MSC-EV treatment. Total HDM-IgE levels were significantly higher in the AAI model and HDM-treated groups compared to the other treatment groups. Both the No-EVs and Hy-EVs-treated groups showed significantly lower levels of total HDM-IgE and IgG compared to the AAI model and HDM-treated groups.

[0120] Lung histopathology: After BALF collection, lung tissue samples were obtained and fixed in 4% neutral buffered formalin for 48 hours, dehydrated, and embedded in paraffin. Paraffin-embedded sections (4 μm thick, 3 sections per animal, 8 animals per group) were then stained with hematoxylin and eosin (H&E) and a periodic acid-Schiff staining kit (PAS, Beijing Solebao Co., Ltd.) to assess lung inflammation and collagen fiber levels. HE staining was observed using an upright microscope and lung inflammation was scored in a blinded manner. Inflammation was graded as follows: grade 0 (no inflammatory cells observed), grade 1 (occasionally inflammatory cells observed), grade 2 (mucosa surrounded by 1–3 layers of inflammatory cells), grade 3 (4–5 layers of inflammatory cells around the mucosa or blood vessels), and grade 4 (most mucosa or blood vessels surrounded by more than 5 layers of inflammatory cells).

[0121] Figure 7 HE and PAS staining images of lung tissue after MSC-EVs treatment. The AAI model showed a large amount of inflammatory infiltration around the bronchi and blood vessels, and obvious mucus production in the epithelial layer. Compared with the AAI model and HDM treatment, both No-EVs and Hy-EVs treatments significantly reduced allergic inflammation and inhibited mucus secretion (HE scores decreased by 38.46% and 61.54% compared with HDM treatment, and PAS scores decreased by 38.24% and 70.59%, respectively); In addition, the inflammation and PAS scores of mice treated with Hy-EVs were much lower than those of mice treated with No-EVs (decreased by 37.5% and 52.38%, respectively). These data indicate that Hy-EVs can better alleviate chronic airway inflammation.

[0122] Example 7: Mesenchymal stem cell-derived extracellular vesicles induce tolerogenic dendritic cells

[0123] 50 mL of normal human peripheral blood was collected and peripheral blood mononuclear cells (PBMCs) were obtained after density gradient centrifugation with lymphocyte separation medium. PBMCs were resuspended in RPMI 1640 medium and counted. 6×10 6PBMCs were added to 6-well plates, 3 mL per well, and incubated in a 5% CO2, 37°C incubator for 1.5 hours. Adherent monocytes were cultured in complete medium (RPMI 1640, Gibco, supplemented with 100 ng / mL recombinant human Fms-related tyrosine kinase 3 ligand, Flt3-L, Peprotech, USA) to induce immature DCs (imDCs) for 4 days. On day 4, imDCs were induced to generate Tol-DCs and then supplemented with No-MSC-EVs or Hy-MSC-EVs (40 μg / mL) for 48 hours. Simultaneously, on day 4, HDM (5 μg / mL) was added to prime imDCs for 48 hours. EV-induced Tol-DCs or HDM-primed DCs were harvested on day 7 and used in subsequent experiments.

[0124] By inducing mesenchymal stem cell-derived exosomes, we have further understood the immunomodulatory properties of tolerogenic dendritic cells (Tol-DCs). In in vitro experiments, we used MSC-EVs to induce Tol-DCs in peripheral blood. Simultaneously, dendritic cells induced by lipopolysaccharide (LPS) (LPS-DCs) served as a normal control, and dendritic cells induced by IL-10 and TGF-β1 (IL10 / TGF-DCs) served as a positive control. Numerous studies have demonstrated that both IL-10 and TGF-β1 have the ability to induce the generation of Tol-DCs.

[0125] Tolerogenic dendritic cells were collected, resuspended in RPMI 1640 containing 10% FBS, and counted. 0.4×10 6 Cells were stained in the dark at 4°C for 30 minutes with four flow cytometry antibody panels: Panel 1: FITC-CD11c, PE-CD80, and PerCP-HLA-DA; Panel 2: FITC-CD11c, PE-CD83, and PerCP-CD44; Panel 3: FITC-CD1α, PE-CD80, and PerCP-CD40; and Panel 4: isotype controls FITC-mouse IgG1, PE-mouse IgG1, and PerCP-mouse IgG1 (all from BioLegend). After staining, the cells were washed twice with 1 mL of PBS, centrifuged at 1500 rpm for 5 minutes, resuspended in 500 μL of PBS, filtered through a 40 μm cell filter, and analyzed on a flow cytometer (FACS Calibur, BD Biosciences, USA) using FlowJo flow cytometry software.

[0126] Figure 8 The results of flow cytometry analysis of the specific phenotypes of dendritic cells induced by No-EVs and Hy-EVs are shown in Figure 2. + CD11c + The proportion of cells decreased significantly. In addition, we also detected the positive rate of dendritic cell co-stimulatory factors, and the results showed that compared with LPS-DCs, the positive rate of CD83 in IL10 / TGF-DCs, No-EV-DCs and Hy-EV-DCs was greatly reduced, among which the reduction in Hy-EV-DCs was particularly prominent and significantly lower than that in No-EV-DCs. Compared with LPS-DCs and IL10 / TGF-DCs, other co-stimulatory factors of No-EV-DCs and Hy-EV-DCs, such as CD86, CD80 and CD40, also showed a downward trend. CD80 in No-EV-DCs and Hy-EV-DCs + The percentage of cells was significantly lower than that of LPS-DCs, and CD40 + The cell percentage decreased.

[0127] Example 8 Co-culture of Tolerogenic Dendritic Cells with Lymphocytes Promotes Polarization of Regulatory Lymphocytes

[0128] The Tol-DCs induced in Example 7 were resuspended in RPMI 1640 containing 10% FBS and counted. 6 Cells were added to 6-well plates in RPMI 1640 containing 10% FBS and plated at a volume of 2.5 × 10 6 The cells were added to PBLs in a volume of 3 mL and then co-cultured in a 5% CO2, 37°C incubator for 3 consecutive days.

[0129] On the third day, the suspended cells were collected and stained with two sets of flow cytometry antibodies in the dark for 30 minutes in a 4°C refrigerator, namely FITC-CD185, PE-PD-1 and PerCP-CD4, or FITC-FR, PE-CD73, PerCP-CD44 and APC-TIGIT (all from BioLegend). Their surface markers were examined according to the above operation. The cells were intracellularly stained with FITC-CD25, PE-Foxp3 and PerCP-CD4 to detect the intracellular expression of Foxp3 according to the above operation. The intracellular control was PE-labeled mouse IgG1, κ isotype. The cells were detected on a flow cytometer and analyzed by FlowJo flow analysis software.

[0130] Figure 9Figure 2 shows the flow cytometry of regulatory T cells after No-EV-DCs and Hy-EV-DCs were co-cultured with PBLs. We observed that both No-EV-DCs and Hy-EV-DCs promoted CD4 + CD25 + Foxp3 + The number of regulatory T cells (Treg cells) increased. Compared with LPS-DCs, the proportion of Treg cells increased to varying degrees. This result strongly confirms that MSC-EVs likely act on dendritic cells, inhibiting the expression of co-stimulatory molecules and upregulating the proliferation of Treg cells. This regulatory effect of Hy-EVs was more significant.

[0131] Example 9: Co-culture of Tolerogenic Dendritic Cells with Lymphocytes Promotes Polarization of Regulatory Lymphocytes

[0132] To confirm whether exosomes can initiate immunomodulatory effects in allergic patients, we recruited four healthy volunteers and four patients with airway inflammation allergic to HDM. Tolerogenic DCs were induced according to Example 7 and co-cultured with lymphocytes according to the method of Example 8. After co-culture of dendritic cells induced by mesenchymal stem cell exosomes with PBLs, MSC-EV-DCs promoted the proliferation of lymphocytes; however, after sensitization with house dust mites, the number of lymphocytes decreased significantly, especially the effect of Hy-EV-DCs, which was more significant, both in healthy donors and patients with allergic airway inflammation (AAI).

[0133] We also detected the expression of key pro-inflammatory cytokines IL-4 and IL-17 in AAI patients allergic to house dust mites by flow cytometry. Figure 10 Figure 2 shows the flow cytometry results of IL-4 and IL-17 after co-culture of HDM-sensitized DCs and lymphocytes after MSC-EV induction. + IL-4 + Th2 cells and CD4 + IL-17 + However, regardless of whether they were sensitized to house dust mites, the mean fluorescence intensity (MFI) of the dendritic cells induced by untreated exosomes (No-EV-DCs) and Hy-EV-DCs was greatly reduced. In addition, Hy-EV-DCs could significantly inhibit the proliferation of CD4 + IL-4 + Th2 cells and CD4 + IL-17+ The proliferation of Th17 cells was much stronger than that of No-EV-DCs, especially after sensitization with house dust mites.

[0134] We also examined the effect of mesenchymal stem cell exosome-induced dendritic cells (MSC-EV-DCs) on the initial CD4 + CD4 during cell polarization + CXCR5 + The production of follicular helper T cells (Tfh cells). Figure 11 Figure 2 shows the flow cytometry results of Tfh cells after co-culture of HDM-sensitized DCs and lymphocytes after MSC-EV induction. In AAI patients, both lipopolysaccharide-induced dendritic cells (LPS-DCs) and HDM-DCs promoted CD4 + CXCR5 + Tfh cell proliferation was higher than that of No-EV-DCs and Hy-EV-DCs, regardless of whether they were sensitized to house dust mites. Furthermore, the number of Tfh cells was lower after sensitization to house dust mites. Similar observations were also observed in healthy donors. However, after house dust mite sensitization, MSC-EV-DCs exhibited a more pronounced inhibitory effect on Tfh cell generation than HDM-DCs.

[0135] At the same time, we also analyzed the expression of programmed cell death protein 1 (PD-1) in dendritic cells that stimulated peripheral blood lymphocytes. Figure 12 The results of flow cytometry analysis of PD-1 after co-culture of HDM-sensitized DCs and lymphocytes after MSC-EV induction. + PD-1 + In addition, there was a significant difference between MSC-EV-DCs and HDM-DCs in patients with AAI. However, this result was not observed in healthy donors, although CD4 + PD-1 + The percentage of cells also increased slightly.

[0136] In addition, we further detected the generation of anergic T cells (Tan cells) in AAI patients. Figure 13 Figure 2 shows the flow cytometry results of Tan cells after co-culture of HDM-sensitized DCs and lymphocytes after MSC-EV induction. It was found that in MSC-EV-DCs, regardless of whether they were sensitized by house dust mites, CD44+ CD73 + TIGIT + Tan cells increased. However, there was no statistical difference between No-EV / HDM-DCs and sHy-EV / HDM-DCs. In addition, we did not observe similar results when analyzing Tan cells from healthy donors. We only found that dendritic cells induced by mesenchymal stem cell exosomes increased CD73 + The number of cells increased, but there was no statistical difference. This indicates that the dendritic cells induced by No-EVs and Hy-EVs can inhibit the generation of Th2, Th17 cells and Tfh cells, and promote the proliferation of CD4 + PD-1 + cells and CD44 + CD73 + TIGIT + The proliferation of immune regulatory cells, including Tan cells, was stimulated, thereby initiating an immunomodulatory effect on patients with house dust mite allergy in vitro.

[0137] Example 10 In vivo experiment on the treatment of asthma model with mesenchymal stem cell-derived extracellular vesicles and induced tolerogenic dendritic cells

[0138] Dendritic cells were generated from bone marrow mononuclear cells of normal mice. Monocytes were cultured in complete medium (RPMI 1640, Gibco) supplemented with 100 ng / mL recombinant mouse Flt3-L (Peprotech, USA) and cultured continuously for 4 days in a 5% CO2, 37°C incubator. On day 4, imDCs were induced to produce Tol-DCs by adding No-EV and Hy-EV (40 μg / mL) and culturing for 48 hours. At the same time, HDM (5 μg / mL) was added on day 4 and cultured for 48 hours to sensitize imDCs. Tol-DCs induced by MSC-EVs or DCs sensitized by HDM were harvested on day 7 and used for subsequent experiments.

[0139] Six-week-old female Balb / c mice were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (Changsha, China). The mice were randomly divided into the following five groups (n = 8 mice per group): normal control group, AAI model (PBS treatment), HDM-sensitized DCs treatment group (HDM), No-EVs prepared in Example 7 induced and HDM-sensitized Tol-DCs treatment (No-EV / HDM-DCs treatment), Hy-EVs prepared in Example 7 induced and HDM-sensitized Tol-DCs treatment (Hy-EV / HDM-DCs treatment. According to previous reports, chronic AAI was induced. Briefly, from day 0, mice were sensitized with 50 μg HDM (Greerlabs, USA) and 5 mg aluminum hydroxide (Sigma) in 100 μL PBS by intraperitoneal injection, once every 2 weeks for a total of two times, except for the control group. DCs (10 5 The mice were injected intranasally with 25 μg of HDM (25 μg / 25 μL PBS) every other day for a total of 3 times. Starting from day 47, HDM (25 μg / 25 μL PBS) was intranasally challenged every other day for a total of 6 times. The mice were sacrificed on day 58.

[0140] AHR analysis of experimental mice: The specific steps were carried out according to Example 6. Figure 14 The statistical analysis of the results of AHR after treatment with Tol-DCs induced by MSC-EVs prepared in Example 7 was measured by a small animal respiratory function detector. We found that it was most effective in the Hy-EV / HDM-DCs transplantation group, but compared with the AAI model and HDM-DCs transplantation, No-EV / HDM-DCs transplantation also significantly reduced Rrs and Ers, and improved the Crs of AHR. At a concentration of 100 mg / ml of methancholine, compared with HDM-DCs transplantation, the Rrs and Ers of No-EV / HDM-DCs implantation were significantly reduced (decreased by 18.77% and 24.39%, respectively), and the Rrs and Ers of Hy-EV / HDM-DCs transplantation decreased sharply by 48.66% and 49.8%, respectively. In addition, compared with HDM-DCs transplantation, the Crs of No-EV / HDM-DCs and Hy-EV / HDM-DCs transplantation increased significantly by 1.28 and 1.90 times, respectively. These results not only indicate that DCs play a key role in HDM-induced allergic airway inflammation but also demonstrate the role of MSC-EVs, especially Hy-EVs, in inducing tolerance to DCs in vivo.

[0141] Bronchoalveolar lavage fluid (BALF) was collected after the last HDM intranasal challenge. After euthanasia, the trachea was ligated at the upper layer, and 1 mL of cold PBS was gently dripped into the nasopharynx through a 21-gauge catheter. The collected BALF was centrifuged at 3000 rpm for 5 minutes at 4°C. The cells in the BALF were resuspended in 50 μL of PBS, and then the immune cells, including monocytes, basophils, eosinophils, and neutrophils, were counted by a small animal blood routine test (Nanjing Huaren Biotechnology).

[0142] Figure 15 This is a graph of immune cell count analysis of small animal blood routine in alveolar lavage fluid after treatment with MSC-EVs-induced Tol-DCs prepared in Example 7. It was observed that HDM-DCs transplantation caused strong allergic airway inflammation, which may be more serious than the AAI model. Compared with the AAI model and HDM-DCs transplantation groups, both No-EV / HDM-DCs and Hy-EV / HDM-DCs transplantation were better able to prevent the increase in the number of inflammatory cells in BALF. Even more surprisingly, by comparing the AAI model and HDM-DCs transplantation, MSC-EV / HDM-DCs not only suppressed the number of granulocytes, but also produced an inhibitory effect on lymphocytes and intermediate cells. Among them, the Hy-EV / HDM-DCs transplantation group produced a more effective inhibitory effect than the No-EV / HDM-DCs transplantation group. Compared with the HDM-DC transplantation group, No-EV / HDM-DCs transplantation reduced the number of immune cells in BALF, including a 52.9% decrease in white blood cells, a 57.4% decrease in lymphocytes, a 46.43% decrease in intermediate cells, and a 48.97% decrease in granulocytes. Hy-EV / HDM-DCs transplantation reduced white blood cells by 84.8%, lymphocytes by 83.2%, intermediate cells by 89.29%, and granulocytes by 85.57%, essentially the same as in the normal control group.

[0143] Detection of cytokine secretion levels in mouse serum: After the experimental mice were sacrificed, the pro-inflammatory factors IL-4, IL-5 and IL-17, and the anti-inflammatory factor IL-10 were measured using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Jingmei Biotechnology).

[0144] Figure 16The ELISA test shows the secretion level of cytokines in the serum of mice after treatment with Tol-DCs induced by MSC-EVs prepared in Example 7. It was found that the IL-4 secretion of No-EV / HDM-DCs and Hy-EV / HDM-DCs transplantation was significantly reduced and lower than that of HDM-DCs transplantation (decreased by 9.99% and 22.72%, respectively). In addition, the transplantation of Hy-EV / HDM-DCs was more obvious than that of No-EV / HDM-DCs transplantation (decreased by 14.14%). We did not observe a downregulation of IL-5 secretion in serum by MSC-EV / HDM-DCs transplantation. Compared with HDM-DCs transplantation, Hy-EV / HDM-DCs transplantation significantly reduced the secretion of IL-17 in serum (decreased by 11.20% and 37.73%, respectively). The No-EV / HDM-DCs and Hy-EV / HDM-DCs transplant groups showed improved serum IL-10 secretion, significantly different from the AAI model and HDM-DCs transplant groups (increased by 1.36 and 1.43 times compared to the AAI model, and by 1.09 and 1.14 times compared to HDM-DCs, respectively). Transplantation of MSC-EV / HDM-DCs can better block the secretion of pro-inflammatory cytokines (IL-4 and IL-17) and promote the production of anti-inflammatory cytokines (IL-10).

Claims

1. A method for preparing extracellular vesicles with immunomodulatory function, characterized in that: The extracellular vesicles are prepared by separating and purifying the conditioned medium collected from large-scale culture of stem cells under low-oxygen atmospheric conditions.

2. The method for preparing extracellular vesicles with immunomodulatory function according to claim 1, characterized in that: The method comprises: (1) simulating a hypoxic atmospheric environment of the body, preferably with an oxygen atmosphere concentration of 1-10%, more preferably 1-5% (volume ratio); (2) then passage the stem cells in serum-free conditioned medium from the primary culture to the P30 generation; and collecting the conditioned medium from each passage; Preferably, in step (2), the stem cells are cultured in a three-dimensional culture system of hydrogel or microcarrier.

3. The method for preparing extracellular vesicles with immunomodulatory function according to claim 1, characterized in that: The extracellular vesicles are preferably capable of inducing CD11c + PD-1 + Generation of tolerogenic dendritic cells; and / or The extracellular vesicles are preferably capable of activating CD44 + CD73 + TIGIT + anergic T cells and CD4 + CD25 + Foxp3 + Regulatory T lymphocyte proliferation; and / or The extracellular vesicles are preferably capable of inhibiting the expression of pro-inflammatory molecule 1, promoting the expression of anti-inflammatory molecule 2, and inhibiting CD4 + CXCR5 + Follicular helper T cells effectively control the polarization of Th0 to Th2 / Th17; further preferably, the pro-inflammatory molecule 1 is one or more of IL-4, IL-17 and IL-5; the anti-inflammatory molecule 2 is one or more of IL-10 and TGF-β1; and / or The extracellular vesicles and the tolerance-inducing dendritic cells thereof produce good anti-allergic and immune tolerance activities in vivo and in vitro.

4. The method for preparing extracellular vesicles with immunomodulatory function according to any one of claims 1 to 3, characterized in that: The extracellular vesicles are prepared by isolating and purifying the conditioned medium prepared by stem cell culture; the particle size analysis shows that the particles are between 40 and 150 μm; and the protein blotting analysis shows that the extracellular vesicle markers CD9, CD63 and TSG101 are expressed; Preferably, the extracellular vesicles are added to lymphocytes for co-culture to inhibit CD3 + CD4 + 、CD3 + CD8 + and CD3 - CD56 / 16 + Lymphocyte proliferation, promotes the proliferation of regulatory T lymphocytes.

5. The method for preparing extracellular vesicles with immunomodulatory function according to any one of claims 1 to 3, characterized in that: The culture medium contains one or more of recombinant human serum albumin, serotonin, recombinant human transferrin, recombinant human insulin, ethanolamine, sodium selenite, β-mercaptoethanol, non-essential amino acids, alanyl-glutamine, a lipid concentrate with defined ingredients, L-ascorbic acid-2-phosphate, progesterone, taurine, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human insulin-like growth factor, recombinant human platelet-derived growth factor, lipostatin-1, 2-hydroxy-D-glutamic acid disodium salt and activator polypeptide 740Y-P, Iscove's modified Dulbecco's medium, alpha low-limit Eagle's medium and keratinocyte basal medium; preferably contains lipostatin-1, 2-hydroxy-D-glutamic acid disodium salt and activator polypeptide 740Y-P.

6. The method for preparing extracellular vesicles with immunomodulatory function according to claim 2, characterized in that: The stem cells are one or more of mesenchymal stem cells, epithelial progenitor cells and endothelial progenitor cells; Preferably, the source includes discarded human umbilical cord, cord blood, placenta, amniotic membrane, urine, fat, bone marrow, skin, menstrual blood, dental pulp, synovial fluid or synovial membrane.

7. Extracellular vesicles with immunomodulatory function prepared according to the preparation method according to any one of claims 1 to 6.

8. A method for preparing tolerogenic dendritic cells induced by extracellular vesicles with immunomodulatory function, characterized in that: The steps include: In the first step, human peripheral blood mononuclear cells were isolated and adherent cells were induced with cytokines to prepare immature dendritic cells. In step 2, the extracellular vesicles with immunomodulatory function prepared by the preparation method according to any one of claims 1 to 6 or the extracellular vesicles with immunomodulatory function according to claim 7 are added to the immature dendritic cells for further induction to prepare tolerant dendritic cells.

9. The method for preparing tolerogenic dendritic cells induced by extracellular vesicles with immunomodulatory function according to claim 8, characterized in that: The steps include: In step 1, mononuclear cells are prepared from human peripheral blood by density gradient centrifugation or apheresis using a blood cell separator, and the adherent cells are induced with cytokines GM-CSF and IL-4 or Flt-3L for 4-7 days to prepare immature dendritic cells; preferably, the concentration of the cytokines GM-CSF, IL-4, or Flt-3L is 100-2000 IU / mL, 50-1000 IU / mL, or 50-1000 IU / mL, respectively; In step 2, the immature dendritic cells are induced with extracellular vesicles for a further 12-96 hours to prepare tolerant dendritic cells; preferably, the concentration of the extracellular vesicles is 1 μg-200 μg / mL.

10. Tolerogenic dendritic cells prepared according to the method according to any one of claims 8-9.

11. The tolerogenic dendritic cell according to claim 10, characterized in that Preferably, the tolerogenic dendritic cells can downregulate CD11c molecules and costimulatory molecule 1 and express the immunosuppressive molecule PD-1, thereby promoting the proliferation of regulatory T lymphocytes; more preferably, the costimulatory molecule 1 is one or more of CD83, CD86, CD80 and CD40; and / or The tolerogenic dendritic cells are co-cultured with lymphocytes and can inhibit CD3 + CD4 + cells, and the expression of IL-4 and IL-17; and / or The tolerogenic dendritic cells can suppress CD4 + CXCR5 + Follicular helper T cells, which control the polarization of Th0 to Th2 / Th17; and / or, The tolerogenic dendritic cells can promote the expression of PD-1 and activate CD44 + CD73+TIGIT + Anergic T cells and enhance the differentiation of regulatory T lymphocytes.

12. A composition, characterized in that It comprises the extracellular vesicles with immunomodulatory function prepared by the preparation method according to any one of claims 1 to 6; and / or, The extracellular vesicle with immunomodulatory function according to claim 7; and / or, Tolerogenic dendritic cells prepared by the preparation method according to any one of claims 8 to 9; and / or, The tolerogenic dendritic cell according to any one of claims 10 to 11.

13. An extracellular vesicle with immunomodulatory function prepared by the method according to any one of claims 1 to 6; and / or, The extracellular vesicle with immunomodulatory function according to claim 7; and / or, Tolerogenic dendritic cells prepared by the preparation method according to any one of claims 8 to 9; and / or, The tolerogenic dendritic cell according to any one of claims 10 to 11; and / or, Use of the composition according to claim 12 in medicine for treating allergic diseases.

Images