Amniotic membrane mesenchymal stem cell-derived exosomes and their uses

Abstract

The present invention relates to amniotic mesenchymal stem cell-derived exosomes and their uses. On the one hand, the present invention relates to the use of exosomes isolated and extracted from amniotic mesenchymal stem cells in the preparation of a medicament for treating inflammatory diseases, wherein the exosomes have an average particle size of 50 to 200 nm, the positive expression rate of the exosome membrane protein CD9 is greater than 70%, and the positive expression rate of the membrane protein CD81 is greater than 80%. The exosomes of the present invention are obtained by treating amniotic mesenchymal stem cells with IL-1β and culturing them under low oxygen partial pressure conditions. It also relates to a method for regulating the level of TNF-α secretion by peripheral blood mononuclear cells (PBMC) using exosomes, as well as exosomes isolated and extracted using amniotic mesenchymal stem cells and their preparation method. The exosomes of the present invention have biological regulatory properties.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for obtaining exosomes by culturing mesenchymal stem cells. Specifically, it relates to a method for inducing the secretion of exosomes from mesenchymal stem cells by culturing them in the presence of inflammatory factors and a hypoxic environment, and extracting the exosomes. The exosomes obtained by this method can significantly enhance the anti-inflammatory activity of the cells.

[0002] The mesenchymal stem cells of the present invention are obtained by separation from amniotic membrane and subculture. Background Art

[0003] Mesenchymal stem cells (MSCs) are a type of non-hematopoietic multipotent stem cells derived from the mesoderm. It has been confirmed that MSCs not only have the potential for self-renewal, self-replication and multidirectional differentiation, but also have strong anti-inflammatory and inhibitory abilities for various immune cells, and can induce peripheral immune tolerance.Studies have shown that MSCs suppress the function of immune cells (such as T cells, B cells, NK cells, antigen-presenting cells, etc.) by secreting a variety of immunoregulatory factors, such as IFN-γ and PGE2 (see: Polchert D, Sobinsky J, Douglas G, Kidd M, Moadsiri A, Reina E, et al. IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. European journal of immunology. 2008; 38(6): 1745-55. and Spaggiari GM, Abdelrazik H, Becchetti F, Moretta L. MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2. Blood. 2009; 113(26): 6576-83.). E, Giunti D, Cappiello V, Cazzanti F, et al. Human mesenchymal stem cellsmodulate B-cell functions. Blood. 2006;107(1):367-72. And Di Nicola M, CarloStella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bonemarrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99(10):3838-43.).

[0004] Mesenchymal stem cells have low immunogenicity. MSC-based cell therapy has been successfully applied to treat cardiovascular diseases, bone and cartilage defects, diabetes, and other conditions. MSCs release a variety of cytokines and growth factors through paracrine and autocrine pathways. These secreted bioactive factors can inhibit fibrosis and apoptosis, enhance angiogenesis, and participate in tissue repair and regeneration.

[0005] Mesenchymal stem cells are abundant in sources and can be obtained from tissues such as umbilical cord, placenta, bone marrow, umbilical cord blood, fat, and amniotic membrane.

[0006] Exosomes are membrane-bound microvesicles secreted by cells, with diameters ranging from approximately 30 to 200 nm and a density ranging from 1.13 to 1.19 g / ml. Exosomes can carry a variety of proteins, mRNAs, and miRNAs similar to those in the source cells, and participate in processes such as immune regulation, cell communication, cell migration, and angiogenesis (see: Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles. 2015;4:27066). Lai et al. found that MSC-derived exosomes can reduce myocardial ischemia-reperfusion injury and confirmed that exosome miRNA can promote angiogenesis. Therefore, exosomes may become a new direction for the treatment of cardiovascular diseases (see: Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS, et al. Exosome secreted by MSC reduces myocardial ischemia / reperfusion injury. Stem cell research. 2010; 4(3): 214-22.). Xin et al. found that MSC-derived exosomes can promote the growth of axons by transferring miR-133b to neural cells (see: Xin H, Li Y, Buller B, Katakowski M, Zhang Y, Wang X, et al. Exosome mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth. Stem cells. 2012; 30(7): 1556-64.).Filipazzi et al. found that tumor cell-derived exosomes can inhibit the cytotoxicity of T cells and NK cells through the NK cell-activating receptor NKG2D (natural killer group 2, member D), thereby affecting the host immune system (see: Filipazzi P, Burdek M, Villa A, Rivoltini L, Huber V. Recent advances on the role of tumor exosomes in immunosuppression and disease progression. Seminars in cancer biology. 2012; 22(4): 342-9).

[0007] Exosomes, membrane vesicles secreted by living cells, were first discovered in 1983. As research deepens, they have been shown to carry out protein and nucleic acid transport, specifically target receptor cells, exchange proteins and lipids, trigger downstream signaling events, and participate in intercellular communication, thus gaining increasing attention. Exosomes carry two types of proteins: those that are non-specific to the source cell and those that are specific to the source cell. The former may be related to the biogenesis and biological effects of exosomes, and primarily include cytosolic proteins, proteins involved in intracellular signaling, various metabolic enzymes, heat shock proteins, and tetraspanins. The other type is specialized proteins, which are only present in exosomes secreted by specific cell types. These specific cell-derived exosomes are closely linked to their biological functions. For example, exosomes derived from molecular sources contain MHC class II molecules. Therefore, exosomes from different cell sources carry different signaling molecules and perform different functions. For example, exosomes secreted by tumor cells can mediate angiogenesis, tumor cell proliferation, and immune evasion, while exosomes derived from dendritic cells can induce effective anti-tumor immune responses. Existing studies have found that exosomes contain protein rRNA and microRNA related to their cell origin, and that exosomes can pass through biological barriers and transfer functional nucleic acid molecules between cells, thereby exerting various biological functions. Therefore, exosomes are expected to become a new drug delivery route and gene therapy vector.

[0008] Exosomes are nanoscale lipid-encapsulated structures that encapsulate proteins, mRNA, microRNA, and other substances. Exosomes are naturally present in body fluids, including blood, saliva, urine, and breast milk. Exosomes are membrane-bound vesicles secreted by living cells and originate from late endosomes (also known as multivesicular bodies). Almost all cells, including tumors, can produce and release exosomes. Exosomes are secreted and released by cells, disseminate within body fluids such as blood, and are ultimately ingested by other cells, serving as crucial mediators of intercellular communication. Exosomes secreted by both host and tumor cells participate in cell growth, proliferation, metabolism, and regulation. Immune cells and tumor cells can also exchange information through exosomes, a communication mechanism that plays a dual role in regulating tumor immunity: exosomes can induce anti-tumor responses by suppressing immune cells (such as DCs, NK cells, and CD4+ and CD8+ T cells), and can also induce immunosuppression by immunosuppressive or regulatory cell populations (MDSCs, Tregs, and Bregs).

[0009] Current research has found that mesenchymal stem cell exosomes carry a variety of potent cytokines, proteins, and small nucleic acid molecules that can effectively mediate cell proliferation, apoptosis, and regulate function. Studies have shown that exosomes contain vascular endothelial growth factor, fibroblast growth factor, platelet proliferation factor, tumor necrosis factor, and tumor growth factor, among other proteins, which have the potential to inhibit apoptosis and fibrosis, promote vascular mitosis, and mediate immune responses. Experiments have demonstrated that insulin-like growth factor and vascular endothelial growth factor carried in exosomes secreted by mesenchymal stem cells are key players in the treatment of acute kidney injury. In immunology, the lipid membrane surface of mesenchymal stem cell exosomes expresses a variety of membrane proteins, such as coagulation factors, tumor necrosis factor, MHC I / II molecules, and CCR5 chemokine receptors. These lipid membrane surface proteins play a crucial role in combating inflammation.

[0010] Currently, there are a variety of methods and approaches for extracting exosomes. Existing methods primarily rely on ultracentrifugation or expensive column-based methods using reagent kits. However, ultracentrifugation yields uneven exosome quality, cannot guarantee the exact amount of exosomes recovered, and requires lengthy, step-by-step centrifugation processes. Sometimes, due to factors like centrifugation time or speed, separation is ultimately impossible, resulting in a waste of time and cost. Column-based methods using reagent kits are generally only suitable for obtaining smaller quantities of exosomes and are expensive.

[0011] There are already some reports on the isolation and extraction methods of exosomes in the prior art. For example, CN106282107A (Chinese patent application number 201610779165.2) discloses a method for isolating exosomes from human placental mesenchymal stem cells, CN105708861A (Chinese patent application number 201610149852.6) discloses the use of exosomes derived from bone marrow mesenchymal stem cells in the preparation of drugs for treating ankylosing spondylitis, CN105267240A (Chinese patent application number 201410781765.3) discloses the use of exosomes derived from mesenchymal stem cells, CN104382827A (Chinese patent application number 201410705462.3) discloses the use of exosomes from human amniotic mesenchymal stem cells, and CN103767985A (Chinese patent application number 201210402915.6) discloses the preparation and use of exosomes secreted by human blood or mesenchymal stem cells. However, in the existing technology, exosomes are extracted using culture supernatants of mesenchymal stem cells from various sources. These methods have shortcomings such as low yield and complex processes.

[0012] Therefore, the art still needs new methods to isolate and extract exosomes, especially to extract exosomes secreted by mesenchymal stem cells in a simple, easy-to-obtain and high-yield manner. In addition, the art also needs to provide a method for treating inflammatory diseases, such as a method for treating inflammatory diseases by using exosomes.

[0013] In particular, the present invention is particularly expected to provide a method for treating inflammatory diseases by using exosomes isolated from human amniotic mesenchymal stem cells. Summary of the Invention

[0014] The present invention aims to provide a novel method for isolating and extracting exosomes, particularly a method that can extract exosomes secreted by mesenchymal stem cells in a simple, accessible, and high-yield manner. It has been unexpectedly discovered that the method of the present invention can advantageously achieve the aforementioned objectives. The present invention is based on this discovery.

[0015] The mesenchymal stem cells involved in the present invention are derived from amnion.

[0016] To this end, the first aspect of the present invention provides exosomes isolated and extracted using amniotic mesenchymal stem cells, which have an average particle size of 50 to 200 nm, for example, an average particle size of 75 to 150 nm.

[0017] The exosomes according to the first aspect of the present invention express membrane protein CD9 and membrane protein CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0018] According to the first aspect of the present invention, the exosomes, the amniotic mesenchymal stem cells are prepared by a method comprising the following steps:

[0019] (a) Remove the placenta from the placenta sample collection box, place it in a white porcelain dish, and repeatedly rinse the surface with a basic balanced salt solution to disinfect the placenta;

[0020] (b) Gently remove the outer layer of the amniotic membrane using surgical forceps and place it in a 150 mm glass dish. Repeatedly clean the surface with basic balanced salt solution to remove blood, then cut the amniotic membrane into small pieces with a diameter of 5 mm. Filter through a 300-mesh filter, rinse the remaining blood with normal saline, and transfer the tissue pieces to a 50 ml centrifuge tube.

[0021] (c) Tissue digestion: Digest the tissue block with 1 volume of mixed enzyme digestion solution at 100 rpm in a 37°C incubator for 30-60 min. After digestion, add 2 ml of fetal bovine serum and mix thoroughly. Filter through a 200-mesh filter and rinse with plenty of physiological saline. Collect the filtrate.

[0022] (d) Perform cell culture on the filtrate and tissue pieces respectively:

[0023] Filtrate: The collected filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in physiological saline for washing; the cell pellet was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in complete culture medium; the nucleated cells were counted using a counter and the cell viability was determined by trypan blue staining; 2×10 6 Cells were seeded in 75 cm 2 Add 20 ml of complete culture medium to the culture flask, shake well, and culture in a 5% CO2, 37°C incubator; change the medium regularly; after 10-15 days of culture, subculture to the P1 generation and continue culturing with complete culture medium; the complete culture medium includes: DMEM-F12 medium, 10% FBS, 100 μg / mL penicillin, 50 μg / mL streptomycin, 0.01-0.05% thiamine nitrate, and 0.05-0.1% maltose, and the DMEM-F12 medium is a medium prepared with DMEM-F12 at a volume ratio of 1:1;

[0024] Tissue block section: Place the collected tissue blocks in a 75cm 2 In a culture flask, allow the tissue to cover half of the flask's culture area. Add complete medium to cover the tissue, shake well, and culture in a 5% CO2, 37°C incubator. After 2 days, add 10 ml of complete medium and continue culturing. Remove the tissue after most mesenchymal stem cells have crawled out, and change the medium regularly. After 10-15 days of culture, subculture to P1 and continue culturing with complete medium.

[0025] (e) Harvesting P1 cells: The combined P1 cells from the two portions in the previous step are digested with trypsin, and the cells are collected, counted, and cell viability determined. The cells are then frozen to obtain P1 amniotic mesenchymal stem cells.

[0026] (f) Purification, culture and passage: 5000 to 15000 cells / cm 2 The amniotic membrane P1 cells were seeded at a seeding density in a T75 culture flask and cultured with complete medium. The medium was completely replaced on the 3rd to 4th day. The culture was continued until the 11th day. The cells were digested with trypsin digestion solution (containing 0.25% trypsin and 0.02% EDTA), and the digestion was terminated with complete medium. The cells were centrifuged at 1400 rpm for 5 minutes, and the precipitate was collected to obtain the P2 amniotic membrane mesenchymal stem cells.

[0027] (g) The P2 cells are purified, cultured, and passaged in the same manner as in step (f) to obtain P3 cells; and so on to obtain P3 to P8 mesenchymal stem cells.

[0028] In the above method for preparing amniotic mesenchymal stem cells, the basic balanced salt solution is prepared as follows: 0.4 g of KCl, 0.06 g of KH2PO4, 0.132 g of Na2HPO4.12H2O, 8 g of NaCl, 0.35 g of NaHCO3, 1.0 g of D-glucose, 0.10 g of streptomycin, and 0.06 g of penicillin are dissolved in water and the volume is adjusted to 1 L of solution.

[0029] In the above method for preparing amniotic mesenchymal stem cells, the mixed enzyme digestion solution contains: 0.1 mg / ml type I collagenase, 0.3 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase and 0.05 mg / ml neutral protease.

[0030] In step (e) of the above method for preparing amniotic mesenchymal stem cells, the trypsin digestion solution is a digestion solution comprising 0.25% trypsin and 0.02% EDTA.

[0031] According to the first aspect of the present invention, the exosomes are prepared using amniotic mesenchymal stem cells by a method comprising the following steps:

[0032] (1) Mesenchymal stem cells were seeded into culture flasks, and MSC complete medium was added. The cells were cultured in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall. IL-1β was then added to the culture medium to a concentration of 8-12 ng / mL, and the culture was continued.

[0033] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0034] (3) Aspirate the culture medium, wash with PBS, add complete MSC culture medium, and culture in a 37°C, 2% O2, 5% CO2 incubator for 42 to 56 hours;

[0035] (4) Pipette the cell supernatant into a centrifuge tube and centrifuge as follows:

[0036] Centrifuge at 250-350g and 4°C for 8-12 minutes, and transfer the supernatant to another centrifuge tube;

[0037] Centrifuge at 1500-2500g and 4°C for 18-25 minutes, and transfer the supernatant to another centrifuge tube;

[0038] Centrifuge at 8000-12000g and 4°C for 25-35 minutes, filter the supernatant with a 0.22 μm filter membrane and place it in another centrifuge tube;

[0039] Centrifuge at 80,000–120,000 g and 4°C for 75–120 min and discard the supernatant.

[0040] (5) Sterile PBS was added to the centrifuge tube to resuspend the exosome pellet, and the pellet was centrifuged at 80,000–120,000 g and 4°C for 75–120 min. The supernatant was discarded and sterile PBS was added to resuspend the exosomes to obtain an exosome suspension.

[0041] According to the exosomes of the first aspect of the present invention, in step (1), the mesenchymal stem cells are cells of passages P2 to P8, for example, cells of passages P3 to P6.

[0042] According to the exosomes of the first aspect of the present invention, in step (1), cells are added to the culture flask at a density of (0.5-5)×10^4 cells / cm^2, for example, at a density of (0.5-2)×10^4 cells / cm^2. In one embodiment, step (1) uses a T75 culture flask, with each flask inoculated with (2-10)×10^5 cells, for example, 6×10^5 cells, and 10-20 ml of culture medium is added.

[0043] According to the exosomes of the first aspect of the present invention, in step (1), the cells are cultured for 20 to 30 hours, for example, 24 hours, to allow the cells to adhere.

[0044] According to the exosomes of the first aspect of the present invention, in step (1), IL-1β is added and cultured for 20 to 30 hours, for example, 24 hours.

[0045] According to the first aspect of the present invention, the exosomes are cultured in a 37°C, 2% O2, 5% CO2 incubator for 48 hours in step (3);

[0046] According to the exosomes of the first aspect of the present invention, in step (4), the exosomes are first centrifuged at 250g and 4°C for 12 minutes, then at 2500g and 4°C for 18 minutes, and then at 8000g and 4°C for 35 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 120,000g and 4°C for 75 minutes.

[0047] According to the exosomes of the first aspect of the present invention, in step (4), the exosomes are first centrifuged at 350g and 4°C for 8 minutes, then at 1500g and 4°C for 25 minutes, and then at 12000g and 4°C for 25 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0048] According to the exosomes of the first aspect of the present invention, in step (4), the exosomes are first centrifuged at 300g and 4°C for 10 minutes, then at 2000g and 4°C for 20 minutes, and then at 10000g and 4°C for 30 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 100000g and 4°C for 90 minutes.

[0049] According to the exosomes of the first aspect of the present invention, in step (5), the exosomes are centrifuged at 100,000 g and 4° C. for 90 minutes, or at 80,000 g and 4° C. for 120 minutes, or at 120,000 g and 4° C. for 75 minutes.

[0050] According to the exosomes of the first aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0051] According to the exosomes of the first aspect of the present invention, in step (5), the exosomes obtained from 1.2×10^6 cells in step (1) are resuspended with 0.5-5 ml of sterile PBS, for example, with 0.5-2 ml of sterile PBS, for example, with 1 ml of sterile PBS.

[0052] According to the exosomes of the first aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture medium at the same time as IL-1β, and the concentrations of the two are 0.1-0.2 mg / mL, for example, 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, for example, 0.15 mg / mL and 2.2 mg / mL, respectively.

[0053] Furthermore, a second aspect of the present invention provides a method for isolating and extracting exosomes using amniotic mesenchymal stem cells, the method comprising the following steps:

[0054] (1) Mesenchymal stem cells were seeded into culture flasks, and MSC complete medium was added. The cells were cultured in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall. IL-1β was then added to the culture medium to a concentration of 8-12 ng / mL, and the culture was continued.

[0055] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0056] (3) Aspirate the culture medium, wash with PBS, add complete MSC culture medium, and culture in a 37°C, 2% O2, 5% CO2 incubator for 42 to 56 hours;

[0057] (4) Pipette the cell supernatant into a centrifuge tube and centrifuge as follows:

[0058] Centrifuge at 250-350g and 4°C for 8-12 minutes, and transfer the supernatant to another centrifuge tube;

[0059] Centrifuge at 1500-2500g and 4°C for 18-25 minutes, and transfer the supernatant to another centrifuge tube;

[0060] Centrifuge at 8000-12000g and 4°C for 25-35 minutes, filter the supernatant with a 0.22 μm filter membrane and place it in another centrifuge tube;

[0061] Centrifuge at 80,000–120,000 g and 4°C for 75–120 min and discard the supernatant.

[0062] (5) Sterile PBS was added to the centrifuge tube to resuspend the exosome pellet, and the pellet was centrifuged at 80,000–120,000 g and 4°C for 75–120 min. The supernatant was discarded and sterile PBS was added to resuspend the exosomes to obtain an exosome suspension.

[0063] According to the method of the second aspect of the present invention, in step (1), the mesenchymal stem cells are cells of passages P2 to P8, for example, cells of passages P3 to P6.

[0064] According to the method of the second aspect of the present invention, in step (1), cells are added to the culture flask at a density of (0.5-5)×10^4 cells / cm^2, for example, at a density of (0.5-2)×10^4 cells / cm^2. In one embodiment, step (1) uses a T75 culture flask, with each flask inoculated with (2-10)×10^5 cells, for example, 6×10^5 cells, and 10-20 ml of culture medium is added.

[0065] According to the method of the second aspect of the present invention, in step (1), the cells are cultured for 20 to 30 hours, for example, 24 hours, to allow the cells to adhere to the wall.

[0066] According to the method of the second aspect of the present invention, in step (1), culturing is continued for 20 to 30 hours, for example, 24 hours, after adding IL-1β.

[0067] According to the method of the second aspect of the present invention, in step (3), the culture is carried out in an incubator at 37° C., 2% O 2 , 5% CO 2 for 48 hours;

[0068] According to the method of the second aspect of the present invention, in step (4), the mixture is first centrifuged at 250g and 4°C for 12 minutes, then centrifuged at 2500g and 4°C for 18 minutes, then centrifuged at 8000g and 4°C for 35 minutes, and the supernatant is filtered with a 0.22μm filter membrane and centrifuged at 120000g and 4°C for 75 minutes.

[0069] According to the method of the second aspect of the present invention, in step (4), the mixture is first centrifuged at 350g and 4°C for 8 minutes, then centrifuged at 1500g and 4°C for 25 minutes, and then centrifuged at 12000g and 4°C for 25 minutes. The supernatant is filtered with a 0.22μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0070] According to the method of the second aspect of the present invention, in step (4), the mixture is first centrifuged at 300g and 4°C for 10 minutes, then at 2000g and 4°C for 20 minutes, and then at 10000g and 4°C for 30 minutes. The supernatant is filtered with a 0.22μm filter membrane and then centrifuged at 100000g and 4°C for 90 minutes.

[0071] According to the method of the second aspect of the present invention, in step (5), centrifugation is performed at 100,000 g and 4° C. for 90 minutes, or at 80,000 g and 4° C. for 120 minutes, or at 120,000 g and 4° C. for 75 minutes.

[0072] According to the method of the second aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0073] According to the method of the second aspect of the present invention, in step (5), the exosomes obtained from 1.2×10^6 cells in step (1) are resuspended with 0.5-5 ml of sterile PBS, for example, with 0.5-2 ml of sterile PBS, for example, with 1 ml of sterile PBS.

[0074] According to the method of the second aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture medium at the same time as IL-1β is added, and the concentrations of the two are 0.1-0.2 mg / mL, for example, 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, for example, 0.15 mg / mL and 2.2 mg / mL, respectively.

[0075] According to the method of the second aspect of the present invention, the average particle size of the obtained exosomes is 50 to 200 nm, for example, the average particle size is 75 to 150 nm.

[0076] According to the method of the second aspect of the present invention, the exosomes obtained express membrane proteins CD9 and CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0077] According to the method of the second aspect of the present invention, the amniotic mesenchymal stem cells are prepared by a method comprising the following steps:

[0078] (a) Remove the placenta from the placenta sample collection box, place it in a white porcelain dish, and repeatedly rinse the surface with a basic balanced salt solution to disinfect the placenta;

[0079] (b) Gently remove the outer layer of the amniotic membrane using surgical forceps and place it in a 150 mm glass dish. Repeatedly clean the surface with basic balanced salt solution to remove blood, then cut the amniotic membrane into small pieces with a diameter of 5 mm. Filter through a 300-mesh filter, rinse the remaining blood with normal saline, and transfer the tissue pieces to a 50 ml centrifuge tube.

[0080] (c) Tissue digestion: Digest the tissue block with 1 volume of mixed enzyme digestion solution at 100 rpm in a 37°C incubator for 30-60 min. After digestion, add 2 ml of fetal bovine serum and mix thoroughly. Filter through a 200-mesh filter and rinse with plenty of physiological saline. Collect the filtrate.

[0081] (d) Perform cell culture on the filtrate and tissue pieces respectively:

[0082] Filtrate: The collected filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in physiological saline for washing; the cell pellet was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in complete culture medium; the nucleated cells were counted using a counter and the cell viability was determined by trypan blue staining; 2×10 6 Cells were seeded in 75 cm 2 Add 20 ml of complete culture medium to the culture flask, shake well, and culture in a 5% CO2, 37°C incubator; change the medium regularly; after 10-15 days of culture, subculture to the P1 generation and continue culturing with complete culture medium; the complete culture medium includes: DMEM-F12 medium, 10% FBS, 100 μg / mL penicillin, 50 μg / mL streptomycin, 0.01-0.05% thiamine nitrate, and 0.05-0.1% maltose, and the DMEM-F12 medium is a medium prepared with DMEM-F12 at a volume ratio of 1:1;

[0083] Tissue block section: Place the collected tissue blocks in a 75cm 2In a culture flask, allow the tissue to cover half of the flask's culture area. Add complete medium to cover the tissue, shake well, and culture in a 5% CO2, 37°C incubator. After 2 days, add 10 ml of complete medium and continue culturing. Remove the tissue after most mesenchymal stem cells have crawled out, and change the medium regularly. After 10-15 days of culture, subculture to P1 and continue culturing with complete medium.

[0084] (e) Harvesting P1 cells: The combined P1 cells from the two portions in the previous step are digested with trypsin, and the cells are collected, counted, and cell viability determined. The cells are then frozen to obtain P1 amniotic mesenchymal stem cells.

[0085] (f) Purification, culture and passage: 5000 to 15000 cells / cm 2 The amniotic membrane P1 cells were seeded at a seeding density in a T75 culture flask and cultured with complete medium. The medium was completely replaced on the 3rd to 4th day. The culture was continued until the 11th day. The cells were digested with trypsin digestion solution (containing 0.25% trypsin and 0.02% EDTA), and the digestion was terminated with complete medium. The cells were centrifuged at 1400 rpm for 5 minutes, and the precipitate was collected to obtain the P2 amniotic membrane mesenchymal stem cells.

[0086] (g) The P2 cells are purified, cultured, and passaged in the same manner as in step (f) to obtain P3 cells; and so on to obtain P3 to P8 mesenchymal stem cells.

[0087] In the above method for preparing amniotic mesenchymal stem cells, the basic balanced salt solution is prepared as follows: 0.4 g of KCl, 0.06 g of KH2PO4, 0.132 g of Na2HPO4.12H2O, 8 g of NaCl, 0.35 g of NaHCO3, 1.0 g of D-glucose, 0.10 g of streptomycin, and 0.06 g of penicillin are dissolved in water and the volume is adjusted to 1 L of solution.

[0088] In the above method for preparing amniotic mesenchymal stem cells, the mixed enzyme digestion solution contains: 0.1 mg / ml type I collagenase, 0.3 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase and 0.05 mg / ml neutral protease.

[0089] In step (e) of the above method for preparing amniotic mesenchymal stem cells, the trypsin digestion solution is a digestion solution comprising 0.25% trypsin and 0.02% EDTA.

[0090] Furthermore, a third aspect of the present invention provides the use of exosomes isolated and extracted from amniotic mesenchymal stem cells in the preparation of a medicament for treating inflammatory diseases, wherein the exosomes have an average particle size of 50 to 200 nm, for example, an average particle size of 75 to 150 nm.

[0091] According to the third aspect of the present invention, the exosomes express membrane proteins CD9 and CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0092] According to the third aspect of the present invention, the exosomes are prepared using amniotic mesenchymal stem cells by a method comprising the following steps:

[0093] (1) Mesenchymal stem cells were seeded into a culture flask, MSC complete medium was added, and the cells were cultured in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall. IL-1β was then added to the culture medium to a concentration of 8 to 12 ng / mL, and the culture was continued. The amniotic mesenchymal stem cells were prepared by a method comprising the following steps:

[0094] (a) Remove the placenta from the placenta sample collection box, place it in a white porcelain dish, and repeatedly rinse the surface with a basic balanced salt solution to disinfect the placenta;

[0095] (b) Gently remove the outer layer of the amniotic membrane using surgical forceps and place it in a 150 mm glass dish. Repeatedly clean the surface with basic balanced salt solution to remove blood, then cut the amniotic membrane into small pieces with a diameter of 5 mm. Filter through a 300-mesh filter, rinse the remaining blood with normal saline, and transfer the tissue pieces to a 50 ml centrifuge tube.

[0096] (c) Tissue digestion: Digest the tissue block with 1 volume of mixed enzyme digestion solution at 100 rpm in a 37°C incubator for 30-60 min. After digestion, add 2 ml of fetal bovine serum and mix thoroughly. Filter through a 200-mesh filter and rinse with plenty of physiological saline. Collect the filtrate.

[0097] (d) Perform cell culture on the filtrate and tissue pieces respectively:

[0098] Filtrate: The collected filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in physiological saline for washing; the cell pellet was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in complete culture medium; the nucleated cells were counted using a counter and the cell viability was determined by trypan blue staining; 2×10 6 Cells were seeded in 75 cm 2Add 20 ml of complete culture medium to the culture flask, shake well, and culture in a 5% CO2, 37°C incubator; change the medium regularly; after 10-15 days of culture, subculture to the P1 generation and continue culturing with complete culture medium; the complete culture medium includes: DMEM-F12 medium, 10% FBS, 100 μg / mL penicillin, 50 μg / mL streptomycin, 0.01-0.05% thiamine nitrate, and 0.05-0.1% maltose, and the DMEM-F12 medium is a medium prepared with DMEM-F12 at a volume ratio of 1:1;

[0099] Tissue block section: Place the collected tissue blocks in a 75cm 2 In a culture flask, allow the tissue to cover half of the flask's culture area. Add complete medium to cover the tissue, shake well, and culture in a 5% CO2, 37°C incubator. After 2 days, add 10 ml of complete medium and continue culturing. Remove the tissue after most mesenchymal stem cells have crawled out, and change the medium regularly. After 10-15 days of culture, subculture to P1 and continue culturing with complete medium.

[0100] (e) Harvesting P1 cells: The combined P1 cells from the two portions in the previous step are digested with trypsin, and the cells are collected, counted, and cell viability determined. The cells are then frozen to obtain P1 amniotic mesenchymal stem cells.

[0101] (f) Purification, culture and passage: 5000 to 15000 cells / cm 2 The amniotic membrane P1 cells were seeded at a seeding density in a T75 culture flask and cultured with complete medium. The medium was completely replaced on the 3rd to 4th day. The culture was continued until the 11th day. The cells were digested with trypsin digestion solution (containing 0.25% trypsin and 0.02% EDTA), and the digestion was terminated with complete medium. The cells were centrifuged at 1400 rpm for 5 minutes, and the precipitate was collected to obtain the P2 amniotic membrane mesenchymal stem cells.

[0102] (g) The P2 cells are purified, cultured, and passaged in the same manner as in step (f) to obtain P3 cells; and so on, to obtain P3 to P8 mesenchymal stem cells in sequence;

[0103] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0104] (3) Aspirate the culture medium, wash with PBS, add complete MSC culture medium, and culture in a 37°C, 2% O2, 5% CO2 incubator for 42 to 56 hours;

[0105] (4) Pipette the cell supernatant into a centrifuge tube and centrifuge as follows:

[0106] Centrifuge at 250-350g and 4°C for 8-12 minutes, and transfer the supernatant to another centrifuge tube;

[0107] Centrifuge at 1500-2500g and 4°C for 18-25 minutes, and transfer the supernatant to another centrifuge tube;

[0108] Centrifuge at 8000-12000g and 4°C for 25-35 minutes, filter the supernatant with a 0.22 μm filter membrane and place it in another centrifuge tube;

[0109] Centrifuge at 80,000–120,000 g and 4°C for 75–120 min and discard the supernatant.

[0110] (5) Sterile PBS was added to the centrifuge tube to resuspend the exosome pellet, and the pellet was centrifuged at 80,000–120,000 g and 4°C for 75–120 min. The supernatant was discarded and sterile PBS was added to resuspend the exosomes to obtain an exosome suspension.

[0111] According to the third aspect of the present invention, in the above method for preparing amniotic mesenchymal stem cells, the basic balanced salt solution is prepared as follows: 0.4 g of KCl, 0.06 g of KH2PO4, 0.132 g of Na2HPO4.12H2O, 8 g of NaCl, 0.35 g of NaHCO3, 1.0 g of D-glucose, 0.10 g of streptomycin, and 0.06 g of penicillin are dissolved in water and the volume is adjusted to 1 L of solution.

[0112] According to the third aspect of the present invention, in the above method for preparing amniotic mesenchymal stem cells, the mixed enzyme digestion solution contains: 0.1 mg / ml type I collagenase, 0.3 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase and 0.05 mg / ml neutral protease.

[0113] According to the third aspect of the present invention, in step (e) of the above method for preparing amniotic mesenchymal stem cells, the trypsin digestion solution is a digestion solution comprising 0.25% trypsin and 0.02% EDTA.

[0114] According to the use of the third aspect of the present invention, in step (1), the mesenchymal stem cells are cells of passages P2 to P8, for example, cells of passages P3 to P6.

[0115] According to the use of the third aspect of the present invention, in step (1), cells are added to the culture flask at a density of (0.5-5)×10^4 cells / cm^2, for example, at a density of (0.5-2)×10^4 cells / cm^2. In one embodiment, step (1) uses a T75 culture flask, with each flask inoculated with (2-10)×10^5 cells, for example, 6×10^5 cells, and 10-20 ml of culture medium is added.

[0116] According to the use of the third aspect of the present invention, in step (1), the cells are cultured for 20 to 30 hours, for example, 24 hours, to allow the cells to adhere to the wall.

[0117] According to the use of the third aspect of the present invention, in step (1), the culture is continued for 20 to 30 hours after adding IL-1β, for example, 24 hours.

[0118] According to the use of the third aspect of the present invention, in step (3), the culture is carried out in an incubator at 37°C, 2% O2, 5% CO2 for 48 hours;

[0119] According to the use of the third aspect of the present invention, in step (4), the mixture is first centrifuged at 250g and 4°C for 12 minutes, then centrifuged at 2500g and 4°C for 18 minutes, and then centrifuged at 8000g and 4°C for 35 minutes. The supernatant is filtered with a 0.22μm filter membrane and then centrifuged at 120000g and 4°C for 75 minutes.

[0120] According to the use of the third aspect of the present invention, in step (4), the mixture is first centrifuged at 350g and 4°C for 8 minutes, then centrifuged at 1500g and 4°C for 25 minutes, and then centrifuged at 12000g and 4°C for 25 minutes. The supernatant is filtered with a 0.22μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0121] According to the use of the third aspect of the present invention, in step (4), first centrifuge at 300g and 4°C for 10 minutes, then centrifuge at 2000g and 4°C for 20 minutes, then centrifuge at 10000g and 4°C for 30 minutes, and the supernatant is filtered with a 0.22μm filter membrane and centrifuged at 100000g and 4°C for 90 minutes.

[0122] According to the use of the third aspect of the present invention, in step (5), centrifugation is performed at 100,000 g and 4° C. for 90 minutes, or at 80,000 g and 4° C. for 120 minutes, or at 120,000 g and 4° C. for 75 minutes.

[0123] According to the use of the third aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0124] According to the use of the third aspect of the present invention, in step (5), the exosomes obtained from 1.2×10^6 cells in step (1) are resuspended with 0.5-5 ml of sterile PBS, for example, with 0.5-2 ml of sterile PBS, for example, with 1 ml of sterile PBS.

[0125] According to the third aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture medium at the same time as IL-1β, and their concentrations are 0.1-0.2 mg / mL, for example, 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, for example, 0.15 mg / mL and 2.2 mg / mL, respectively.

[0126] Furthermore, a fourth aspect of the present invention provides a method for regulating the level of TNF-α secretion by peripheral blood mononuclear cells (PBMCs) using exosomes, the method comprising exposing the peripheral blood mononuclear cells to the exosomes; wherein the exosomes have an average particle size of 50 to 200 nm, for example, an average particle size of 75 to 150 nm, and the exosomes express membrane protein CD9 and membrane protein CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0127] According to the method of the fourth aspect of the present invention, the exosomes are prepared using amniotic mesenchymal stem cells by a method comprising the following steps:

[0128] (1) Mesenchymal stem cells were seeded into a culture flask, MSC complete medium was added, and the cells were cultured in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall. IL-1β was then added to the culture medium to a concentration of 8 to 12 ng / mL, and the culture was continued. The amniotic mesenchymal stem cells were prepared by a method comprising the following steps:

[0129] (a) Remove the placenta from the placenta sample collection box, place it in a white porcelain dish, and repeatedly rinse the surface with a basic balanced salt solution to disinfect the placenta;

[0130] (b) Gently remove the outer layer of the amniotic membrane using surgical forceps and place it in a 150 mm glass dish. Repeatedly clean the surface with basic balanced salt solution to remove blood, then cut the amniotic membrane into small pieces with a diameter of 5 mm. Filter through a 300-mesh filter, rinse the remaining blood with normal saline, and transfer the tissue pieces to a 50 ml centrifuge tube.

[0131] (c) Tissue digestion: Digest the tissue block with 1 volume of mixed enzyme digestion solution at 100 rpm in a 37°C incubator for 30-60 min. After digestion, add 2 ml of fetal bovine serum and mix thoroughly. Filter through a 200-mesh filter and rinse with plenty of physiological saline. Collect the filtrate.

[0132] (d) Perform cell culture on the filtrate and tissue pieces respectively:

[0133] Filtrate: The collected filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in physiological saline for washing; the cell pellet was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in complete culture medium; the nucleated cells were counted using a counter and the cell viability was determined by trypan blue staining; 2×10 6 Cells were seeded in 75 cm 2 Add 20 ml of complete culture medium to the culture flask, shake well, and culture in a 5% CO2, 37°C incubator; change the medium regularly; after 10-15 days of culture, subculture to the P1 generation and continue culturing with complete culture medium; the complete culture medium includes: DMEM-F12 medium, 10% FBS, 100 μg / mL penicillin, 50 μg / mL streptomycin, 0.01-0.05% thiamine nitrate, and 0.05-0.1% maltose, and the DMEM-F12 medium is a medium prepared with DMEM-F12 at a volume ratio of 1:1;

[0134] Tissue block section: Place the collected tissue blocks in a 75cm 2 In a culture flask, allow the tissue to cover half of the flask's culture area. Add complete medium to cover the tissue, shake well, and culture in a 5% CO2, 37°C incubator. After 2 days, add 10 ml of complete medium and continue culturing. Remove the tissue after most mesenchymal stem cells have crawled out, and change the medium regularly. After 10-15 days of culture, subculture to P1 and continue culturing with complete medium.

[0135] (e) Harvesting P1 cells: The combined P1 cells from the two portions in the previous step are digested with trypsin, and the cells are collected, counted, and cell viability determined. The cells are then frozen to obtain P1 amniotic mesenchymal stem cells.

[0136] (f) Purification, culture and passage: 5000 to 15000 cells / cm 2 The amniotic membrane P1 cells were seeded at a seeding density in a T75 culture flask and cultured with complete medium. The medium was completely replaced on the 3rd to 4th day. The culture was continued until the 11th day. The cells were digested with trypsin digestion solution (containing 0.25% trypsin and 0.02% EDTA), and the digestion was terminated with complete medium. The cells were centrifuged at 1400 rpm for 5 minutes, and the precipitate was collected to obtain the P2 amniotic membrane mesenchymal stem cells.

[0137] (g) The P2 cells are purified, cultured, and passaged in the same manner as in step (f) to obtain P3 cells; and so on, to obtain P3 to P8 mesenchymal stem cells in sequence;

[0138] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0139] (3) Aspirate the culture medium, wash with PBS, add complete MSC culture medium, and culture in a 37°C, 2% O2, 5% CO2 incubator for 42 to 56 hours;

[0140] (4) Pipette the cell supernatant into a centrifuge tube and centrifuge as follows:

[0141] Centrifuge at 250-350g and 4°C for 8-12 minutes, and transfer the supernatant to another centrifuge tube;

[0142] Centrifuge at 1500-2500g and 4°C for 18-25 minutes, and transfer the supernatant to another centrifuge tube;

[0143] Centrifuge at 8000-12000g and 4°C for 25-35 minutes, filter the supernatant with a 0.22 μm filter membrane and place it in another centrifuge tube;

[0144] Centrifuge at 80,000–120,000 g and 4°C for 75–120 min and discard the supernatant.

[0145] (5) Sterile PBS was added to the centrifuge tube to resuspend the exosome pellet, and the pellet was centrifuged at 80,000–120,000 g and 4°C for 75–120 min. The supernatant was discarded and sterile PBS was added to resuspend the exosomes to obtain an exosome suspension.

[0146] According to the method of the fourth aspect of the present invention, in the above method for preparing amniotic mesenchymal stem cells, the basic balanced salt solution is prepared as follows: 0.4 g of KCl, 0.06 g of KH2PO4, 0.132 g of Na2HPO4.12H2O, 8 g of NaCl, 0.35 g of NaHCO3, 1.0 g of D-glucose, 0.10 g of streptomycin, and 0.06 g of penicillin are dissolved in water and the volume is adjusted to 1 L of solution.

[0147] According to the fourth aspect of the present invention, in the above method for preparing amniotic mesenchymal stem cells, the mixed enzyme digestion solution contains: 0.1 mg / ml type I collagenase, 0.3 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase and 0.05 mg / ml neutral protease.

[0148] According to the method of the fourth aspect of the present invention, in step (e) of the above method for preparing amniotic mesenchymal stem cells, the trypsin digestion solution is a digestion solution comprising 0.25% trypsin and 0.02% EDTA.

[0149] According to the method of the fourth aspect of the present invention, in step (1), the mesenchymal stem cells are cells of passages P2 to P8, for example, cells of passages P3 to P6.

[0150] According to the method of the fourth aspect of the present invention, in step (1), cells are added to the culture flask at a density of (0.5-5)×10^4 cells / cm^2, for example, at a density of (0.5-2)×10^4 cells / cm^2. In one embodiment, step (1) uses a T75 culture flask, with each flask inoculated with (2-10)×10^5 cells, for example, 6×10^5 cells, and 10-20 ml of culture medium is added.

[0151] According to the method of the fourth aspect of the present invention, in step (1), the cells are cultured for 20 to 30 hours, for example, 24 hours, to allow the cells to adhere to the wall.

[0152] According to the method of the fourth aspect of the present invention, in step (1), culturing is continued for 20 to 30 hours, for example, 24 hours, after adding IL-1β.

[0153] According to the method of the fourth aspect of the present invention, in step (3), the culture is carried out in an incubator at 37° C., 2% O 2 , 5% CO 2 for 48 hours;

[0154] According to the method of the fourth aspect of the present invention, in step (4), the mixture is first centrifuged at 250g and 4°C for 12 minutes, then centrifuged at 2500g and 4°C for 18 minutes, then centrifuged at 8000g and 4°C for 35 minutes, and the supernatant is filtered with a 0.22μm filter membrane and centrifuged at 120000g and 4°C for 75 minutes.

[0155] According to the method of the fourth aspect of the present invention, in step (4), the mixture is first centrifuged at 350g and 4°C for 8 minutes, then centrifuged at 1500g and 4°C for 25 minutes, and then centrifuged at 12000g and 4°C for 25 minutes. The supernatant is filtered with a 0.22μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0156] According to the method of the fourth aspect of the present invention, in step (4), the mixture is first centrifuged at 300g and 4°C for 10 minutes, then centrifuged at 2000g and 4°C for 20 minutes, and then centrifuged at 10000g and 4°C for 30 minutes. The supernatant is filtered with a 0.22μm filter membrane and then centrifuged at 100000g and 4°C for 90 minutes.

[0157] According to the method of the fourth aspect of the present invention, in step (5), centrifugation is performed at 100,000 g and 4° C. for 90 minutes, or at 80,000 g and 4° C. for 120 minutes, or at 120,000 g and 4° C. for 75 minutes.

[0158] According to the method of the fourth aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0159] According to the method of the fourth aspect of the present invention, in step (5), the exosomes obtained from 1.2×10^6 cells in step (1) are resuspended with 0.5-5 ml of sterile PBS, for example, with 0.5-2 ml of sterile PBS, for example, with 1 ml of sterile PBS.

[0160] According to the method of the fourth aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture medium at the same time as IL-1β is added, and the concentrations of the two are 0.1-0.2 mg / mL, for example, 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, for example, the concentrations of the two are 0.15 mg / mL and 2.2 mg / mL, respectively.

[0161] In the various operating steps described above in the present invention, although the specific steps described therein are different from the steps described in the preparation examples in the specific implementation method section below in some details or language descriptions, those skilled in the art can fully summarize the above-mentioned method steps based on the detailed disclosure of the full text of the present invention.

[0162] Any embodiment of any aspect of the present invention may be combined with other embodiments, as long as they do not conflict. In addition, any technical feature in any embodiment of any aspect of the present invention may be applicable to the same technical feature in other embodiments, as long as they do not conflict. The present invention is further described below.

[0163] All documents cited herein are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with those of the present invention, the present invention shall prevail. In addition, various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still intends to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail.

[0164] Although MSC-derived exosomes have many advantages, such as promoting angiogenesis, cell proliferation, growth, and migration, most MSC-derived exosomes cultured using conventional methods currently have limited efficacy in mediating inflammation regulation. Based on this, the present invention designed a method for inducing MSC culture using low oxygen partial pressure combined with inflammatory cytokine stimulation and isolating and purifying the exosomes secreted by them, demonstrating excellent inflammation regulation in vitro, providing a new approach for exosome-based treatment of inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0165] Figure 1 : Electron micrograph of amniotic mesenchymal stem cell exosomes obtained in Example 2.

[0166] Figure 2 : Electron micrograph of amniotic mesenchymal stem cell exosomes obtained in Example 3.

[0167] Figure 3 A scatter plot of the particle size distribution of exosomes in Example 3 is shown.

[0168] Figure 4 The expression level of the exosome membrane protein CD9 obtained in Example 3 is shown.

[0169] Figure 5 The expression level of the exosome membrane protein CD81 obtained in Example 3 is shown. DETAILED DESCRIPTION

[0170] The present invention can be further described by the following examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various changes and modifications may be made to the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods employed for achieving the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible herein.

[0171] Unless otherwise specified, some reagents used in the present invention are conventional in the art or readily commercially available. For example, serum-free culture medium (Gibco) was purchased from ThermoFisher Scientific; human platelet lysate was purchased from Precicion BioMedicals; phosphate buffer (pH 6.8, PBS) was prepared by adding 118 ml of 0.2 mol / L sodium hydroxide solution to 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, diluting to 1000 ml with water, shaking, and sterilizing at 121°C for 15 minutes; and tissue rinse solution (PRS-TCR-1) was purchased from Precion Biosciences. The complete MSC culture medium described herein is a serum-free medium containing 2% human platelet lysate.

[0172] Example 1: Primary and Subculture of Amniotic Mesenchymal Stem Cells

[0173] Methods for obtaining primary and passaged mesenchymal stem cells through amniotic membrane have been reported in many literatures. The mesenchymal stem cells used to prepare exosomes in the present invention can be obtained using these literature methods. Although the key core of the technology of the present invention is not in this, for example, the present invention is still willing to describe a method for preparing mesenchymal stem cells here.

[0174] This example refers to the method described in Chinese Patent No. ZL2016111295528 (Example 4) of the invention team of this application to prepare amniotic mesenchymal stem cells. The materials and sources used are also referenced in the patent document, and the entire content of the patent document is incorporated herein by reference.

[0175] (1) Remove the placenta from the placenta sample collection box, place it in a white porcelain dish, and use a basic balanced salt solution (0.4 g KCl, 0.06 g KH2PO4, 0.132 g Na2HPO4.12H2O, 8 g NaCl, 0.35 g NaHCO3, 1.0 g D-glucose, 0.10 g streptomycin, and 0.06 g penicillin dissolved in water and diluted to 1 L) to repeatedly rinse the surface and disinfect the placenta;

[0176] (2) Use surgical forceps to slowly tear off the outer layer of the amniotic membrane and place it in a 150 mm glass dish. Use basic balanced salt solution to repeatedly clean the surface blood, and cut it into small pieces of amniotic tissue with a diameter of 5 mm; filter it with a 300-mesh filter, rinse the residual blood with physiological saline, and transfer the tissue pieces to a 50 ml centrifuge tube;

[0177] (3) Tissue digestion: In a 37°C constant temperature shaker, the tissue block was digested with a mixed enzyme digest solution (containing: 0.1 mg / ml type I collagenase, 0.3 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase and 0.05 mg / ml neutral proteinase) at 100 rpm for 40 min; after the digestion was completed, 2 ml of fetal bovine serum was added and mixed to terminate the digestion; the tissue block was filtered through a 200-mesh filter and washed with a large amount of physiological saline, and the filtrate was collected;

[0178] (4) Perform cell culture on the filtrate and tissue block respectively:

[0179] Filtrate: The collected filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in physiological saline for washing; the cell pellet was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in complete culture medium (the specific formula used in this example is different from the complete culture medium used in other occasions of the present invention, and its formula is: DMEM-F12 (1:1) culture medium, 10% FBS, 100 μg / mL penicillin, 50 μg / mL streptomycin, 0.02% thiamine nitrate, 0.085% maltose); the number of nucleated cells was counted using a counter and the cell viability was determined by trypan blue staining (usually greater than 1×10 7 cells, cell viability greater than 95%); 2×10 6 Cells were seeded in 75 cm 2 Add 20 ml of complete medium to the culture flask, shake well, and culture in a 5% CO2, 37°C incubator; change the medium regularly; after 12 days of culture, subculture to the P1 generation and continue to culture with complete medium;

[0180] Tissue block section: Place the collected tissue blocks in a 75cm 2 In a culture flask, allow the tissue to cover half of the flask's culture area. Add complete medium to cover the tissue, shake well, and culture in a 5% CO2, 37°C incubator. After 2 days, add 10 ml of complete medium and continue culturing. Remove the tissue after most mesenchymal stem cells have crawled out, and change the medium regularly. After 12 days of culture, subculture to P1 and continue culturing with complete medium.

[0181] (5) Harvesting P1 cells: Digest the combined P1 cells from the two parts in the previous step with trypsin digestion solution (containing 0.25% trypsin and 0.02% EDTA), collect the cells, count and determine the cell viability, and freeze to obtain P1 amniotic mesenchymal stem cells;

[0182] (6) Purification, culture and passage: 5000 to 15000 cells / cm 2 The amniotic membrane P1 cells were seeded at a seeding density in a T75 culture flask and cultured with complete medium. The medium was completely replaced on the 3rd to 4th day. The culture was continued until the 11th day. The cells were digested with trypsin digestion solution (containing 0.25% trypsin and 0.02% EDTA), and the digestion was terminated with complete medium. The cells were centrifuged at 1400 rpm for 5 minutes, and the precipitate was collected to obtain the P2 amniotic membrane mesenchymal stem cells.

[0183] (7) The P2 generation cells are purified, cultured and passaged in the same manner as in step (6) to obtain P3 generation cells; and so on, to obtain P3 to P8 generation mesenchymal stem cells in sequence.

[0184] As described in ZL2016111295528, the human amniotic mesenchymal stem cells obtained by the above method exhibit excellent performance in terms of cell morphology, cell phenotype, differentiation potential, etc., for example, they have excellent induction differentiation into osteoblasts, cartilages, and adipocytes.

[0185] Example 2: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes using differential centrifugation

[0186] (1) The mesenchymal stem cells (P4 generation) obtained in Example 1 were seeded into T75 culture flasks at a density of 6×10^5 cells. 15 mL of MSC complete medium was added to each flask, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere. IL-1β was then added to the culture medium to a concentration of 10 ng / mL, and the culture was continued for 24 hours. [Those skilled in the art will appreciate that culture flasks of other specifications, such as T25 or T75 flasks, may be used. The number of cells seeded and / or the amount of culture medium added may be varied depending on the culture area. For example, using the above concentration and adding approximately 15 mL of culture medium during MSC culture is a common practice in the art.]

[0187] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0188] (3) Aspirate the culture medium, wash three times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then culture in a 37°C, 2% O2, 5% CO2 incubator for 48 hours;

[0189] [The atmosphere in the aforementioned 2% O2, 5% CO2 incubator refers to an atmosphere balanced with nitrogen, while the atmosphere in the aforementioned 5% CO2 incubator refers to an atmosphere balanced with 21% oxygen and the remainder nitrogen. The meanings of these expressions are generally recognized by those skilled in the art.]

[0190] (4) Pipette the cell supernatant (30 ml, from the supernatant of two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0191] Centrifuge at 300 g and 4°C for 10 min (to remove dead cells and larger cell debris), and transfer the supernatant to another 50 ml centrifuge tube;

[0192] Centrifuge at 2000 g and 4°C for 20 min (to further remove impurities such as cell debris), and transfer the supernatant to another (high-speed) centrifuge tube;

[0193] Centrifuge at 10,000 g and 4°C for 30 min (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22 μm filter membrane (PES filter, Millipore) and place it in another (ultracentrifuge) tube;

[0194] Centrifuge at 100,000 g and 4°C for 90 min and discard the supernatant;

[0195] (5) Add 20 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 100,000 g and 4°C for 90 min, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes to obtain 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0196] The centrifuge tubes and centrifuges with different centrifugal force requirements used in the above steps were purchased from Beckman Coulter.

[0197] Example 2a: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0198] (1) The mesenchymal stem cells (P3 generation) obtained in Example 1 were seeded into T75 culture flasks at a density of 6 × 10^5 cells. 17 mL of MSC complete medium was added to each flask and cultured in a 37°C, 5% CO2 incubator for 20 hours to allow the cells to adhere. IL-1β was then added to the culture medium to a concentration of 8 ng / mL and cultured for another 24 hours.

[0199] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0200] (3) Aspirate the culture medium, wash three times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then culture in a 37°C, 2% O2, 5% CO2 incubator for 48 hours;

[0201] (4) Pipette the cell supernatant (30 ml, from the supernatant of two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0202] Centrifuge at 250 g and 4°C for 12 min (to remove dead cells and larger cell debris), and transfer the supernatant to another 50 ml centrifuge tube;

[0203] Centrifuge at 2500 g and 4°C for 18 min (to further remove impurities such as cell debris), and transfer the supernatant to another (high-speed) centrifuge tube;

[0204] Centrifuge at 8000 g and 4°C for 35 min (to further remove smaller cell debris and impurities), and filter the supernatant with a 0.22 μm filter membrane (PES filter, Millipore) and place it in another (ultracentrifuge) tube;

[0205] Centrifuge at 120,000 g and 4°C for 75 min and discard the supernatant;

[0206] (5) Add 15 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 80,000 g and 4°C for 120 min, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes to obtain 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0207] Example 2b: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0208] (1) The mesenchymal stem cells (P6 generation) obtained in Example 1 were seeded into T75 culture flasks at a density of 6 × 10^5 cells. 13 mL of MSC complete medium was added to each flask and cultured in a 37°C, 5% CO2 incubator for 30 hours to allow the cells to adhere. IL-1β was then added to the culture medium to a concentration of 12 ng / mL and cultured for another 24 hours.

[0209] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0210] (3) Aspirate the culture medium, wash three times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then culture in a 37°C, 2% O2, 5% CO2 incubator for 48 hours;

[0211] (4) Pipette the cell supernatant (30 ml, from the supernatant of two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0212] Centrifuge at 350 g and 4°C for 8 min (to remove dead cells and larger cell debris), and transfer the supernatant to another 50 ml centrifuge tube;

[0213] Centrifuge at 1500 g and 4°C for 25 min (to further remove impurities such as cell debris), and transfer the supernatant to another (high-speed) centrifuge tube;

[0214] Centrifuge at 12000g and 4°C for 25 min (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22 μm filter membrane (PES filter, Millipore) and place it in another (ultracentrifuge) tube;

[0215] Centrifuge at 80,000 g and 4°C for 120 min and discard the supernatant;

[0216] (5) Add 25 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 120,000 g and 4°C for 75 min, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes to obtain 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0217] Example 3: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes by differential centrifugation

[0218] (1) The mesenchymal stem cells (P4 generation) obtained in Example 1 were seeded into T75 culture flasks at a density of 6 × 10^5 cells. 15 mL of MSC complete medium was added to each flask, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere. IL-1β, sodium tartrate, and lysine were then added to the culture medium to a concentration of 10 ng / mL, 0.15 mg / mL, and 2.2 mg / mL, respectively, and the culture was continued for 24 hours.

[0219] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0220] (3) Aspirate the culture medium, wash three times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then culture in a 37°C, 2% O2, 5% CO2 incubator for 48 hours;

[0221] [The atmosphere in the aforementioned 2% O2, 5% CO2 incubator refers to an atmosphere balanced with nitrogen, while the atmosphere in the aforementioned 5% CO2 incubator refers to an atmosphere balanced with 21% oxygen and the remainder nitrogen. The meanings of these expressions are generally recognized by those skilled in the art.]

[0222] (4) Pipette the cell supernatant (30 ml, from the supernatant of two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0223] Centrifuge at 300 g and 4°C for 10 min (to remove dead cells and larger cell debris), and transfer the supernatant to another 50 ml centrifuge tube;

[0224] Centrifuge at 2000 g and 4°C for 20 min (to further remove impurities such as cell debris), and transfer the supernatant to another (high-speed) centrifuge tube;

[0225] Centrifuge at 10,000 g and 4°C for 30 min (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22 μm filter membrane (PES filter, Millipore) and place it in another (ultracentrifuge) tube;

[0226] Centrifuge at 100,000 g and 4°C for 90 min and discard the supernatant;

[0227] (5) Add 20 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 100,000 g and 4°C for 90 min, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes to obtain 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0228] Example 3a: Treatment of mesenchymal stem cells with IL-1β and isolation and purification of exosomes by differential centrifugation

[0229] (1) The mesenchymal stem cells (P3 generation) obtained in Example 1 were seeded into T75 culture flasks at a density of 6 × 10^5 cells. 17 mL of MSC complete medium was added to each flask and cultured in a 37°C, 5% CO2 incubator for 20 hours to allow the cells to adhere. IL-1β, sodium tartrate, and lysine hydrochloride were then added to the culture medium to a concentration of 8 ng / mL, 0.175 mg / mL, and 2 mg / mL, respectively, and the culture was continued for 24 hours.

[0230] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0231] (3) Aspirate the culture medium, wash three times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then culture in a 37°C, 2% O2, 5% CO2 incubator for 48 hours;

[0232] (4) Pipette the cell supernatant (30 ml, from the supernatant of two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0233] Centrifuge at 250 g and 4°C for 12 min (to remove dead cells and larger cell debris), and transfer the supernatant to another 50 ml centrifuge tube;

[0234] Centrifuge at 2500 g and 4°C for 18 min (to further remove impurities such as cell debris), and transfer the supernatant to another (high-speed) centrifuge tube;

[0235] Centrifuge at 8000 g and 4°C for 35 min (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22 μm filter membrane (PES filter, Millipore) and place it in another (ultracentrifuge) tube;

[0236] Centrifuge at 120,000 g and 4°C for 75 min and discard the supernatant;

[0237] (5) Add 15 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 80,000 g and 4°C for 120 min, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes to obtain 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0238] Example 3b: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0239] (1) The mesenchymal stem cells (P6 generation) obtained in Example 1 were seeded into T75 culture flasks at a density of 6 × 10^5 cells. 13 mL of MSC complete medium was added to each flask, and the cells were cultured in a 37°C, 5% CO2 incubator for 30 hours to allow the cells to adhere. IL-1β, sodium tartrate, and lysine hydrochloride were then added to the culture medium to a concentration of 12 ng / mL, 0.125 mg / mL, and 2.5 mg / mL, respectively, and the culture was continued for 24 hours.

[0240] (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0241] (3) Aspirate the culture medium, wash three times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then culture in a 37°C, 2% O2, 5% CO2 incubator for 48 hours;

[0242] (4) Pipette the cell supernatant (30 ml, from the supernatant of two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0243] Centrifuge at 350 g and 4°C for 8 min (to remove dead cells and larger cell debris), and transfer the supernatant to another 50 ml centrifuge tube;

[0244] Centrifuge at 1500 g and 4°C for 25 min (to further remove impurities such as cell debris), and transfer the supernatant to another (high-speed) centrifuge tube;

[0245] Centrifuge at 12000g and 4°C for 25 min (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22 μm filter membrane (PES filter, Millipore) and place it in another (ultracentrifuge) tube;

[0246] Centrifuge at 80,000 g and 4°C for 120 min and discard the supernatant;

[0247] (5) Add 25 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 120,000 g and 4°C for 75 min, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes to obtain 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0248] Example 3c: Referring to Example 3, Example 3a and Example 3b, respectively, the only difference is that sodium tartrate is not added in step (1), and three batches of exosomes are obtained, which can be respectively recorded as Example 3c1, Example 3c2 and Example 3c3.

[0249] Example 3d: Referring to Example 3, Example 3a and Example 3b, respectively, the only difference is that lysine hydrochloride is not added in step (1), and three batches of exosomes are obtained, which can be respectively recorded as Example 3d1, Example 3d2 and Example 3d3.

[0250] Example 4: Transmission electron microscopy observation of exosome particles

[0251] 1. Take 50 μl of the isolated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and place in a 4°C refrigerator for 1 hour;

[0252] 2. Add 20 μl of the fixed exosome suspension onto the front of the copper mesh and let it stand for 20 minutes;

[0253] 3. Carefully absorb the excess solution with absorbent filter paper; then wash the copper mesh with ultrapure water 5 times, each time for 30 seconds, and dry it with filter paper;

[0254] 4. Add 1 drop of 2% uranyl acetate staining solution to the front of the copper mesh and stain for 1 minute. Then use filter paper to absorb the excess stain along the edge of the copper mesh.

[0255] 5. Place the copper mesh in air at room temperature to dry naturally. After drying, observe it on the machine and calculate and count the particle size and distribution of exosomes.

[0256] The above method was used to detect the exosomes (suspension) obtained in each embodiment of the present invention. The number of exosomes obtained from 1.2×10^6 cells (2 culture flasks) was calculated, and the results were as follows: the number of exosome particles in Example 2, Example 2a, and Example 2b were 5.3×10^9, 6.1×10^9, and 4.6×10^9, respectively; the number of exosome particles in Example 3, Example 3a, and Example 3b were 64.4×10^9, 76.2×10^9, and 60.7×10^9, respectively; the number of exosome particles in Example 3c1, Example 3c2, and Example 3c3 were 6.7×10^9, 4.9×10^9, and 6.2×10^9, respectively; and the number of exosome particles in Example 3d1, Example 3d2, and Example 3d3 were 7.3×10^9, 4.4×10^9, and 6.3×10^9, respectively. Taking the exosomes from Example 2 as an example, the initial MSC cell count was 1.2×10^6 cells, and the exosome volume obtained was 1 ml. The measured exosome concentration was 5.3×10^9 particles / mL, equivalent to obtaining 5.3×10^9 exosomes from 1.2×10^6 cells. The above "5.3×10^9 exosomes" represents 5.3 times 10 to the power of 9 exosomes, and other similar expressions have similar meanings.

[0257] Figure 1 and Figure 2 Electron micrographs of amniotic mesenchymal stem cell exosomes obtained in Examples 2 and 3 are shown. Measurements showed that the average particle size of all exosomes obtained in Examples 2, 2a, and 2b, 3, 3a, and 3b, and 3c and 3d was within the range of 94 to 142 nm. For example, the exosomes in Example 3 had a peak particle size of 106.3 ± 3.6 nm and an average particle size of 98.4 ± 1.2 nm. Figure 3 A scatter plot of the particle size distribution of exosomes in Example 3 is shown.

[0258] Example 5: Detection of exosome surface protein expression by flow cytometry

[0259] 1. Mix the Thermofisher CD63 magnetic beads (Cat. No. 10606D) by inversion for 10 minutes; pipette 20 μl of the magnetic bead suspension into a 1.5 mL round-bottom EP tube;

[0260] 2. Add 200 μl of magnetic bead cleaning solution to the EP tube and mix thoroughly with a pipette tip;

[0261] 3. Place the EP on a magnetic stand for 1 minute; then discard the supernatant;

[0262] 4. Take 50 μl of the extracted plasma exosome suspension, add 50 μl of washing solution to a final volume of 100 μl, and mix thoroughly;

[0263] 5. Place the exosome-washing solution mixture on a rotary mixer, set the speed to 10 rpm, and incubate at 2-8°C overnight;

[0264] 6. The next day, centrifuge the sample for 3 to 5 seconds to collect the precipitate;

[0265] 7. Add 300 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds;

[0266] 8. Place the sample on the magnetic stand for about 1 minute and discard the supernatant;

[0267] 9. Add 400 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds;

[0268] 10. Place the sample on a magnetic stand for about 1 minute, discard the supernatant, and resuspend in 300 μl of washing solution;

[0269] 11. Take 100 μl of sample, add CD9-PE and CD81-FITC flow cytometry antibodies to the samples respectively, and incubate at 4°C in the dark for 30 minutes.

[0270] 12. Place the incubated sample on a magnetic rack for about 1 minute, discard the supernatant, and add 300 μl of washing solution for washing;

[0271] 13. Repeat step 12 once, resuspend the sample in 300 μl PB and then test on the instrument.

[0272] Figure 4 The expression level of exosome membrane protein CD9 obtained in Example 3 is shown. Figure 5 The expression of membrane protein CD81 in the exosomes obtained in Example 3 is shown. The positive expression rate of CD9 is 78.3%, and the positive expression rate of CD81 is 93.7%. The expression of membrane protein CD9 and CD81 in the exosomes obtained in other examples is similar to that in Figure 4 and Figure 5 There is no significant difference.

[0273] Example 6: Detection of exosome protein content

[0274] This example uses Pierce TM Protein quantification kit (Cat. No. 23225, Thermo Scientific) was used for the test.

[0275] 1. Take 10 μL of 5 mg / mL BCA (2,2-biquinoline-4,4-dicarboxylic acid disodium) standard and dilute it with PBS to a final concentration of 0.5 mg / mL as the BCA standard solution. Add 0, 2, 4, 6, 8, 12, 16, and 20 μL of this standard solution to the protein standard wells of a 96-well plate and add PBS to make up to 20 μL.

[0276] 2. Dilute the exosome sample appropriately and add 20 μL to the sample wells of a 96-well plate. Because pipettes can have large errors when taking small samples, points before the standard line may not be very accurate. Therefore, try to place the sample concentration point halfway after the standard line.

[0277] 3. Add 200 μL of BCA working solution to each well and incubate at 37°C for 15-30 minutes. Measure the OD value at A562nm using a microplate reader and calculate the protein concentration based on the standard curve.

[0278] The standard curve is y = 0.9999x-0.0488, where x is the OD value and y is the exosome protein concentration in mg / ml.

[0279] The above method was used to detect the exosomes (suspensions) obtained in each embodiment of the present invention. The protein content of the exosomes obtained from 1.2×10^6 cells in each embodiment was calculated. The results were: the protein content of Example 2, Example 2a, and Example 2b were 52.6μg, 72.3μg, and 59.3μg, respectively; the protein content of Example 3, Example 3a, and Example 3b were 297.4μg, 322.6μg, and 283.8μg, respectively; and the protein content of the six exosome samples in Example 3c and Example 3d was in the range of 47 to 71μg.

[0280] Example 7: Elisa assay for immunomodulatory effects of exosomes on TNF-α secretion by PBMCs

[0281] Tumor Necrosis Factor-α (TNF-α) is a proinflammatory cytokine involved in normal inflammatory and immune responses. It is primarily produced by activated monocytes and macrophages and acts in vivo in two forms: transmembrane TNF (tmTNF) and secretory TNF (sTNF). tmTNF is distributed as a membrane protein on cells secreting TNF-α and is cleaved by TACE (TNF-α-converting enzyme) to produce sTNF. There are two types of TNFα receptors (TNFR I and TNFR II) present on the surfaces of various cells. Binding of TNFα to TNFRs typically induces apoptosis, inflammation, and tumorigenesis.

[0282] The exosomes secreted by mesenchymal stem cells in the method of the present invention can inhibit the release of TNF-α by lymphocytes. In this experiment, the exosomes extracted and purified from mesenchymal stem cells were co-cultured with PBMCs in a certain ratio, and then the TNF-α expression level in the cell supernatant was detected by ELISA. The inhibitory ability of exosomes on the release of TNF-α by lymphocytes was detected by analyzing the changes in TNF-α expression levels.

[0283] 1. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult peripheral blood using the Ficoll method. PBMCs were resuspended in immune cell serum-free medium (Miltenyi, containing 2.5% serum replacement) to a density of 4 × 10^5 / ml. Then, 50 μl of CD3 / CD28 magnetic beads (Thermofisher) were added to each ml of PBMC suspension. After mixing the PBMCs and magnetic beads, 0.5 ml of PBMCs were added to a well of a 24-well plate, and 50 μl of a suspension containing 1 × 10^7 exosomes was added to the well. Experimental wells (PBMCs activated with magnetic beads and supplemented with exosomes), control wells (PBMCs activated with magnetic beads but not supplemented with exosomes), negative wells (PBMCs not activated with magnetic beads), and blank wells (containing only immune cell serum-free medium) were set up.

[0284] 2. After 5 days of co-culture, collect the supernatant of the co-culture medium; then centrifuge at 2000 rpm for 5 minutes; collect the supernatant;

[0285] 3. Remove the TNF-α ELISA kit (R&D Systems) and reagents from the refrigerator and place at room temperature (equilibrate to room temperature before use). Remove the microwell strips and place them in the microwell strip holder. Place the remaining microwell strips back into the foil bag, seal it, and return it to the refrigerator.

[0286] 4. Remove one vial of TNF-α standard, add 0.95ml of deionized water, and gently pipette several times with a pipette tip to dissolve the PGE2 to prepare a standard stock solution with a concentration of 10,000pg / ml. Leave the dissolved PGE2 standard at room temperature for 10 minutes, and shake the TNF-α standard by hand 2-3 times every 4-5 minutes. Then, take seven 1.5ml EP tubes and dilute the TNF-α standard with Calibrator Diluent RD6-12 to different concentrations: 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.3pg / ml, and 15.6pg / ml.

[0287] 5. Sample release: For each test sample, take an appropriate amount of the sample to be tested and dilute it with Calibrator Diluent RD6-12 to an exosome protein concentration of 5 μg / ml;

[0288] 6. Add samples and incubate: Add 50 μl of Assay Diluent RD1F to each well of the microplate; add 50 μl of culture medium to the microplate, repeat for 3 wells as a control; add 50 μl of standard solution of different concentrations to the microplate, repeat for 3 wells; add 50 μl of diluted sample to the microplate, repeat for 3 wells; carefully cover the microplate with film and shake at 450 rpm at room temperature for 2 hours;

[0289] 7. Carefully remove the film, pour out the liquid in the microplate, and tap the microplate upside down on absorbent paper several times; add 300μl 1× Wash Buffer to each well, then pour out the 1× Wash Buffer, and tap the microplate upside down on absorbent paper several times; repeat 3 times;

[0290] 8. Add 200 μl of TNF-α Conjugate to each well, carefully cover with a new film, and incubate at room temperature for 2 hours;

[0291] 9. Carefully remove the film, pour out the liquid in the microplate, and tap the microplate upside down on absorbent paper several times; add 300μl 1× Wash Buffer to each well, then pour out the 1× Wash Buffer, and tap the microplate upside down on absorbent paper several times; repeat 3 times;

[0292] 10. Add 200 μl of Substrate Solution to each well and incubate at room temperature in the dark for 30 minutes. Then add 50 μl of Stop Solution to each well.

[0293] 11. Read the OD value: Place the microplate into a microplate reader (Thermofisher, Multiscan), then set the microplate reader program to detect absorbance at a wavelength of 450 nm and perform data analysis;

[0294] 12. After subtracting the average OD value of the culture medium blank group from the results, use Origin software, with the theoretical concentration of each point on the standard curve as the X-axis and the absorbance value as the Y-axis, perform logarithmic transformation on both sides, fit with the four-parameter method, draw the standard curve, input the sample absorbance value, and calculate the TNF-α concentration of the sample.

[0295] The above method was used to measure the level of exosomes inhibiting PBMC secretion of TNF-α. The results were as follows:

[0296] The TNF-α level in the negative group was 802.2 pg / ml.

[0297] The TNF-α level in the control group was 5203.7 pg / ml.

[0298] The TNF-α levels of the test groups of Example 2, Example 2a, and Example 2b were 1193 to 1386 pg / ml. For example, the TNF-α level of the test group of Example 2 was 1237.4 pg / ml.

[0299] The TNF-α levels of the test groups of Example 3, Example 3a, and Example 3b were 1142 to 1292 pg / ml. For example, the TNF-α level of the test group of Example 3 was 1174.3 pg / ml.

[0300] The TNF-α levels of the test groups of Example 3c and Example 3d were in the range of 1246 to 1423 pg / ml.

[0301] Example 8: RT-PCR analysis of the inhibitory effect of exosomes on TNF-α gene expression levels in PBMCs

[0302] 1. Extraction of total PBMC RNA: 1 ml of PBMC cells from each group (experimental group, control group, and negative group) after 5 days of co-culture in Step 2 of Example 7 was aspirated from a 24-well plate and placed in a 1.5 ml EP tube. The cells were centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The pellet was added with 1 ml of Trizol reagent (Life Technologies) and allowed to stand at room temperature for 5 minutes for complete lysis.

[0303] 2. Place the EP tube in a high-speed centrifuge, centrifuge at 4°C, 12,000 rpm for 5 minutes, and discard the precipitate.

[0304] 3. Add 200 μl of chloroform, shake and mix well, then let it stand at room temperature for 15 minutes;

[0305] 4. Place the EP tube in a high-speed centrifuge at 12,000 rpm for 15 minutes;

[0306] 5. Pipette the upper aqueous phase into another centrifuge tube; add 0.5 ml of isopropanol solution, mix well, and let it stand at room temperature for 10 minutes;

[0307] 6. Place the EP tube in a high-speed centrifuge, centrifuge at 4°C, 12,000 rpm for 10 minutes, discard the supernatant, and the RNA will precipitate at the bottom of the tube;

[0308] 7. Add 1 mL of 75% ethanol solution and gently shake the centrifuge tube to suspend the precipitate;

[0309] 8. Place the EP tube in a high-speed centrifuge at 4°C, 8000 rpm, and centrifuge for 5 minutes. Discard the supernatant; air dry at room temperature for 5-10 minutes, and resuspend the RNA in 20 μl of sterile deionized water.

[0310] 9. Detect RNA concentration using nanodrop instrument;

[0311] 10. Reverse transcription of cDNA: RNA was reverse transcribed into cDNA according to the instructions of the Bestar qPCR kit (Cat. No.: DBI-2220);

[0312] 11. Detection of TNF-α gene expression level by qPCR: Real-time fluorescence quantitative PCR was performed according to the operating instructions of the Bestar qPCR (SyberGreen) kit (Cat. No.: DBI-2043) to detect the expression level of TNF-α gene.

[0313] Real-time fluorescence quantitative PCR detection of TNF-α gene expression levels was expressed as relative mRNA levels (fold). The relative mRNA level of the negative group was 1, and the relative mRNA levels of the remaining groups were calculated. The results were as follows:

[0314] The relative mRNA level of the control group was 114.6 times,

[0315] The relative mRNA levels of the test groups of Example 2, Example 2a, and Example 2b were 59 to 71 times. For example, the relative mRNA level of the test group of Example 2 was 68.3 times.

[0316] The relative mRNA levels of the test groups of Example 3, Example 3a, and Example 3b were 65 to 77 times. For example, the relative mRNA level of the test group of Example 3 was 72.4 times.

[0317] The relative mRNA levels of the test groups of Example 3c and Example 3d were 62 to 75 times.

[0318] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A method for isolating and extracting exosomes using amniotic mesenchymal stem cells, the method comprising the following steps: (1) P3 to P8 amniotic mesenchymal stem cells were inoculated into T75 culture flasks, with (2 to 10) × 10 5 cells, adding 10-20 ml of MSC complete medium, culturing in a 37°C, 5% CO2 incubator for 20-30 hours to allow the cells to adhere, then adding IL-1β to a concentration of 8-12 ng / mL, sodium tartrate 0.125-0.175 mg / mL, and lysine hydrochloride 2.0-2.5 mg / mL to the culture medium, and continuing to culture for 20-30 hours; the amniotic membrane mesenchymal stem cells are prepared by a method comprising the following steps: (a) The placenta was removed from the placenta sample collection kit, placed in a white porcelain dish, and repeatedly rinsed with a basic balanced salt solution to disinfect the placenta; the basic balanced salt solution was prepared by dissolving 0.4 g of KCl, 0.06 g of KH2PO4, 0.132 g of Na2HPO4·12H2O, 8 g of NaCl, 0.35 g of NaHCO3, 1.0 g of D-glucose, 0.10 g of streptomycin, and 0.06 g of penicillin in water and diluting the volume to 1 L; (b) Gently remove the outer layer of the amniotic membrane using surgical forceps and place it in a 150 mm glass dish. Repeatedly clean the surface with basic balanced salt solution to remove blood, then cut the amniotic membrane into small pieces with a diameter of 5 mm. Filter through a 300-mesh filter, rinse the remaining blood with normal saline, and transfer the tissue pieces to a 50 ml centrifuge tube. (c) Tissue Digestion: Tissue blocks were digested in a shaker at 37°C with 1x the volume of tissue in a mixed enzyme digestion solution at 100 rpm for 30-60 min. After digestion, 2 ml of fetal bovine serum was added and mixed thoroughly. The tissue blocks were filtered through a 200-mesh filter and washed with a large amount of physiological saline, and the filtrate was collected. The mixed enzyme digestion solution consisted of 0.1 mg / ml type I collagenase, 0.3 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase, and 0.05 mg / ml neutral protease. (d) Perform cell culture on the filtrate and tissue pieces respectively: Filtrate: The collected filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in normal saline to wash; the collected filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in complete culture medium; The nucleated cells were counted using a counter and the cell viability was determined by trypan blue staining; 2×10 6 Cells were seeded in 75 cm 2 Add 20 ml of complete medium to the culture flask, shake well, and culture in a 5% CO2, 37°C incubator; change the medium regularly; after 10-15 days of culture, subculture to the P1 generation and continue to culture with complete medium; the complete medium includes: DMEM-F12 medium, 10% FBS, 100 μg / mL penicillin, 50 μg / mL streptomycin, 0.01-0.05% thiamine nitrate, 0.05-0.1% maltose, wherein the DMEM-F12 medium is a medium prepared by DMEM-F12 at a volume ratio of 1:1; Tissue block section: Place the collected tissue blocks in a 75cm 2 In a culture flask, allow the tissue to cover half of the flask's culture area. Add complete medium to cover the tissue, shake well, and culture in a 5% CO2, 37°C incubator. After 2 days, add 10 ml of complete medium and continue culturing. Remove the tissue after most mesenchymal stem cells have crawled out, and change the medium regularly. After 10-15 days of culture, subculture to P1 and continue culturing with complete medium. (e) Harvesting P1 cells: The combined P1 cells from the two portions in the previous step are digested with trypsin digestion solution, the cells are collected, the cell viability is determined, and the cells are frozen to obtain P1 amniotic mesenchymal stem cells; the trypsin digestion solution comprises 0.25% trypsin and 0.02% EDTA; (f) Purification, culture and passage: 5000~15000 cells / cm 2 The amniotic membrane P1 cells were inoculated at a seeding density in a T75 culture flask and cultured with complete medium. The medium was completely replaced on the 3rd to 4th day. The culture was continued until the 11th day, and the cells were digested with trypsin digestion solution. The digestion was terminated with complete medium and centrifuged at 1400 rpm for 5 minutes. The precipitate was collected to obtain the P2 amniotic membrane mesenchymal stem cells. The trypsin digestion solution contained 0.25% trypsin and 0.02% EDTA. (g) The P2 cells are purified, cultured, and passaged in the same manner as in step (f) to obtain P3 cells; and so on, to obtain P3 to P8 mesenchymal stem cells in sequence; (2) Aspirate the culture medium and replace it with fresh MSC complete culture medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%; (3) Aspirate the culture medium, wash with PBS, add MSC complete culture medium, and culture in a 37°C, 2% O2, 5% CO2 incubator for 42-56 hours; (4) Pipette the cell supernatant into a centrifuge tube and centrifuge as follows: centrifuge at 250-350g and 4°C for 8-12 minutes, and transfer the supernatant to another centrifuge tube; centrifuge at 1500-2500g and 4°C for 18-25 minutes, and transfer the supernatant to another centrifuge tube; centrifuge at 8000-12000g and 4°C for 25-35 minutes, filter the supernatant with a 0.22 μm filter membrane, and transfer it to another centrifuge tube; centrifuge at 80000-120000g and 4°C for 75-120 minutes, and discard the supernatant; (5) Sterile PBS was added to the centrifuge tube to resuspend the exosome pellet, and the pellet was centrifuged at 80,000-120,000 g and 4°C for 75-120 minutes. The supernatant was discarded and sterile PBS was added to resuspend the exosomes to obtain exosomes in the form of a suspension. The obtained exosomes expressed membrane protein CD9 and membrane protein CD81, with the positive expression rate of membrane protein CD9 being greater than 70%, and the positive expression rate of membrane protein CD81 being greater than 80%.

2. The method according to claim 1, wherein the exosomes have an average particle size of 50 to 200 nm.

3. The method according to claim 1, wherein the exosomes have an average particle size of 75 to 150 nm.

4. The method according to claim 1, wherein the positive expression rate of the exosomal membrane protein CD9 is greater than 75%.

5. The method according to claim 1, wherein the positive expression rate of the exosomal membrane protein CD81 is greater than 85%.

6. The method according to claim 1, wherein in step (1), the mesenchymal stem cells are cells of passages P3 to P6.

7. The method according to claim 1, wherein in step (1), the cells are cultured for 24 hours to allow the cells to adhere to the wall.

8. The method according to claim 1, wherein in step (1), culturing is continued for 24 hours after adding IL-1β.

9. The method according to claim 1, wherein in step (3), the culture is carried out in an incubator at 37°C, 2% O2, and 5% CO2 for 48 hours.

10. The method according to claim 1, wherein in step (4), the mixture is first centrifuged at 250 g and 4° C. for 12 minutes, then at 2500 g and 4° C. for 18 minutes, and then at 8000 g and 4° C. for 35 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 120,000 g and 4° C. for 75 minutes.

11. The method according to claim 1, wherein in step (4), the mixture is first centrifuged at 350 g and 4° C. for 8 minutes, then at 1500 g and 4° C. for 25 minutes, and then at 12,000 g and 4° C. for 25 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 80,000 g and 4° C. for 120 minutes.

12. The method according to claim 1, wherein in step (4), the mixture is first centrifuged at 300 g and 4° C. for 10 minutes, then at 2000 g and 4° C. for 20 minutes, and then at 10,000 g and 4° C. for 30 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 100,000 g and 4° C. for 90 minutes.

13. The method according to claim 1, wherein in step (5), the centrifugation is performed at 100,000 g and 4°C for 90 minutes, or at 80,000 g and 4°C for 120 minutes, or at 120,000 g and 4°C for 75 minutes.

14. The method according to claim 1, wherein in step (5), the 1.2×10 6 The exosomes obtained from each cell were resuspended in 0.5-2 ml sterile PBS.

15. The method according to claim 1, wherein in step (1), the concentrations of sodium tartrate and lysine hydrochloride added are 0.15 mg / mL and 2.2 mg / mL, respectively.

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