Exosomes Prepared from Placenta Mesenchymal Stem Cells and Their Uses

Abstract

The present invention relates to exosomes prepared from placenta mesenchymal stem cells and their uses. On the one hand, the present invention relates to exosomes isolated and extracted using placenta mesenchymal stem cells, which have an average particle size of 50-200 nm, and which express membrane protein CD9 and membrane protein CD81. The positive expression rate of the exosome membrane protein CD9 is greater than 65%, and the positive expression rate of the membrane protein CD81 is greater than 75%. The preparation method of the exosomes is as follows: inoculate mesenchymal stem cells into a culture flask, culture to make the cells adhere to the wall, and then add IL-1β to the culture medium for culture; after replacing the complete culture medium, culture in an incubator at 37°C, 2% O2, and 5% CO2; subject the cells to differential centrifugation to obtain exosomes in the form of a suspension. The present invention also relates to the use of the prepared exosomes in the preparation of drugs for treating inflammatory diseases. The method of the present invention has the advantages of high exosome yield, high activity, etc.

Description

Technical Field

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

[0002] The mesenchymal stem cells of the present invention are obtained by separating and subculturing from the placenta. Background Art

[0003] Mesenchymal stem cells (MSCs) are a type of non-hematopoietic system pluripotent stem cells derived from the mesoderm. It has now been confirmed that in addition to having the ability of self-renewal, self-replication and multi-directional differentiation potential, MSCs also have strong anti-inflammatory ability and the ability to inhibit a variety of immune cells, and can induce peripheral immune tolerance.Studies have shown that MSCs inhibit the functions of immune cells (such as T cells, B cells, NK cells, antigen-presenting cells, etc.) by secreting a variety of immunomodulatory factors, such as IFN-γ, PGE2, etc. (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.), (see: Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, Cazzanti F, et al. Human mesenchymal stem cells modulate B-cell functions. Blood. 2006;107(1):367-72. and Di Nicola M, Carlo Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bone marrow 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. MSCs-based cell therapy has been successfully applied to the treatment of cardiovascular diseases, bone and cartilage defects, diabetes and other diseases. MSCs release a variety of cytokines and growth factors through paracrine and autocrine, and these secreted bioactive factors can inhibit fibrosis and apoptosis, enhance angiogenesis, and participate in tissue repair and regeneration.

[0005] Mesenchymal stem cells are rich in sources and can be obtained from tissues such as umbilical cord, placenta, bone marrow, umbilical cord blood, and adipose tissue.

[0006] Exosomes are membranous microvesicles secreted by cells, with a diameter of approximately 30 - 200 nm and a density range between 1.13 - 1.19 g / ml. Exosomes can carry a variety of proteins, mRNAs, and miRNAs similar to the source cells and are involved 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 exosomes derived from MSCs can reduce myocardial ischemia-reperfusion injury and confirmed that the miRNAs in exosomes 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 exosomes derived from MSCs can promote neurite outgrowth by transferring miR-133b to nerve 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 exosomes derived from tumor cells 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, which are membrane vesicles secreted by living cells, were first discovered in 1983. With the deepening of research, it has been found that they have functions such as transporting proteins and nucleic acids, specifically targeting recipient cells, exchanging proteins and lipids or triggering downstream signaling events, and participating in cell-to-cell communication, and have been continuously valued. The proteins carried by exosomes include two types of protein molecules: non-specific and cell-source-specific proteins of the source cell. The former may be related to the biogenesis and biological functions of exosomes, mainly including: cytosolic proteins, proteins involved in intracellular signal transduction, various metabolic enzymes, heat shock proteins and tetraspanins; the other type is special proteins, which only exist in exosomes secreted by a certain special cell, and the exosomes from these specific cell sources are closely related to their biological functions. For example, exosomes from dendritic cell sources contain MHC class II molecules. Therefore, the signal molecules carried by exosomes from different cell sources are different, and the functions they play are also not the same. For example: exosomes secreted by tumor cells can mediate angiogenesis, tumor cell proliferation and immune escape, while exosomes derived from dendritic cells can induce an effective anti-tumor immune response in the body. Existing studies have found that exosomes contain proteins, rRNA and microRNA related to cell sources, and exosomes can cross 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] As a nanoscale lipid-encapsulated structure, exosomes encapsulate substances such as proteins, mRNAs, and microRNAs inside. Exosomes naturally exist in body fluids, including blood, saliva, urine, breast milk, etc. Exosomes are membranous vesicles secreted by living cells and derived 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, spread in body fluids such as blood, and can finally be phagocytosed by other cells, serving as an important medium for cell communication. Exosomes secreted by host cells or tumor cells are involved in cell growth, proliferation, metabolism, and regulation. Information exchange can also occur between immune cells and tumor cells through exosomes, and this communication method plays a dual role in regulating tumor immunity: exosomes can trigger anti-tumor responses by inhibiting immune cells (DCs, NK cells, CD4+, CD8+ T cells, etc.), and can also induce immunosuppression or regulate the immunosuppressive effects of cell populations (MDSCs, Tregs, Bregs).

[0009] Current research has found that exosomes of mesenchymal stem cells carry a variety of effective cytokines, proteins, and small molecule nucleic acid substances, and can effectively mediate cell proliferation, apoptosis, and functional regulation. It has been found that exosomes contain vascular endothelial growth factor, fibroblast growth factor, platelet proliferation factor, tumor necrosis factor, and tumor growth factor, etc., and have functions such as inhibiting cell apoptosis, the degree of cell fibrosis, promoting angiogenesis mitosis, and mediating immune responses. Experiments have proved that insulin-like growth factor and vascular endothelial growth factor carried by exosomes secreted by mesenchymal stem cells are the key leading factors in the treatment of acute kidney injury. In terms of immunology, various membrane proteins are expressed on the lipid membrane surface of exosomes secreted by mesenchymal stem cells, such as: coagulation factors, tumor necrosis factors, MHC I / II molecules, and CCR5 chemokine receptors, etc., and these lipid membrane surface proteins play an important role in resisting inflammation.

[0010] Currently, there are also various methods and approaches for exosome extraction. The existing exosome extraction methods mainly use ultracentrifugation or the method of passing through columns with expensive kits. However, the exosomes obtained by ultracentrifugation have non-uniform quality, cannot guarantee the amount of exosomes obtained, the step-by-step centrifugation time is long, and sometimes they cannot be finally separated due to centrifugation time or centrifugation speed reasons, wasting time and cost. The method of passing through columns with kits is usually only applicable to obtaining a small amount of exosomes and is expensive.

[0011] There have been some reports on the methods for isolating and extracting exosomes in the prior art. For example, CN106282107A (Chinese Patent Application No. 201610779165.2) discloses a method for isolating exosomes from human placenta mesenchymal stem cell sources, CN105708861A (Chinese Patent Application No. 201610149852.6) discloses the application of exosomes derived from bone marrow mesenchymal stem cells in the preparation of drugs for treating ankylosing spondylitis, CN105267240A (Chinese Patent Application No. 201410781765.3) discloses the uses of exosomes derived from mesenchymal stem cells, CN104382827A (Chinese Patent Application No. 201410705462.3) discloses the uses of exosomes from human amniotic mesenchymal stem cells, and CN103767985A (Chinese Patent Application No. 201210402915.6) discloses the preparation and application of exosomes secreted by human blood or mesenchymal stem cells. However, in the prior art, when using culture supernatants of mesenchymal stem cells from various sources to extract exosomes, these methods all have deficiencies such as low yield and complex processes.

[0012] Therefore, there is still an expectation in the art for new methods to isolate and extract exosomes, especially to extract exosomes secreted by mesenchymal stem cells in a simple, easily obtainable and high-yield manner. In addition, there is also an expectation in the art to provide a method for treating inflammatory diseases, such as a method for treating inflammatory diseases by using exosomes. Summary of the Invention

[0013] The object of the present invention is to provide a new method for isolating and extracting exosomes, especially a method that can extract exosomes secreted by mesenchymal stem cells in a simple, easily obtainable and high-yield manner. It has been unexpectedly found that the above object can be beneficially achieved by the method of the present invention. The present invention is completed based on this discovery.

[0014] The mesenchymal stem cells involved in the present invention are placenta-derived.

[0015] To this end, in the first aspect of the present invention, there is provided an exosome isolated and extracted using placenta mesenchymal stem cells, which has an average particle size of 50 - 200 nm, for example, it has an average particle size of 75 - 150 nm.

[0016] For the exosome according to the first aspect of the present invention, it expresses membrane protein CD9 and membrane protein CD81. In one embodiment, the positive expression rate of exosome membrane protein CD9 is greater than 65%, for example, greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosome membrane protein CD81 is greater than 75%, for example, greater than 80%, for example, greater than 85%.

[0017] The exosomes according to the first aspect of the present invention are prepared by using placental mesenchymal stem cells through a method comprising the following steps:

[0018] (1) Inoculate mesenchymal stem cells into a culture flask, add MSC complete medium, and culture in an incubator at 37°C and 5% CO2 to allow the cells to adhere. Then add IL-1β to the culture medium to a concentration of 8 - 12 ng / mL and continue culturing;

[0019] (2) Aspirate the culture medium and replace it with fresh MSC complete medium, and continue culturing the cells in an incubator at 37°C and 5% CO2 until the cell confluence ≥ 80%;

[0020] (3) Aspirate the culture medium, wash with PBS, then add MSC complete medium, and culture in an incubator at 37°C, 2% O2, and 5% CO2 for 42 - 56 hours;

[0021] (4) Aspirate the cell supernatant into a centrifuge tube and perform the following centrifugation treatment:

[0022] Centrifuge at 250 - 350 g and 4°C for 8 - 12 minutes, and aspirate the supernatant into another centrifuge tube;

[0023] Centrifuge at 1500 - 2500 g and 4°C for 18 - 25 minutes, and aspirate the supernatant into another centrifuge tube;

[0024] Centrifuge at 8000 - 12000 g and 4°C for 25 - 35 minutes, filter the supernatant through a 0.22 μm filter membrane and place it in another centrifuge tube;

[0025] Centrifuge at 80000 - 120000 g and 4°C for 75 - 120 minutes, and discard the supernatant;

[0026] (5) Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80000 - 120000 g and 4°C for 75 - 120 minutes, discard the supernatant, add sterile PBS to resuspend the exosomes, and obtain exosomes in the form of a suspension.

[0027] For the exosomes according to the first aspect of the present invention, in step (1), the mesenchymal stem cells are cells of passage P2 - P8, for example, cells of passage P3 - P6.

[0028] For the exosomes according to 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, in step (1), a T75 culture flask is used, and (2 - 10)×10^5 cells, for example, 7.5×10^5 cells, are inoculated into each flask, and 10 - 20 ml of culture medium is added.

[0029] For the exosomes according to the first aspect of the present invention, in step (1), culture for 20 to 30 hours, for example, culture for 24 hours to allow the cells to adhere to the wall.

[0030] For the exosomes according to the first aspect of the present invention, in step (1), after adding IL-1β, continue to culture for 20 to 30 hours, for example, culture for 24 hours.

[0031] For the exosomes according to the first aspect of the present invention, in step (3), culture in an incubator at 37°C, 2% O2, and 5% CO2 for 48 hours;

[0032] For the exosomes according to the first aspect of the present invention, in step (4), first centrifuge at 250 g and 4°C for 12 minutes, then centrifuge at 2500 g and 4°C for 18 minutes, then centrifuge at 8000 g and 4°C for 35 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 120000 g and 4°C for 75 minutes.

[0033] For the exosomes according to the first aspect of the present invention, in step (4), first centrifuge at 350 g and 4°C for 8 minutes, then centrifuge at 1500 g and 4°C for 25 minutes, then centrifuge at 12000 g and 4°C for 25 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 80000 g and 4°C for 120 minutes.

[0034] For the exosomes according to the first aspect of the present invention, in step (4), first centrifuge at 300 g and 4°C for 10 minutes, then centrifuge at 2000 g and 4°C for 20 minutes, then centrifuge at 10000 g and 4°C for 30 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 100000 g and 4°C for 90 minutes.

[0035] For the exosomes according to the first aspect of the present invention, in step (5), centrifuge at 100000 g and 4°C for 90 minutes, or centrifuge at 80000 g and 4°C for 120 minutes, or centrifuge at 120000 g and 4°C for 75 minutes.

[0036] For the exosomes according to the first aspect of the present invention, in step (5), store the obtained exosome suspension at -80°C.

[0037] For the exosomes according to the first aspect of the present invention, in step (5), the exosomes obtained from 1.5×10^6 cells in step (1) are resuspended with 0.5 to 5 ml of sterile PBS, for example, resuspended with 0.5 to 2 ml of sterile PBS, for example, resuspended with 1 ml of sterile PBS.

[0038] For the exosomes according to the first aspect of the present invention, in step (1), while adding IL-1β, sodium tartrate and lysine are also added to the culture medium, and their concentrations are 0.15 - 0.2 mg / mL and 2.0 - 2.5 mg / mL respectively. For example, their concentrations are 0.15 mg / mL and 2.2 mg / mL respectively.

[0039] For the exosomes according to the first aspect of the present invention, the placental mesenchymal stem cells are prepared by a method comprising the following steps:

[0040] (a) Treating the placental tissue

[0041] Remove the amnion from the human placenta, cut the surface membrane-like tissue of the placental lobules, and wash it with physiological saline;

[0042] Cut the surface membrane-like tissue of the placental lobules into tissue fragments with a volume of 0.2 cm 3 of tissue fragments;

[0043] Place the tissue fragments in a centrifuge tube, add an appropriate amount of 0.9% physiological saline, filter through a 300-mesh filter, and wash twice with an appropriate amount of 0.9% physiological saline until the filtrate is clear;

[0044] Add the washed tissue to HBSS digestive solution containing 0.005% Liberase MNP-S enzyme and 0.05% DNA type I enzyme, mix well, and digest in a shaker at 37°C and 100 rpm for 30 min;

[0045] (b) Obtaining placental primitive cells

[0046] After digestion, add fetal bovine serum to the centrifuge tube to terminate digestion, mix well, dilute with 50 ml of physiological saline containing 5% dextran, 2.5% human serum albumin, and 200 U of DNase I, filter through a 300-mesh filter, and wash the tissue with 100 ml of physiological saline multiple times, and collect the filtrate;

[0047] Centrifuge the obtained filtrate at 1400 rpm for 5 min, discard the supernatant and collect the precipitate, resuspend with physiological saline and centrifuge again, and collect the precipitate;

[0048] Resuspend the precipitated cells with DMEM / F12, take a sample for counting, and obtain P0 generation cells;

[0049] (c) Purification and culture

[0050] Culture conditions: DMEM / F12 medium containing 10% fetal bovine serum and 2 mM L-glutamine, culture in a constant temperature and humidity incubator at 37°C and 5% CO2;

[0051] Digestion conditions: 0.25% trypsin, digest at 37°C for 2 minutes;

[0052] Harvesting conditions: After terminating digestion with complete medium, centrifuge at 1400 rpm for 5 minutes and collect the precipitate.

[0053] Inoculate placental P0 cells into a T75 culture flask at an inoculation density of 5000 - 15000 cells / cm 2 . Replace the culture medium completely on the 3rd - 4th day; cell clones appear on the 6th - 7th day, and confluent spindle-shaped cells growing in a swirling pattern appear on the 10th - 11th day, which are P1-generation placental mesenchymal stem cells. After collection, they can be passaged.

[0054] (d) Purify and passage the P1 cells in the same way as in step (c) to obtain P2 cells; and so on, successively obtaining P3 - P8 generations of mesenchymal stem cells.

[0055] Furthermore, the second aspect of the present invention provides a method for isolating and extracting exosomes using placental mesenchymal stem cells, and the method includes the following steps:

[0056] (1) Inoculate mesenchymal stem cells into a culture flask, add MSC complete medium, and culture in an incubator at 37°C and 5% CO2 until the cells adhere. Then add IL-1β to the culture medium to a concentration of 8 - 12 ng / mL and continue culturing.

[0057] (2) Aspirate the culture medium and replace it with fresh MSC complete medium, and continue culturing the cells in an incubator at 37°C and 5% CO2 until the cell confluence ≥ 80%.

[0058] (3) Aspirate the culture medium, wash with PBS, then add MSC complete medium, and culture in an incubator at 37°C, 2% O2, and 5% CO2 for 42 - 56 hours.

[0059] (4) Aspirate the cell supernatant into a centrifuge tube and perform the following centrifugation treatment:

[0060] Centrifuge at 250 - 350 g and 4°C for 8 - 12 minutes, and aspirate the supernatant into another centrifuge tube.

[0061] Centrifuge at 1500 - 2500 g and 4°C for 18 - 25 minutes, and aspirate the supernatant into another centrifuge tube.

[0062] Centrifuge at 8000 - 12000 g and 4°C for 25 - 35 minutes. Filter the supernatant through a 0.22 μm filter membrane and place it in another centrifuge tube.

[0063] Centrifuge at 80000 - 120000 g and 4°C for 75 - 120 minutes, and discard the supernatant.

[0064] (5) Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80,000 - 120,000 g for 75 - 120 minutes at 4°C, discard the supernatant, add sterile PBS to resuspend the exosomes, and obtain exosomes in the form of a suspension.

[0065] According to the method of the second aspect of the present invention, in step (1), the mesenchymal stem cells are cells at passages P2 - P8, for example, cells at passages P3 - P6.

[0066] 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, in step (1), a T75 culture flask is used, and (2 - 10) × 10^5 cells, for example, 7.5 × 10^5 cells, are inoculated per flask, and 10 - 20 ml of culture medium is added.

[0067] According to the method of the second aspect of the present invention, in step (1), the cells are cultured for 20 - 30 hours, for example, cultured for 24 hours to allow the cells to adhere to the wall.

[0068] According to the method of the second aspect of the present invention, in step (1), after adding IL - 1β, the cells are further cultured for 20 - 30 hours, for example, cultured for 24 hours.

[0069] According to the method of the second aspect of the present invention, in step (3), the cells are cultured in an incubator at 37°C, 2% O2, and 5% CO2 for 48 hours;

[0070] According to the method of the second aspect of the present invention, in step (4), first centrifuge at 250 g for 12 minutes at 4°C, then centrifuge at 2500 g for 18 minutes at 4°C, then centrifuge at 8000 g for 35 minutes at 4°C, filter the supernatant through a 0.22 μm filter membrane, and then centrifuge at 120,000 g for 75 minutes at 4°C.

[0071] According to the method of the second aspect of the present invention, in step (4), first centrifuge at 350 g for 8 minutes at 4°C, then centrifuge at 1500 g for 25 minutes at 4°C, then centrifuge at 12000 g for 25 minutes at 4°C, filter the supernatant through a 0.22 μm filter membrane, and then centrifuge at 80,000 g for 120 minutes at 4°C.

[0072] According to the method of the second aspect of the present invention, in step (4), first centrifuge at 300 g for 10 minutes at 4°C, then centrifuge at 2000 g for 20 minutes at 4°C, then centrifuge at 10000 g for 30 minutes at 4°C, filter the supernatant through a 0.22 μm filter membrane, and then centrifuge at 100,000 g for 90 minutes at 4°C.

[0073] According to the method of the second aspect of the present invention, in step (5), centrifuge at 100,000 g for 90 minutes at 4°C, or centrifuge at 80,000 g for 120 minutes at 4°C, or centrifuge at 120,000 g for 75 minutes at 4°C.

[0074] According to the method of the second aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0075] According to the method of the second aspect of the present invention, in step (5), the exosomes obtained from 1.5×10^6 cells in step (1) are resuspended in 0.5 - 5 ml of sterile PBS, for example, resuspended in 0.5 - 2 ml of sterile PBS, for example, resuspended in 1 ml of sterile PBS.

[0076] According to the method of the second aspect of the present invention, in step (1), while adding IL-1β, sodium tartrate and lysine are also added to the culture medium, and their concentrations are 0.15 - 0.2 mg / mL and 2.0 - 2.5 mg / mL respectively, for example, their concentrations are 0.15 mg / mL and 2.2 mg / mL respectively.

[0077] According to the method of the second aspect of the present invention, the average particle size of the obtained exosomes is 50 - 200 nm, for example, the average particle size is 75 - 150 nm.

[0078] According to the method of the second aspect of the present invention, the obtained exosomes express membrane protein CD9 and membrane protein CD81. In one embodiment, the positive expression rate of exosome membrane protein CD9 is greater than 65%, for example, greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosome membrane protein CD81 is greater than 75%, for example, greater than 80%, for example, greater than 85%.

[0079] According to the method of the second aspect of the present invention, the placental mesenchymal stem cells are prepared by a method comprising the following steps:

[0080] (a) Treat the placental tissue

[0081] Remove the amnion from the human placenta, cut the surface membrane-like tissue of the placental lobules, and wash it with physiological saline;

[0082] Cut the surface membrane-like tissue of the placental lobules into tissue fragments with a volume of 0.2 cm 3 of tissue fragments;

[0083] Place the tissue fragments in a centrifuge tube, add an appropriate amount of 0.9% physiological saline, filter through a 300-mesh filter, and wash twice with an appropriate amount of 0.9% physiological saline until the filtrate is clear;

[0084] Add the washed tissue to HBSS digestive solution containing 0.005% Liberase MNP-S enzyme and 0.05% DNA type I enzyme. After thorough mixing, incubate in a shaker at 37 °C and 100 rpm for 30 min for digestion.

[0085] (b) Obtain placental primitive cells

[0086] After digestion, add fetal bovine serum to the centrifuge tube to terminate digestion. After mixing, dilute with 50 ml of normal saline containing 5% dextran, 2.5% human serum albumin, and 200 U of DNase I, filter through a 300-mesh filter, and then wash the tissue with 100 ml of normal saline multiple times. Collect the filtrate.

[0087] Centrifuge the obtained filtrate at 1400 rpm for 5 min, discard the supernatant and collect the precipitate. Resuspend the precipitate with normal saline and centrifuge again to collect the precipitate.

[0088] Resuspend the precipitated cells with DMEM / F12, take a sample for counting to obtain P0 generation cells.

[0089] (c) Purification and culture

[0090] Culture conditions: DMEM / F12 medium containing 10% fetal bovine serum and 2 mM L-glutamine, culture in a constant temperature and humidity incubator at 37 °C and 5% CO2.

[0091] Digestion conditions: 0.25% trypsin, digest at 37 °C for 2 minutes.

[0092] Harvest conditions: After terminating digestion with complete medium, centrifuge at 1400 rpm for 5 min and collect the precipitate.

[0093] Inoculate P0 generation placental cells into a T75 culture flask at an inoculation density of 5000 - 15000 cells / cm 2 . Replace the culture medium completely on the 3rd - 4th day; cell clones appear on the 6th - 7th day, and spindle-shaped cells growing in a swirling pattern appear in patches on the 10th - 11th day, which are P1 generation placental mesenchymal stem cells. After collection, they can be passaged.

[0094] (d) Purify and culture and passage the P1 generation cells in the same way as in step (c) to obtain P2 generation cells; and so on, successively obtaining P3 - P8 generation mesenchymal stem cells.

[0095] Furthermore, the third aspect of the present invention provides the use of exosomes isolated and extracted from placental mesenchymal stem cells in the preparation of drugs for treating inflammatory diseases. The exosomes have an average particle size of 50 - 200 nm, for example, an average particle size of 75 - 150 nm.

[0096] For the use according to the third aspect of the present invention, the exosomes express the membrane protein CD9 and the membrane protein CD81. In one embodiment, the positive expression rate of the exosome membrane protein CD9 is greater than 65%, for example greater than 70%, for example greater than 75%. In one embodiment, the positive expression rate of the exosome membrane protein CD81 is greater than 75%, for example greater than 80%, for example greater than 85%.

[0097] For the use according to the third aspect of the present invention, the exosomes are prepared by using placental mesenchymal stem cells through a method comprising the following steps:

[0098] (1) Inoculate the mesenchymal stem cells into a culture flask, add MSC complete medium, and culture in an incubator at 37°C and 5% CO2 to allow the cells to adhere. Then add IL-1β to the culture medium to a concentration of 8 - 12 ng / mL and continue culturing;

[0099] (2) Aspirate the culture medium and replace it with fresh MSC complete medium, and continue to culture the cells in an incubator at 37°C and 5% CO2 until the cell confluence ≥ 80%;

[0100] (3) Aspirate the culture medium, wash with PBS, then add MSC complete medium, and culture in an incubator at 37°C, 2% O2, and 5% CO2 for 42 - 56 hours;

[0101] (4) Aspirate the cell supernatant into a centrifuge tube and perform the following centrifugation:

[0102] Centrifuge at 250 - 350 g and 4°C for 8 - 12 minutes, aspirate the supernatant into another centrifuge tube;

[0103] Centrifuge at 1500 - 2500 g and 4°C for 18 - 25 minutes, aspirate the supernatant into another centrifuge tube;

[0104] Centrifuge at 8000 - 12000 g and 4°C for 25 - 35 minutes, filter the supernatant through a 0.22 μm filter membrane and place it in another centrifuge tube;

[0105] Centrifuge at 80000 - 120000 g and 4°C for 75 - 120 minutes, discard the supernatant;

[0106] (5) Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80000 - 120000 g and 4°C for 75 - 120 minutes, discard the supernatant, and add sterile PBS to resuspend the exosomes to obtain exosomes in the form of a suspension.

[0107] For the use according to the third aspect of the present invention, in step (1), the mesenchymal stem cells are cells of passage P2 - P8, for example cells of passage P3 - P6.

[0108] For the use according to 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, added at a density of (0.5 - 2)×10^4 cells / cm^2. In one embodiment, a T75 culture flask is used in step (1), and (2 - 10)×10^5 cells, for example, 7.5×10^5 cells are inoculated into each flask, and 10 - 20 ml of culture medium is added.

[0109] For the use according to the third aspect of the present invention, in step (1), the cells are cultured for 20 - 30 hours, for example, cultured for 24 hours to allow the cells to adhere to the wall.

[0110] For the use according to the third aspect of the present invention, in step (1), after adding IL-1β, the cells are further cultured for 20 - 30 hours, for example, cultured for 24 hours.

[0111] For the use according to the third aspect of the present invention, in step (3), the cells are cultured in an incubator at 37°C, 2% O2, and 5% CO2 for 48 hours;

[0112] For the use according to the third aspect of the present invention, in step (4), first centrifuge at 250 g and 4°C for 12 minutes, then centrifuge at 2500 g and 4°C for 18 minutes, then centrifuge at 8000 g and 4°C for 35 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 120000 g and 4°C for 75 minutes.

[0113] For the use according to the third aspect of the present invention, in step (4), first centrifuge at 350 g and 4°C for 8 minutes, then centrifuge at 1500 g and 4°C for 25 minutes, then centrifuge at 12000 g and 4°C for 25 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 80000 g and 4°C for 120 minutes.

[0114] For the use according to the third aspect of the present invention, in step (4), first centrifuge at 300 g and 4°C for 10 minutes, then centrifuge at 2000 g and 4°C for 20 minutes, then centrifuge at 10000 g and 4°C for 30 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 100000 g and 4°C for 90 minutes.

[0115] For the use according to the third aspect of the present invention, in step (5), centrifuge at 100000 g and 4°C for 90 minutes, or centrifuge at 80000 g and 4°C for 120 minutes, or centrifuge at 120000 g and 4°C for 75 minutes.

[0116] For the use according to the third aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0117] For the use according to the third aspect of the present invention, in step (5), the exosomes obtained from 1.5×10^6 cells in step (1) are resuspended in 0.5 - 5 ml of sterile PBS, for example, resuspended in 0.5 - 2 ml of sterile PBS, for example, resuspended in 1 ml of sterile PBS.

[0118] For the use according to the third aspect of the present invention, in step (1), while adding IL-1β, sodium tartrate and lysine are also added to the culture medium, and their concentrations are 0.15 - 0.2 mg / mL and 2.0 - 2.5 mg / mL respectively, for example, their concentrations are 0.15 mg / mL and 2.2 mg / mL respectively.

[0119] For the use according to the third aspect of the present invention, the placental mesenchymal stem cells are prepared by a method comprising the following steps:

[0120] (a) Treating the placental tissue

[0121] Remove the amnion from the human placenta, cut the surface membrane-like tissue of the placental lobules, and wash it with physiological saline;

[0122] Cut the surface membrane-like tissue of the placental lobules into tissue fragments with a volume of 0.2 cm 3 tissue fragments;

[0123] Place the tissue fragments in a centrifuge tube, add an appropriate amount of 0.9% physiological saline, filter through a 300-mesh sieve, and wash twice with an appropriate amount of 0.9% physiological saline until the filtrate is clear;

[0124] Add the washed tissue to HBSS digestive solution containing 0.005% Liberase MNP-S enzyme and 0.05% DNA type I enzyme, mix well, and digest on a shaker at 37°C and 100 rpm for 30 min;

[0125] (b) Obtaining placental primitive cells

[0126] After digestion, add fetal bovine serum to the centrifuge tube to terminate digestion, mix well, dilute with 50 ml of physiological saline containing 5% dextran, 2.5% human serum albumin, and 200 U of DNase I, filter through a 300-mesh sieve, and wash the tissue with 100 ml of physiological saline multiple times, and collect the filtrate;

[0127] Centrifuge the obtained filtrate at 1400 rpm for 5 min, discard the supernatant and collect the precipitate, resuspend it with physiological saline and centrifuge again, and collect the precipitate;

[0128] Resuspend the precipitated cells with DMEM / F12, take a sample for counting, and obtain P0 generation cells;

[0129] (c) Purification and culture

[0130] Culture conditions: DMEM / F12 medium containing 10% fetal bovine serum and 2mM L-glutamine, cultured in a constant temperature and humidified incubator at 37°C and 5% CO2;

[0131] Digestion conditions: 0.25% trypsin, 37°C for 2 minutes;

[0132] Harvest conditions: After terminating digestion with complete medium, centrifuge at 1400 rpm for 5 minutes to collect the precipitate;

[0133] 5000~15000 pieces / cm 2 Placental P0 cells were inoculated into T75 culture flasks at an inoculation density of 1.50 μg / cm2, and the medium was completely replaced on the 3rd to 4th day. Cell clones appeared on the 6th to 7th day, and cells with a fusiform and spiral growth were formed on the 10th to 11th day, which were P1 placental mesenchymal stem cells, and they could be collected and passaged.

[0134] (d) The P1 cells are purified, cultured and passaged in the same manner as in step (c) to obtain P2 cells; and so on to obtain P3 to P8 mesenchymal stem cells.

[0135] In the various operation 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 mode section below in some details or in the language description, those skilled in the art can fully summarize the above-described method steps based on the detailed disclosure of the full text of the present invention.

[0136] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not conflict. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the technical features in other embodiments, as long as they do not conflict. The present invention is further described below.

[0137] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the 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 hopes to provide a more detailed description and explanation of these terms and phrases. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail.

[0138] Although MSC-derived exosomes have many advantages such as promoting angiogenesis, cell proliferation, growth, migration, etc., the role of exosomes derived from MSCs cultured by most conventional methods in mediating inflammation regulation is not obvious at present. Based on this, the present invention designs a method of inducing MSC culture and isolating and purifying exosomes secreted by it by combining low oxygen partial pressure and inflammatory factor stimulation, which shows good inflammation regulation effect in vitro, providing a new scheme for the treatment of inflammatory diseases with exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 : Electron micrograph of placenta mesenchymal stem cell exosomes.

[0140] Figure 2 : Expression level of exosome membrane protein CD9.

[0141] Figure 3 : Expression level of exosome membrane protein CD81. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0142] The present invention can be further described by the following embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiments. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them in as much detail as possible here.

[0143] Unless otherwise specified, some reagents and drugs used in the present invention are conventional in the art or can be easily obtained from commercial sources. For example, serum-free medium (Gibco) is purchased from ThermoFisher Scientific; human platelet lysate is purchased from Precicion BioMedicals; the preparation method of phosphate buffer solution (pH 6.8, PBS) is as follows: take 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, add 118 ml of 0.2 mol / L sodium hydroxide solution, dilute with water to 1000 ml, shake well, sterilize at 121 °C for 15 min to obtain; tissue cleaning solution (PRS-TCR-1) is purchased from Purisheng. The MSC complete medium described in this article is a serum-free medium containing 2% human platelet lysate.

[0144] Example 1: Primary and subculture of placental mesenchymal stem cells

[0145] Methods for obtaining primary and passage mesenchymal stem cells through the placenta have been reported in many documents. The mesenchymal stem cells used in the preparation of exosomes in the present invention can be obtained by using the methods in these documents. Although the key core of the technology of the present invention does not lie in this, for example, the present invention still wishes to describe a method for preparing mesenchymal stem cells herein.

[0146] In this example, the method described in Chinese Patent No. ZL 2019108190702 was referred to for preparing placental mesenchymal stem cells, and the materials and sources used can also be referred to the patent literature.

[0147] (1) Treat the placental tissue: Remove the amnion from a fresh human placenta, cut the surface membranous tissue of the placental lobules, and wash it with physiological saline; Cut the surface membranous tissue of the placental lobules into tissue fragments with a volume of about 0.2 cm^3; Place the tissue fragments in a centrifuge tube, add an appropriate amount (100 ml) of 0.9% physiological saline, filter through a 300-mesh sieve, and wash twice with an appropriate amount (100 ml) of 0.9% physiological saline until the filtrate is clear; Add the washed tissue to HBSS digestive solution (100 ml) containing 0.005% Liberase MNP-S enzyme and 0.05% DNA type I enzyme, mix well, and digest on a shaker for 30 min (37 °C, 100 rpm);

[0148] (2) Obtain placental primitive cells: After digestion, add fetal bovine serum (2 ml) to the centrifuge tube to terminate digestion, mix well, dilute with 50 ml of physiological saline containing 5% dextran, 2.5% human serum albumin, and 200 U of DNase I, filter through a 300-mesh sieve, and wash the tissue with 100 ml of physiological saline multiple times, and collect the filtrate; Centrifuge the obtained filtrate at 1400 rpm for 5 min (acceleration 9, deceleration 7), discard the supernatant and collect the precipitate, resuspend with physiological saline (100 ml) and centrifuge again, and collect the precipitate; Resuspend the precipitated cells with DMEM / F12 (100 ml), take a sample for counting to obtain P0 generation cells;

[0149] (3) Purification culture and passage:

[0150] Culture conditions: DMEM / F12 medium containing 10% fetal bovine serum and 2 mM L-glutamine, cultured in a constant temperature and humidity incubator at 37 °C and 5% CO2;

[0151] Digestion conditions: 0.25% trypsin, digest at 37 °C for 2 minutes;

[0152] Harvest conditions: After terminating digestion with complete medium, centrifuge at 1400 rpm for 5 minutes, and collect the precipitate;

[0153] At 5000 - 15000 cells / cm 2Placental P0 cells were inoculated into T75 culture flasks at an inoculation density of 1.50 μg / cm2, and the medium was completely replaced on the 3rd to 4th day. Cell clones appeared on the 6th to 7th day, and cells with a fusiform and spiral growth were formed on the 10th to 11th day, which were P1 placental mesenchymal stem cells, and they could be collected and passaged.

[0154] (4) The P1 cells are purified, cultured and passaged in the same manner as in step (3) to obtain P2 cells; and so on to obtain P3 to P8 mesenchymal stem cells.

[0155] As described in ZL 2019108190702, the cells obtained by the above method show excellent performance in terms of cell morphology, cell phenotype, differentiation potential, etc.

[0156] Example 2: Treatment of mesenchymal stem cells with IL-1β and isolation and purification of exosomes by differential centrifugation

[0157] (1) The mesenchymal stem cells (P4 generation) obtained in Example 1 were inoculated into T75 culture flasks at a density of 7.5×10^5 cells, and 15 mL of MSC complete medium was added to each flask. The flasks were placed in a 37° C., 5% CO2 incubator for 24 hours to allow the cells to adhere to the wall. 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 instead. The number of cells inoculated and / or the amount of culture medium added may be changed according to the culture area. For example, adding about 15 mL of culture medium at the above concentration during MSC culture is a common practice in the art]

[0158] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium, and continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0159] (3) Aspirate the culture medium, wash with PBS three times, 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;

[0160] [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 such expressions are generally recognized by those skilled in the art]

[0161] (4) Pipette the cell supernatant (30 ml, from the supernatant in two culture flasks) into a (50 ml) centrifuge tube and perform the following centrifugation treatment:

[0162] 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;

[0163] Centrifuge at 2000 g and 4 °C for 20 minutes (to further remove impurities such as cell debris), and aspirate the supernatant into another (high-speed) centrifuge tube;

[0164] Centrifuge at 10000 g and 4 °C for 30 minutes (to further remove smaller cell debris and impurities), filter the supernatant through a 0.22 μm filter membrane (PES filter membrane, millipore), and place it in another (ultra-high-speed) centrifuge tube;

[0165] Centrifuge at 100000 g and 4 °C for 90 minutes, and discard the supernatant;

[0166] (5) Add 20 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 100000 g and 4 °C for 90 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosomes, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance measurement.

[0167] The centrifuge tubes and centrifuges with different centrifugal force requirements used in the above steps are all purchased from Beckman Coulter.

[0168] Example 2a: Treatment of mesenchymal stem cells with IL-1β and isolation and purification of exosomes by differential centrifugation

[0169] (1) Inoculate the mesenchymal stem cells (P3 generation) obtained in Example 1 at a density of 7.5×10^5 cells into a T75 culture flask, add 17.5 mL of MSC complete medium to each flask, and culture in an incubator at 37 °C and 5% CO2 for 20 hours to allow the cells to adhere. Then add IL-1β to the culture medium to a concentration of 8 ng / mL and continue culturing for 24 hours;

[0170] (2) Aspirate and discard the culture medium, replace it with fresh MSC complete medium, and continue culturing the cells in an incubator at 37 °C and 5% CO2 until the cell confluence ≥ 80%;

[0171] (3) Aspirate and discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete medium to each flask, and culture in an incubator at 37 °C, 2% O2, and 5% CO2 for 48 hours;

[0172] (4) Aspirate the cell supernatant (30 ml, the supernatant volume from 2 culture flasks) into a (50 ml) centrifuge tube and perform the following centrifugation:

[0173] Centrifuge at 250 g and 4 °C for 12 minutes (to remove dead cells and larger cell debris), and aspirate the supernatant into another (50 ml) centrifuge tube;

[0174] Centrifuge at 2500 g and 4 °C for 18 minutes (to further remove cell debris and other impurities), and aspirate the supernatant into another (high-speed) centrifuge tube;

[0175] Centrifuge at 8000 g and 4 °C for 35 minutes (to further remove smaller cell debris and impurities). Filter the supernatant through a 0.22-μm filter membrane (PES filter membrane, Millipore) and transfer it to another (ultracentrifuge) tube.

[0176] Centrifuge at 120000 g and 4 °C for 75 minutes, and discard the supernatant.

[0177] (5) Add 15 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet. Centrifuge at 80000 g and 4 °C for 120 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosomes, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance determination.

[0178] Example 2b: Treatment of mesenchymal stem cells with IL-1β and isolation and purification of exosomes by differential centrifugation

[0179] (1) Seed the mesenchymal stem cells (P6 generation) obtained in Example 1 at a density of 7.5×10^5 cells per T75 culture flask. Add 12.5 mL of MSC complete medium to each flask and culture in a 37 °C, 5% CO2 incubator for 30 hours to allow the cells to adhere. Then add IL-1β to the culture medium to a concentration of 12 ng / mL and continue culturing for 24 hours.

[0180] (2) Aspirate the culture medium and replace it with fresh MSC complete medium. Continue culturing the cells in a 37 °C, 5% CO2 incubator until the cell confluence is ≥80%.

[0181] (3) Aspirate the culture medium, wash the cells 3 times with PBS, then add 15 mL of MSC complete medium to each flask and culture in a 37 °C, 2% O2, 5% CO2 incubator for 48 hours.

[0182] (4) Collect the cell supernatant (30 ml, the supernatant volume from 2 culture flasks) and transfer it to a (50 ml) centrifuge tube. Perform the following centrifugation steps:

[0183] Centrifuge at 350 g and 4 °C for 8 minutes (to remove dead cells and larger cell debris). Aspirate the supernatant and transfer it to another (50 ml) centrifuge tube.

[0184] Centrifuge at 1500 g and 4 °C for 25 minutes (to further remove cell debris and other impurities). Aspirate the supernatant and transfer it to another (high-speed) centrifuge tube.

[0185] Centrifuge at 12000 g and 4 °C for 25 minutes (to further remove smaller cell debris and impurities). Filter the supernatant through a 0.22-μm filter membrane (PES filter membrane, Millipore) and transfer it to another (ultracentrifuge) tube.

[0186] Centrifuge at 80000g for 120 minutes at 4°C, discard the supernatant;

[0187] (5) Add 25 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate. Centrifuge at 120000g for 75 minutes at 4°C, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosomes, and obtain 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0188] Example 3: Treatment of mesenchymal stem cells with IL-1β and isolation and purification of exosomes by differential centrifugation

[0189] (1) Seed the mesenchymal stem cells (P4 generation) obtained in Example 1 at a density of 7.5×10^5 cells per T75 culture flask. Add 15 mL of MSC complete medium to each flask and culture in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere. Then add IL-1β, sodium tartrate, and lysine to the culture medium to final concentrations of 10 ng / mL, 0.175 mg / mL, and 2.2 mg / mL, respectively, and continue to culture for 24 hours;

[0190] (2) Aspirate the culture medium and replace it with fresh MSC complete medium. Continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence is ≥80%;

[0191] (3) Aspirate the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete medium to each flask and culture in a 37°C, 2% O2, 5% CO2 incubator for 48 hours;

[0192] [The atmosphere in the above 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 remaining nitrogen. The meaning of such expressions is generally recognized by those skilled in the art.]

[0193] (4) Aspirate the cell supernatant (30 ml, the supernatant volume from 2 culture flasks) and transfer it to a (50 ml) centrifuge tube for the following centrifugation:

[0194] Centrifuge at 300g for 10 minutes at 4°C (to remove dead cells and large cell debris), aspirate the supernatant and transfer it to another (50 ml) centrifuge tube;

[0195] Centrifuge at 2000g for 20 minutes at 4°C (to further remove cell debris and other impurities), aspirate the supernatant and transfer it to another (high-speed) centrifuge tube;

[0196] Centrifuge at 10000g for 30 minutes at 4°C (to further remove smaller cell debris and impurities). Filter the supernatant through a 0.22 μm filter membrane (PES filter membrane, millipore) and transfer it to another (ultra-high-speed) centrifuge tube;

[0197] Centrifuge at 100,000g for 90 minutes at 4°C, and discard the supernatant.

[0198] (5) Add 20 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet. Centrifuge at 100,000g for 90 minutes at 4°C, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes, obtaining 1 ml of exosome suspension, which can be stored at -80°C and / or aliquoted for performance measurement.

[0199] Example 3a: Treatment of mesenchymal stem cells with IL-1β and isolation and purification of exosomes by differential centrifugation

[0200] (1) Inoculate the mesenchymal stem cells (P3 generation) obtained in Example 1 into a T75 culture flask at a density of 7.5×10^5 cells per flask. Add 17.5 mL of MSC complete medium to each flask, and culture in an incubator at 37°C and 5% CO2 for 20 hours to allow the cells to adhere. Then, add IL-1β, sodium tartrate, and lysine hydrochloride to the culture medium to final concentrations of 8 ng / mL, 0.2 mg / mL, and 2 mg / mL, respectively, and continue culturing for 24 hours.

[0201] (2) Aspirate and discard the culture medium, and replace it with fresh MSC complete medium. Continue culturing the cells in an incubator at 37°C and 5% CO2 until the cell confluence is ≥80%.

[0202] (3) Aspirate and discard the culture medium, wash the cells 3 times with PBS, then add 15 mL of MSC complete medium to each flask, and culture in an incubator at 37°C, 2% O2, and 5% CO2 for 48 hours.

[0203] (4) Aspirate the cell supernatant (30 ml, the supernatant volume from 2 culture flasks) and transfer it to a (50 ml) centrifuge tube for the following centrifugation steps:

[0204] Centrifuge at 250g for 12 minutes at 4°C (to remove dead cells and large cell debris), and aspirate the supernatant into another (50 ml) centrifuge tube.

[0205] Centrifuge at 2500g for 18 minutes at 4°C (to further remove cell debris and other impurities), and aspirate the supernatant into another (high-speed) centrifuge tube.

[0206] Centrifuge at 8000g for 35 minutes at 4°C (to further remove smaller cell debris and impurities). Filter the supernatant through a 0.22 μm filter membrane (PES filter membrane, millipore) and transfer it to another (ultra-high-speed) centrifuge tube.

[0207] Centrifuge at 120,000g for 75 minutes at 4°C, and discard the supernatant.

[0208] (5) Add 15 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate. Centrifuge at 80,000 g and 4 °C for 120 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosomes, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance measurement.

[0209] Example 3b: Treatment of mesenchymal stem cells with IL-1β and isolation and purification of exosomes by differential centrifugation

[0210] (1) Inoculate the mesenchymal stem cells (P6 generation) obtained in Example 1 at 7.5×10^5 cells into a T75 culture flask, add 12.5 mL of MSC complete medium to each flask, and culture in an incubator at 37 °C and 5% CO2 for 30 hours to allow the cells to adhere. Then add IL-1β, sodium tartrate, and lysine hydrochloride to the culture medium to concentrations of 12 ng / mL, 0.15 mg / mL, and 2.5 mg / mL, respectively, and continue to culture for 24 hours.

[0211] (2) Aspirate and discard the culture medium, replace it with fresh MSC complete medium, and continue to culture the cells in an incubator at 37 °C and 5% CO2 until the cell confluence ≥ 80%.

[0212] (3) Aspirate and discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete medium to each flask, and culture in an incubator at 37 °C, 2% O2, and 5% CO2 for 48 hours.

[0213] (4) Aspirate the cell supernatant (30 ml, the supernatant volume from 2 culture flasks) and place it in a (50 ml) centrifuge tube, and perform the following centrifugation:

[0214] Centrifuge at 350 g and 4 °C for 8 minutes (to remove dead cells and large cell debris), aspirate the supernatant into another (50 ml) centrifuge tube;

[0215] Centrifuge at 1500 g and 4 °C for 25 minutes (to further remove cell debris and other impurities), aspirate the supernatant into another (high-speed) centrifuge tube;

[0216] Centrifuge at 12,000 g and 4 °C for 25 minutes (to further remove smaller cell debris and impurities), filter the supernatant through a 0.22 μm filter membrane (PES filter membrane, millipore) and place it in another (ultra-high-speed) centrifuge tube;

[0217] Centrifuge at 80,000 g and 4 °C for 120 minutes, discard the supernatant;

[0218] (5) Add 25 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate. Centrifuge at 120,000 g for 75 minutes at 4 °C. Discard the supernatant. Add 1 mL of sterile PBS to resuspend the exosomes, obtaining 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance determination.

[0219] Example 3c: Refer to Example 3, Example 3a, and Example 3b respectively. The only difference is that sodium tartrate is not added in step (1), obtaining three batches of exosomes, which can be denoted as Example 3c1, Example 3c2, and Example 3c3 respectively.

[0220] Example 3d: Refer to Example 3, Example 3a, and Example 3b respectively. The only difference is that lysine hydrochloride is not added in step (1), obtaining three batches of exosomes, which can be denoted as Example 3d1, Example 3d2, and Example 3d3 respectively.

[0221] Example 4: Transmission electron microscopy observation of exosome particles

[0222] 1. Take 50 μl of the separated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde thereto, and fix it in a refrigerator at 4 °C for 1 hr;

[0223] 2. Drop 20 μl of the fixed exosome suspension onto the front side of the copper grid and let it stand for 20 min;

[0224] 3. Carefully suck off the excess solution with a filter paper; then wash the copper grid 5 times with ultrapure water, 30 seconds each time, and dry it with a filter paper;

[0225] 4. Drop 1 drop of 2% uranyl acetate staining solution onto the front side of the copper grid, stain for 1 min, and then suck off the excess staining solution along the edge of the copper grid with a filter paper;

[0226] 5. Place the copper grid in the air at room temperature to dry naturally. After drying, put it on the machine for observation, and at the same time calculate and count the particle size and distribution of the exosomes.

[0227] The exosomes (suspensions) obtained from each embodiment of the present invention were detected using the above method. The number of exosomes obtained from each embodiment from 1.5×10^6 cells (2 culture flasks) was calculated. The results were as follows: the number of exosome particles in Example 2, Example 2a, and Example 2b were 7.6×10^9 particles, 6.8×10^9 particles, and 8.2×10^9 particles respectively; the number of exosome particles in Example 3, Example 3a, and Example 3b were 93.3×10^9 particles, 98.7×10^9 particles, and 90.4×10^9 particles respectively; the number of exosome particles in Example 3c1, Example 3c2, and Example 3c3 were 8.3×10^9 particles, 7.7×10^9 particles, and 8.9×10^9 particles respectively; the number of exosome particles in Example 3d1, Example 3d2, and Example 3d3 were 5.4×10^9 particles, 4.8×10^9 particles, and 6.2×10^9 particles respectively. Taking the exosomes of Example 2 as an example, the initial MSC cell amount was 1.5×10^6 cells, the volume of the obtained exosomes was 1 ml, and the measured exosome concentration was 7.6×10^9 particles / mL, which was equivalent to obtaining 7.6×10^9 exosome particles from 1.5×10^6 cells. The above "7.6×10^9 exosome particles" means 7.6 multiplied by 10 to the 9th power of exosomes, and other similar expressions have similar meanings.

[0228] Figure 1 The electron micrograph of the placenta mesenchymal stem cell exosomes obtained in Example 3 is shown. It was measured that the average particle sizes of all the exosomes obtained in the above Example 2, Example 2a, and Example 2b, Example 3, Example 3a, and Example 3b, Example 3c, and Example 3d were all in the range of 98 - 131 nm. For example, the peak particle size of the exosomes in Example 3 was 113.7 ± 4.2 nm, and the average particle size was 107.7 ± 0.8 nm.

[0229] Example 5: Detection of surface protein expression of exosome particles by flow cytometry

[0230] 1. Invert and mix the components of Thermofisher CD63 magnetic beads (product number 10606D) for 10 min; aspirate 20 μl of the magnetic bead suspension into a 1.5 mL round-bottom EP tube;

[0231] 2. Add 200 μl of the magnetic bead washing solution to the EP tube and mix well with a pipette tip;

[0232] 3. Place the EP tube on the magnetic rack for 1 min; then aspirate and discard the supernatant;

[0233] 4. Take 50 μl of the extracted plasma exosome suspension, add 50 μl of the washing solution to a final volume of 100 μl, and mix well;

[0234] 5. Place the exosome-washing solution mixture on a rotary mixer, set the rotation speed to 10 rpm, and incubate it at 2 - 8 °C with rotation overnight.

[0235] 6. The next day, centrifuge the sample quickly for 3 - 5 seconds to collect the precipitate.

[0236] 7. Add 300 μl of washing solution to the sample and mix well with a pipette tip for 30 seconds.

[0237] 8. Place the sample on a magnetic stand for about 1 minute and aspirate the supernatant.

[0238] 9. Add 400 μl of washing solution to the sample and mix well with a pipette tip for 30 seconds.

[0239] 10. Place the sample on a magnetic stand for about 1 minute, aspirate the supernatant, and resuspend it with 300 μl of washing solution.

[0240] 11. Take 100 μl of each sample, add CD9-PE and CD81-FITC flow antibodies to the samples respectively, and incubate them at 4 °C in the dark for 30 minutes.

[0241] 12. Place the incubated sample on a magnetic stand for about 1 minute, aspirate the supernatant, and add 300 μl of washing solution for washing.

[0242] 13. After repeating step 12 once, resuspend the selected sample with 300 μl of PB and then detect it on the machine.

[0243] Figure 2 Shows the expression level of exosomal membrane protein CD9 obtained in Example 3. Figure 3 Shows the expression level of exosomal membrane protein CD81 obtained in Example 3. The positive expression rate of CD9 is 82.7%, and the positive expression rate of CD81 is 94.4%. The expression levels of exosomal membrane proteins CD9 and CD81 obtained in other examples are Figure 2 and Figure 3 There is no significant difference.

[0244] Example 6: Detection of exosome protein content

[0245] This example uses Pierce TM Protein Quantification Kit (Catalog No.: 23225, Thermo Scientific) for testing.

[0246] 1. Take 10 μl of 5 mg / mL BCA (sodium 2,2'-bicinchoninate) standard, dilute it with PBS to a final concentration of 0.5 mg / mL to obtain the BCA standard solution. Add this standard solution to the protein standard wells of a 96-well plate according to 0, 2, 4, 6, 8, 12, 16, 20 μL, and make up to 20 μL with PBS.

[0247] 2. Appropriately dilute the exosome sample and add 20 μL to the sample wells of a 96-well plate. Since the pipette has a relatively large error when taking small amounts of samples, the points in front of the standard curve may not be very accurate. Therefore, try to make the sample concentration points fall behind 1 / 2 of the standard curve as much as possible.

[0248] 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 an ELISA reader and calculate the protein concentration according to the standard curve.

[0249] 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.

[0250] Use the above method to detect the exosomes (suspensions) obtained in each example of the present invention. Calculate the exosome protein content obtained from 1.5 × 10^6 cells. The results are as follows: the protein contents of Example 2, Example 2a, and Example 2b are 59.7 μg, 56.3 μg, and 64.2 μg respectively; the protein contents of Example 3, Example 3a, and Example 3b are 325.3 μg, 346.2 μg, and 337.4 μg respectively; the protein contents of the 6 exosome samples of Example 3c and Example 3d are in the range of 58 - 74 μg.

[0251] Example 7: Immunoregulation experiment of ELISA to detect the level of TNF-α secreted by PBMC cells by exosomes

[0252] Tumor Necrosis Factor-α (TNF-α) is a pro-inflammatory cytokine that participates in normal inflammatory and immune responses. It is mainly produced by activated monocytes and macrophages and acts in two forms in the body: transmembrane type (tmTNF) and secreted type (sTNF). tmTNF is distributed on the cells secreting TNF-α in the form of a membrane protein and is cleaved by the TNF-α-converting enzyme (TACE) to produce sTNF. The TNFα receptor is divided into two types (TNFRⅠ and TNFRⅡ), which exist on the surface of various cells. The binding of TNFα to TNFR usually causes cell apoptosis, inflammation, and tumorigenesis, etc.

[0253] The exosomes secreted by mesenchymal stem cells in the method of the present invention can inhibit lymphocytes from releasing TNF-α. In this experiment, the exosomes extracted and purified from mesenchymal stem cells and PBMC were co-cultured in a certain ratio, and then the ELISA method was used to detect the TNF-α expression level in the cell supernatant. The inhibitory ability of exosomes on lymphocytes releasing TNF-α was detected by analyzing the change in the TNF-α expression level.

[0254] 1. Isolate peripheral blood mononuclear cells (PBMCs) from the peripheral blood of healthy adults using the Ficoll method. Resuspend the PBMCs in serum-free immunocyte medium (Miltenyi, containing 2.5% serum substitute), adjust the density of PBMCs to 4×10^5 / ml, and then add 50 μl of CD3 / CD28 magnetic beads (Thermofisher) to each ml of PBMC suspension. After mixing the PBMCs and magnetic beads, add 0.5 ml of PBMCs to the wells of a 24-well plate, and add 50 μl of a suspension containing 1×10^7 exosomes to this well. Set up experimental group wells (PBMCs activated with magnetic beads and added with exosomes), control group wells (PBMCs activated with magnetic beads and not added with exosomes), negative group wells (PBMCs not activated with magnetic beads), and blank group wells (only containing serum-free immunocyte medium);

[0255] 2. After co-culturing for 5 days, collect the supernatant of the co-culture medium; then centrifuge at a speed of 2000 rpm for 5 minutes; collect the supernatant;

[0256] 3. Take out the ELISA kit (R&D Systems) and reagents for detecting TNF-α from the refrigerator, place them at room temperature (equilibrate to room temperature before use), remove the microplate strips and place them in the microplate strip holder, put the remaining microplate strips back into the foil bag, seal it, and put it back into the refrigerator;

[0257] 4. Take out a TNF-α standard, add 0.95 ml of deionized water, gently pipette it a few times with a pipette tip to dissolve the PGE2, and prepare a standard stock solution with a concentration of 10000 pg / ml; place the dissolved PGE2 standard at room temperature for minutes, shake the TNF-α standard 2 - 3 times by hand every 4 - 5 minutes; then take 7 1.5 ml EP tubes and dilute the TNF-α standard into different concentration gradients with Calibrator Diluent RD6 - 12: 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.3 pg / ml, and 15.6 pg / ml;

[0258] 5. Dilute the samples: 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;

[0259] 6. Sample addition and incubation: 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 the control group; add 50 μl of standard solution at different concentrations to the microplate, repeat for 3 wells respectively; add 50 μl of diluted sample to the microplate, repeat for 3 wells respectively; carefully cover the microplate with a film, and shake at a speed of 450 rpm at room temperature for 2 hours.

[0260] 7. Carefully tear off the film, pour out the liquid in the microplate, and invert the microplate and pat it on the absorbent paper several times; add 300 μl of 1×Wash Buffer to each well, then pour out the 1×Wash Buffer, and invert the microplate and pat it on the absorbent paper several times; repeat 3 times.

[0261] 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.

[0262] 9. Carefully tear off the film, pour out the liquid in the microplate, and invert the microplate and pat it on the absorbent paper several times; add 300 μl of 1×Wash Buffer to each well, then pour out the 1×Wash Buffer, and invert the microplate and pat it on the absorbent paper several times; repeat 3 times.

[0263] 10. Add 200 μl of Substrate Solution to each well, place it in the dark at room temperature for 30 minutes; then add 50 μl of Stop Solution to each well.

[0264] 11. Read the OD value: Put the microplate into the microplate reader (Thermofisher, Multiscan), then set the microplate reader program, detect the absorbance at a wavelength of 450 nm, and perform data analysis.

[0265] 12. After subtracting the average OD value of the culture medium blank group from the result, 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 conversion on both sides at the same time, and fit by the four-parameter method to draw the standard curve. Input the absorbance value of the sample and calculate the TNF-α concentration value of the sample.

[0266] Using the above method to measure the level of exosomes inhibiting PBMC from secreting TNF-α, the results are as follows:

[0267] The TNF-α level in the negative group is 802.2 pg / ml.

[0268] The TNF-α level in the control group is 5203.7 pg / ml.

[0269] The TNF-α levels in the test groups of Example 2, Example 2a, and Example 2b were 1227-1413 pg / ml. For example, the TNF-α level in the test group of Example 2 was 1316.2 pg / ml.

[0270] The TNF-α levels in the test groups of Example 3, Example 3a, and Example 3b were 1087-1348 pg / ml. For example, the TNF-α level in the test group of Example 3 was 1284.7 pg / ml.

[0271] The TNF-α levels in the test groups of Example 3c and Example 3d were in the range of 1232-1446 pg / ml.

[0272] Example 8: RT-PCR analysis of the inhibitory effect of exosomes on the TNF-α gene expression level of PBMC

[0273] 1. Extraction of total PBMC RNA: Aspirate 1 ml of PBMC cells from each group (experimental group, control group, negative group) after co-culture for 5 days in step 2 of Example 7 from the 24-well plate, place them in a 1.5-ml EP tube, centrifuge at 300 g for 5 minutes, then aspirate and discard the supernatant. Add 1 ml of Trizol reagent (Life company) to the precipitate, let it stand at room temperature for 5 minutes to fully lyse.

[0274] 2. Place the EP tube in a high-speed centrifuge, centrifuge at 4°C and 12,000 rpm for 5 minutes, then discard the precipitate.

[0275] 3. Add 200 μl of chloroform, mix well by shaking and let it stand at room temperature for 15 minutes.

[0276] 4. Place the EP tube in a high-speed centrifuge and centrifuge at 12,000 rpm for 15 minutes.

[0277] 5. Aspirate 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.

[0278] 6. Place the EP tube in a high-speed centrifuge, centrifuge at 4°C and 12,000 rpm for 10 minutes, then discard the supernatant. The RNA precipitate is at the bottom of the tube.

[0279] 7. Add 1 mL of 75% ethanol solution, gently shake the centrifuge tube to suspend the precipitate.

[0280] 8. Place the EP tube in a high-speed centrifuge, centrifuge at 4°C and 8,000 rpm for 5 minutes, then discard the supernatant; air-dry at room temperature for 5-10 minutes, and resuspend the RNA with 20 μl of sterile deionized water.

[0281] 9. Detect the RNA concentration with a nanodrop instrument.

[0282] 10. Reverse Transcription cDNA: Reverse transcribe RNA into cDNA according to the operating instructions of the Bestar qPCR kit (product number: DBI-2220).

[0283] 11. qPCR Detection of TNF-α Gene Expression Level: Perform real-time fluorescence quantitative PCR to detect the TNF-α gene expression level according to the operating instructions of the Bestar qPCR (SyberGreen) kit (product number: DBI-2043).

[0284] The TNF-α gene expression level detected by real-time fluorescence quantitative PCR is characterized by the relative mRNA level (fold). The relative mRNA level of the negative group is 1. Calculate the relative mRNA levels of the remaining groups. Results:

[0285] The relative mRNA level of the control group is 114.6-fold.

[0286] The relative mRNA levels of the experimental groups of Example 2, Example 2a, and Example 2b are 64 - 73-fold. For example, the relative mRNA level of the experimental group of Example 2 is 70.6-fold.

[0287] The relative mRNA levels of the experimental groups of Example 3, Example 3a, and Example 3b are 66 - 74-fold. For example, the relative mRNA level of the experimental group of Example 3 is 68.2-fold.

[0288] The relative mRNA levels of the experimental groups of Example 3c and Example 3d are 67 - 79-fold.

[0289] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to encompass all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. An exosome isolated and extracted using placenta mesenchymal stem cells, having an average particle size of 50 - 200 nm, and expressing membrane protein CD9 and membrane protein CD81, with a positive expression rate of membrane protein CD9 greater than 70% and a positive expression rate of membrane protein CD81 greater than 80%; this exosome is prepared using placenta mesenchymal stem cells by a method including the following steps: (1) Inoculate placenta mesenchymal stem cells of passages P2 - P8 at a density of (0.5 - 2)×10^4 cells / cm^2 into a culture flask, add MSC complete medium, place it in an incubator at 37°C and 5% CO2 for 20 - 30 hours to allow the cells to adhere, then add IL-1β to a concentration of 8 - 12 ng / mL, sodium tartrate to a concentration of 0.15 - 0.2 mg / mL, and lysine to a concentration of 2.0 - 2.5 mg / mL to the culture medium, and continue to culture for 20 - 30 hours; (2) Aspirate the culture medium and replace it with fresh MSC complete medium, and continue to culture the cells in an incubator at 37°C and 5% CO2 until the cell confluence ≥ 80%; (3) Aspirate the culture medium, wash with PBS, then add MSC complete medium, and place it in an incubator at 37°C, 2% O2, and 5% CO2 for 42 - 56 hours; (4) Aspirate the cell supernatant into a centrifuge tube and perform the following centrifugation: Centrifuge at 250 - 350 g and 4°C for 8 - 12 minutes, aspirate the supernatant into another centrifuge tube; Centrifuge at 1500 - 2500 g and 4°C for 18 - 25 minutes, aspirate the supernatant into another centrifuge tube; Centrifuge at 8000 - 12000 g and 4°C for 25 - 35 minutes, filter the supernatant through a 0.22 μm filter membrane and place it in another centrifuge tube; Centrifuge at 80000 - 120000 g and 4°C for 75 - 120 minutes, discard the supernatant; (5) Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80000 - 120000 g and 4°C for 75 - 120 minutes, discard the supernatant, add sterile PBS to resuspend the exosome, and obtain the exosome in the form of a suspension.

2. The exosome according to claim 1, having an average particle size of 75 - 150 nm, and a positive expression rate of membrane protein CD9 greater than 75% and a positive expression rate of membrane protein CD81 greater than 85%.

3. The exosome according to claim 1, in step (1), the mesenchymal stem cells are cells of passages P3 - P6.

4. The exosome according to claim 1, in step (1), use a T75 culture flask, inoculate (2 - 10)×10^5 cells per flask, and add 10 - 20 ml of culture medium.

5. The exosome according to claim 1, in step (1), use a T75 culture flask, inoculate 7.5×10^5 cells per flask, and add 10 - 20 ml of culture medium.

6. The exosome according to claim 1, in step (1), continue to culture for 24 hours after adding IL-1β.

7. For the exosomes according to claim 1, in step (1), the concentrations of sodium tartrate and lysine are 0.15 mg / mL and 2.2 mg / mL, respectively.

8. For the exosomes according to claim 1, in step (3), culture in an incubator at 37 °C, 2% O2, and 5% CO2 for 48 hours.

9. For the exosomes according to claim 1, in step (4), First, centrifuge at 250 g and 4 °C for 12 minutes, then centrifuge at 2500 g and 4 °C for 18 minutes, then centrifuge at 8000 g and 4 °C for 35 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 120000 g and 4 °C for 75 minutes; First, centrifuge at 350 g and 4 °C for 8 minutes, then centrifuge at 1500 g and 4 °C for 25 minutes, then centrifuge at 12000 g and 4 °C for 25 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 80000 g and 4 °C for 120 minutes; or, First, centrifuge at 300 g and 4 °C for 10 minutes, then centrifuge at 2000 g and 4 °C for 20 minutes, then centrifuge at 10000 g and 4 °C for 30 minutes. After filtering the supernatant with a 0.22 μm filter membrane, centrifuge at 100000 g and 4 °C for 90 minutes.

10. For the exosomes according to claim 1, in step (5), centrifuge at 100000 g and 4 °C for 90 minutes, or centrifuge at 80000 g and 4 °C for 120 minutes, or centrifuge at 120000 g and 4 °C for 75 minutes.

11. For the exosomes according to claim 1, in step (5), store the obtained exosome suspension at -80 °C.

12. For the exosomes according to claim 1, in step (5), resuspend the exosomes obtained from 1.5×10^6 cells in step (1) with 0.5 - 5 ml of sterile PBS.

13. For the exosomes according to claim 1, the placental mesenchymal stem cells are prepared by a method comprising the following steps: (a) Treat the placental tissue Remove the amnion from the human placenta, cut the surface membrane-like tissue of the placental lobules, and wash with physiological saline; Cut the surface membranous tissue of the placental lobule into tissue fragments with a volume of 0.2 cm 3 ; Place the tissue fragments in a centrifuge tube, add an appropriate amount of 0.9% physiological saline, filter through a 300-mesh sieve, and wash twice with an appropriate amount of 0.9% physiological saline until the filtrate is clear; Add the washed tissue to HBSS digestive solution containing 0.005% Liberase MNP-S enzyme and 0.05% DNA type I enzyme, mix well, and digest on a shaker at 37 °C and 100 rpm for 30 min; (b) Obtain placental primitive cells After digestion, add fetal bovine serum to the centrifuge tube to terminate digestion. Mix well and dilute with 50 ml of physiological saline containing 5% dextran, 2.5% human serum albumin, and 200 U of DNase I. Filter through a 300-mesh sieve, and wash the tissue with 100 ml of physiological saline multiple times. Collect the filtrate; Centrifuge the obtained filtrate at 1400 rpm for 5 min, discard the supernatant and collect the precipitate. Resuspend with physiological saline and centrifuge again to collect the precipitate; Resuspend the precipitated cells with DMEM / F12, take a sample for counting to obtain P0 generation cells; (c) Purify and culture Culture conditions: DMEM / F12 medium containing 10% fetal bovine serum and 2 mM L-glutamine, cultured in a constant temperature and humidity incubator at 37°C and 5% CO2; Digestion conditions: 0.25% trypsin, digested at 37°C for 2 minutes; Harvesting conditions: After terminating digestion with complete medium, centrifuge at 1400 rpm for 5 minutes and collect the precipitate; 5000~15000 pieces / cm 2 Placental P0 cells were inoculated into T75 culture flasks at an inoculation density of 1.50 μg / cm2, and the medium was completely replaced on the 3rd to 4th day. Cell clones appeared on the 6th to 7th day, and cells with a fusiform and spiral growth were formed on the 10th to 11th day, which were P1 placental mesenchymal stem cells, and they could be collected and passaged. (d) The P1 cells are purified and passaged in the same manner as in step (c) to obtain P2 cells; and so on, successively obtaining mesenchymal stem cells of P3 - P8 generations.

14. A method for isolating and extracting exosomes using placental mesenchymal stem cells, the method comprising the following steps: (1) Inoculate placental mesenchymal stem cells of P2 - P8 generations into a culture flask at a density of (0.5 - 2)×10^4 cells / cm^2, add MSC complete medium, and culture in a 37°C, 5% CO2 incubator for 20 - 30 hours to allow the cells to adhere. Then add IL-1β to the culture medium to a concentration of 8 - 12 ng / mL, sodium tartrate to a concentration of 0.15 - 0.2 mg / mL, and lysine to a concentration of 2.0 - 2.5 mg / mL, and continue to culture for 20 - 30 hours; (2) Aspirate the culture medium and replace it with fresh MSC complete medium, and continue to culture the cells in a 37°C, 5% CO2 incubator until the cell confluence ≥ 80%; (3) Aspirate the culture medium, wash with PBS, then add MSC complete medium, and then culture in a 37°C, 2% O2, 5% CO2 incubator for 42 - 56 hours; (4) Aspirate the cell supernatant into a centrifuge tube and perform the following centrifugation treatment: Centrifuge at 250 - 350 g at 4°C for 8 - 12 minutes, aspirate the supernatant into another centrifuge tube; Centrifuge at 1500 - 2500 g at 4°C for 18 - 25 minutes, aspirate the supernatant into another centrifuge tube; Centrifuge at 8000 - 12000 g at 4°C for 25 - 35 minutes, filter the supernatant through a 0.22 μm filter membrane and place it in another centrifuge tube; Centrifuge at 80000 - 120000 g at 4°C for 75 - 120 minutes, discard the supernatant; (5) Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80000 - 120000 g at 4°C for 75 - 120 minutes, discard the supernatant, add sterile PBS to resuspend the exosomes, and obtain exosomes in the form of a suspension.

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