Preparation method of umbilical cord mesenchymal stem cell-derived exosomes with inflammation regulation function

Abstract

The present invention relates to a method for preparing umbilical cord mesenchymal stem cell-derived exosomes with inflammation regulation function. The method for isolating and extracting exosomes from umbilical cord mesenchymal stem cells of the present invention comprises the following steps: inoculating mesenchymal stem cells into a culture bottle, culturing the cells to adhere to the wall, and then adding IL-1β to the culture medium for culturing; replacing the complete culture medium with 37°C, 2% O 2 , 5% CO 2 The invention also relates to the use of the prepared exosomes in the preparation of a drug for treating inflammatory diseases. The method of the invention has the advantages of high exosome yield, etc.

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 by culturing mesenchymal stem cells under inflammatory factors and hypoxic environment and extracting the exosomes, wherein the exosomes obtained by the method can significantly improve the anti-inflammatory activity of the cells. The mesenchymal stem cells of the present invention are obtained by separation from umbilical cord. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of non-hematopoietic multipotent stem cells derived from the mesoderm. It has been confirmed that in addition to the potential for self-renewal, self-replication and multidirectional differentiation, MSCs also have strong anti-inflammatory and inhibitory abilities for multiple immune cells, and can induce peripheral immune tolerance.Studies have shown that MSCs inhibit the function 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 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 cells modulate 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.).

[0003] Mesenchymal stem cells have low immunogenicity. Cell therapy based on MSCs 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 secretion. These secreted bioactive factors can inhibit fibrosis and apoptosis, enhance angiogenesis, and participate in tissue repair and regeneration.

[0004] Mesenchymal stem cells are abundant in sources and can be obtained from tissues such as umbilical cord, placenta, bone marrow, umbilical cord blood, and fat.

[0005] Exosomes are membrane-bound microvesicles secreted by cells with a diameter of about 30 to 200 nm and a density range of 1.13 to 1.19 g / ml. Exosomes can carry a variety of proteins, mRNAs, and miRNAs similar to those of the source cells, and participate in immune regulation, cell communication, cell migration, angiogenesis, and other processes (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 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 exosomes derived from MSCs 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's 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).

[0006] Exosomes, membrane vesicles secreted by living cells, were first discovered in 1983. With the deepening of research, it was found that they have the functions of transporting proteins and nucleic acids, specifically targeting receptor cells, exchanging proteins and lipids or triggering downstream signaling events, and participating in intercellular communication, and they have been continuously valued. Exosomes carry proteins, including two types of protein molecules: source cell nonspecific and source cell specific. The former may be related to the biogenesis and biological effects 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 type of special cells, and these exosomes of specific cell sources are closely related to their biological functions, for example: exosomes of molecular origin contain MHCII molecules. Therefore, exosomes from different cell sources carry different signal molecules and play different functions. For example, exosomes secreted by tumor cells can mediate angiogenesis, tumor cell proliferation and immune escape, while exosomes derived from dendritic cells can induce effective anti-tumor immune responses in the body. Existing studies have found that exosomes contain protein rRNA and microRNA related to the cell source, 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.

[0007] Exosomes are nano-scale lipid inclusion structures that contain proteins, mRNA, microRNA and other substances. Exosomes exist naturally in body fluids, including blood, saliva, urine, breast milk, etc. Exosomes are membrane vesicles derived from late endosomes (also known as multivesicular bodies) secreted by living cells. 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 finally can be engulfed by other cells. They are important mediators of intercellular communication. Exosomes secreted by host cells or tumor cells are involved in cell growth, proliferation, metabolism and regulation. Immune cells and tumor cells can also exchange information through exosomes. This communication method plays a dual role in regulating tumor immunity: exosomes can induce anti-tumor responses by inhibiting immune cells (DCs, NK cells, CD4+, CD8+T cells, etc.), and can also induce immunosuppression or immunosuppression of regulatory cell populations (MDSCs, Tregs, Bregs).

[0008] Current studies have found that the exosomes of mesenchymal stem cells carry a variety of effective cytokines, proteins and small molecule nucleic acid substances, which can effectively mediate cell proliferation, apoptosis and functional regulation. Studies have found that exosomes contain vascular endothelial growth factor, fibroblast growth factor, platelet proliferation factor, tumor necrosis factor and tumor growth factor, which have the functions of inhibiting cell apoptosis, cell fibrosis, promoting vascular mitosis and mediating immune response. Experiments have shown that the 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 immunology, the lipid membrane surface of exosomes secreted by mesenchymal stem cells expresses a variety of membrane proteins, such as coagulation factors, tumor necrosis factor, MHC I / II molecules and CCR5 chemokine receptors, etc. These lipid membrane surface proteins play an important role in resisting inflammation.

[0009] There are many methods and approaches for extracting exosomes. The existing methods for extracting exosomes mainly use ultracentrifugation or expensive kit column method. However, the quality of exosomes obtained by ultracentrifugation is uneven, the amount of exosomes obtained cannot be guaranteed, the step-by-step centrifugation time is long, and sometimes even the centrifugation time or centrifugation speed cannot be used to separate the exosomes, which wastes time and cost. The kit column method is usually only applicable to the acquisition of a small amount of exosomes and is expensive.

[0010] There are some reports on the isolation and extraction methods of exosomes in the prior art. For example, CN106282107A (Chinese patent application No. 201610779165.2) discloses a method for isolating exosomes from human placental mesenchymal stem cells, CN105708861A (Chinese patent application No. 201610149852.6) discloses the use of exosomes derived from bone marrow mesenchymal stem cells in the preparation of drugs for the treatment of ankylosing spondylitis, CN105267240A (Chinese patent application No. 201410781765.3) discloses the use of exosomes derived from mesenchymal stem cells, CN104382827A (Chinese patent application No. 201410705462.3) discloses the use of exosomes from human amniotic mesenchymal stem cells, and CN103767985A (Chinese patent application No. 201210402915.6) discloses the preparation and use of exosomes secreted by human blood or mesenchymal stem cells. However, in the prior art, exosomes are extracted using culture supernatants of mesenchymal stem cells from various sources, and these methods all have the disadvantages of low yield and complex processes.

[0011] Therefore, the art still expects new methods to separate and extract exosomes, especially to extract exosomes by secretion of mesenchymal stem cells in a simple, easy and high-yield manner. In addition, the art also expects to provide a method for treating inflammatory diseases, such as a method for treating inflammatory diseases by using exosomes. Summary of the invention

[0012] The object of the present invention is to provide a new method for isolating and extracting exosomes, in particular, this method can extract exosomes secreted by mesenchymal stem cells in a simple, easy and high-yield manner. It has been unexpectedly found that the above-mentioned object can be beneficially achieved by the method of the present invention. The present invention is completed based on this discovery.

[0013] To this end, the first aspect of the present invention provides a method for isolating and extracting exosomes from umbilical cord mesenchymal stem cells, the method comprising the following steps:

[0014] (1) Mesenchymal stem cells were seeded into culture flasks, and MSC complete medium was added. The culture flasks were incubated at 37°C and 5% CO 2 The cells were cultured in an incubator to allow them to adhere to the wall, and IL-1β was added to the culture medium to a concentration of 8 to 12 ng / mL, and the culture was continued;

[0015] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium. Keep the cells at 37°C and 5% CO. 2 Culture in an incubator until the cell confluence is ≥80%;

[0016] (3) Aspirate the culture medium, wash with PBS, add MSC complete culture medium, and place at 37°C, 2% O 2 , 5% CO 2 Culture in an incubator for 42 to 56 hours;

[0017] (4) Pipette the cell supernatant into a centrifuge tube and perform the following centrifugation treatment:

[0018] Centrifuge at 250-350g and 4°C for 8-12 minutes, and transfer the supernatant to another centrifuge tube;

[0019] Centrifuge at 1500-2500g and 4°C for 18-25 minutes, and transfer the supernatant to another centrifuge tube;

[0020] Centrifuge at 8000-12000g and 4°C for 25-35 minutes, filter the supernatant with a 0.22 μm filter membrane and place in another centrifuge tube;

[0021] Centrifuge at 80,000-120,000 g and 4°C for 75-120 min and discard the supernatant;

[0022] (5) Sterile PBS was added to the centrifuge tube to resuspend the exosome precipitate, and the mixture 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 the exosomes in the form of a suspension.

[0023] According to the method of the first aspect of the present invention, in step (1), the mesenchymal stem cells are cells of generation P2 to P8, for example, cells of generation P3 to P6.

[0024] According to the method 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, each flask is inoculated with 2-10×10^5 cells, for example, 5×10^5 cells, and 10-20 ml of culture medium is added.

[0025] According to the method 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 to the wall.

[0026] According to the method of the first aspect of the present invention, in step (1), culturing is continued for 20 to 30 hours, for example, 24 hours, after adding IL-1β.

[0027] According to the method of the first aspect of the present invention, in step (3), the mixture is heated at 37°C and 2% O 2 , 5% CO 2 Culture in an incubator for 48 hours;

[0028] According to the method of the first aspect of the present invention, 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, then at 8000 g and 4° C. for 35 minutes, and the supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 120,000 g and 4° C. for 75 minutes.

[0029] According to the method of the first aspect of the present invention, in step (4), the mixture is first centrifuged at 350g and 4°C for 8 minutes, then at 1500g and 4°C for 25 minutes, then at 12000g and 4°C for 25 minutes, and the supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0030] According to the method of the first 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, then at 10000g and 4°C for 30 minutes, and the supernatant is filtered through a 0.22 μm filter membrane and then centrifuged at 100000g and 4°C for 90 minutes.

[0031] According to the method of the first 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.

[0032] According to the method of the first aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0033] According to the method of the first aspect of the present invention, in step (5), the exosomes obtained from 1×10^6 cells in step (1) are resuspended with 0.5-5 ml of sterile PBS, for example, with 0.5-2 ml of sterile PBS.

[0034] According to the method of the first aspect of the present invention, in step (1), potassium sodium tartrate and lysine are added to the culture medium at the same time as IL-1β, and the concentrations of the two are 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, respectively. For example, the concentrations of the two are 0.15 mg / mL and 2.2 mg / mL, respectively.

[0035] According to the method of the first 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.

[0036] According to the method of the first 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%, such as greater than 70%, such as greater than 75%. In one embodiment, the positive expression rate of exosome membrane protein CD81 is greater than 75%, such as greater than 80%, such as greater than 85%.

[0037] According to the method of the first aspect of the present invention, the umbilical cord mesenchymal stem cells are prepared by a method comprising the following steps:

[0038] (a) Clean the surface of the placenta with tissue cleaning solution to remove surface blood, cut the umbilical cord and remove the internal blood vessels;

[0039] (b) Remove Wharton's jelly from the umbilical cord, clean it, cut it into small pieces of 2-3 mm^3, and transfer it to a T75 culture flask;

[0040] (c) Slowly adding MSC complete medium to the culture bottle, gently shaking the culture bottle to spread the umbilical cord tissue block in the culture bottle, and culturing it in a 37° C., 5% CO2 incubator. Replenishing the medium every 2-3 days, after 2-3 times of replenishing the medium, remove the tissue block and replace the MSC complete medium in full to continue culturing until the cells grow to a confluency of 70%, and then digest the obtained cells;

[0041] (d) The culture medium in the culture flask was discarded, PBS was aspirated to rinse the culture flask to remove the residual culture medium, 3 mL of recombinant trypsin solution was added for digestion, the culture flask was tapped to make the cells fall off to the bottom of the culture flask, MSC complete culture medium was added to neutralize trypsin to terminate the digestion process, the cells were blown off with a pipette, the cell suspension was collected in a centrifuge tube, centrifuged, and the supernatant was discarded to obtain a cell pellet recorded as cells of the P0 generation;

[0042] (e) The P0 cell pellet was resuspended in MSC complete medium, the cells were counted, and then inoculated into a culture flask, supplemented with the above MSC complete medium, and cultured in a 37° C., 5% CO2 incubator until the cell confluence reached 80%, the medium in the culture flask was discarded, the culture flask was rinsed with PBS to remove the residual medium, a recombinant trypsin solution was added for digestion, the culture flask was tapped to make the cells fall off to the bottom of the culture flask, MSC complete medium was added to neutralize the trypsin to terminate the digestion process, the cells were blown off with a pipette, the cell suspension was collected in a centrifuge tube, centrifuged, and the supernatant was discarded to obtain a cell pellet recorded as P1 cells;

[0043] (f) Repeat step (e) to perform subculture of MSC cells until the cell generations are P3 to P8.

[0044] Furthermore, the second aspect of the present invention provides use of the exosomes prepared by the method described in the first aspect of the present invention in preparing a medicament for treating inflammatory diseases.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Although MSC-derived exosomes have many advantages such as promoting angiogenesis, cell proliferation, growth, and migration, most of the MSC-derived exosomes cultured by conventional methods currently do not have a significant effect in mediating inflammation regulation. Based on this, the present invention designs a method of inducing MSC culture and isolating and purifying the exosomes secreted by MSC by combining low oxygen partial pressure with inflammatory factor stimulation, which shows good inflammation regulation in vitro, providing a new solution for the treatment of inflammatory diseases with exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 :Electron microscopic images of exosomes from umbilical cord mesenchymal stem cells.

[0050] Figure 2 :Particle size of exosomes from umbilical cord mesenchymal stem cells.

[0051] Figure 3 : Expression of exosomal membrane protein CD9.

[0052] Figure 4 : Expression level of exosomal membrane protein CD81.

[0053] Figure 5 : BCA standard curve. DETAILED DESCRIPTION

[0054] 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. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of 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 used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible here.

[0055] Unless otherwise specified, some reagents used in the present invention are conventional in the art or can be easily purchased from commercial sources. 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 taking 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, adding 118 ml of 0.2 mol / L sodium hydroxide solution, diluting with water to 1000 ml, shaking well, and sterilizing at 121°C for 15 min; tissue cleaning solution (PRS-TCR-1) was purchased from Precicion. The MSC complete culture medium described herein is a serum-free culture medium containing 2% human platelet lysate.

[0056] Example 1: Primary and Subculture of Umbilical Cord Mesenchymal Stem Cells

[0057] Methods for obtaining primary and passaged mesenchymal stem cells through the umbilical cord have been reported in many literatures. The mesenchymal stem cells used for preparing exosomes in the present invention can be obtained using these literature methods, and 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.

[0058] 1. Use tissue cleaning solution (0.9% saline containing 1% penicillin and 1% streptomycin) to clean the surface of the placenta to remove surface congestion, cut the umbilical cord and remove the internal blood vessels. This step is specifically performed as follows: Place stainless steel trays, filter screens, stainless steel cups, lunch boxes, tissue cleaning solution (0.9% saline containing 1% penicillin and 1% streptomycin), pipettes and other items in appropriate positions in the biosafety cabinet. Use surgical tweezers to remove the placental tissue and place it in a stainless steel tray, clean the surface of the placenta with tissue cleaning solution, and remove the congestion on the surface of the placenta. Use surgical scissors and tweezers to cut the umbilical cord connected to the placenta, and cut the other end from behind the sterile umbilical cord clamp. Take out the umbilical cord and wash it 3 times with tissue cleaning solution to remove surface congestion and blood clots. Clamp one end of the umbilical cord with tweezers, clamp the umbilical cord with another pair of tweezers and slide along the umbilical cord tissue to remove residual umbilical cord blood in the blood vessels. First, cut the umbilical cord into several sections of 2-3 cm in length, then cut it open along the longitudinal section with scissors, flatten it, remove the three blood vessels inside (two umbilical arteries and one umbilical vein), and transfer it to a new dish.

[0059] 2. Peel off the Wharton's jelly in the umbilical cord, wash it (with tissue cleaning solution), cut it into small pieces of 2-3mm^3, and transfer it to a T75 culture bottle, 1mL per bottle. This step is specifically performed as follows: Use tweezers to peel off the Wharton's jelly, put it into a culture dish filled with tissue cleaning solution, wash it with tissue cleaning solution and tear the Wharton's jelly into long strips. After processing, use tweezers to transfer the Wharton's jelly to a 50mL centrifuge tube, cut it into small pieces of 2-3mm3 with scissors, and transfer it to a T75 culture bottle with a 3mL sterile dropper, 1mL per bottle. The tip of the dropper needs to be cut off with sterile scissors before use.

[0060] 3. Slowly add 10 mL of MSC complete medium (serum-free medium containing 2% human platelet lysate) to the culture bottle, gently shake the culture bottle, so that the umbilical cord tissue block is flattened in the culture bottle, and cultured in a 37°C, 5% CO2 incubator. Replenish the fluid once every 2-3 days (add 3-4 mL of MSC complete medium each time), and after 2-3 times of repletion, remove the tissue block and replace the MSC complete medium in full to continue culturing until the cells grow to a confluency of 70%, and the obtained cells are then digested. This step is specifically performed as follows: Slowly add 10 mL of MSC complete medium (serum-free medium containing 2% human platelet lysate) to the culture bottle, gently shake the culture bottle, so that the umbilical cord tissue block is flattened in the culture bottle. Place the T75 culture bottle in a 37°C, 5% CO2 incubator for static culture. On the third day of culture, remove the culture bottle from the incubator, add 3.5 mL of MSC complete medium, and then rehydrate once every 3 days. After three times of rehydration, cells will crawl out from the bottom of the culture flask. The tissue blocks will be removed and the MSC complete medium will be replaced to continue the culture. When the cells grow to a confluency of 70%, the obtained cells will be digested.

[0061] 4. The culture medium in the culture bottle is discarded, (20 mL) PBS is drawn to rinse the culture bottle to remove the residual culture medium, 3 mL of recombinant trypsin solution is added for digestion, the culture bottle is tapped to make the cells fall off to the bottom of the culture bottle, 10 mL of MSC complete medium is added to neutralize trypsin to terminate the digestion process, the cells are blown away with a pipette, the cell suspension is collected in a centrifuge tube, centrifuged, the supernatant is discarded, and the cell pellet is recorded as the P0 generation of cells. This step is specifically operated as follows: before cell subculture, recombinant trypsin, MSC complete medium (serum-free medium containing 2% human platelet lysate), and PBS are placed at room temperature for equilibrium temperature. The culture bottle is taken out of the incubator, the culture medium in the culture bottle is discarded with a pipette, and 20 mL of PBS is drawn to rinse the culture bottle to remove the residual culture medium. Add 3mL of recombinant trypsin solution (GIBCO) to the culture flask for digestion, gently shake the culture flask to allow trypsin to fully cover the cell surface, digest for about 1min, observe the cells shrink into a circle under a microscope, gently tap the culture flask to make the cells fall off to the bottom of the culture flask in a quicksand-like state, add 10mL of MSC complete medium to neutralize trypsin to terminate the digestion process, blow the cells with a pipette, and collect the cell suspension in a 15mL centrifuge tube. Place the centrifuge tube in a centrifuge, centrifuge at 1400rpm for 5min, remove the supernatant, and the precipitated cells are recorded as P0 generation.

[0062] 5. Resuspend the P0 cell pellet in 10 mL of MSC complete medium, count the cells, and then inoculate at a density of 1×10^ 4MSC cells / cm^2 were inoculated into T75 culture flasks, supplemented with the above-mentioned MSC complete medium to 10 mL, and cultured in a 37°C, 5% CO2 incubator for more than 72 h until the cell confluence reached 80%, the medium in the culture flask was discarded, (20 mL) PBS was aspirated to rinse the culture flask to remove the residual medium, 3 mL of recombinant trypsin solution was added for digestion, the culture flask was tapped to make the cells fall off to the bottom of the culture flask, 10 mL of MSC complete medium was added to neutralize the trypsin to terminate the digestion process, the cells were blown off with a pipette, the cell suspension was collected in a centrifuge tube, centrifuged, the supernatant was discarded, and the cell pellet was recorded as P1 cells.

[0063] 6. Repeat step 5 to subculture MSC cells until the cell generations are P3 to P8.

[0064] Example 2: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes by differential centrifugation

[0065] (1) The mesenchymal stem cells (P4 generation) obtained in Example 1 were inoculated into T75 culture flasks at a density of 5 × 10^5 cells, and 15 mL of MSC complete medium was added to each flask. The flask was placed at 37°C and 5% CO. 2 The cells were cultured in an incubator (24 hours) to allow the cells to adhere to the wall, and IL-1β was 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 other specifications of culture flasks, such as T25 and T75 flasks, may be used instead, and the number of cells seeded and / or the amount of culture medium added may be changed according to the culture area. For example, when culturing MSCs, it is a common practice in the art to use the above concentration and add about 15 ml of culture medium]

[0066] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium. Keep the cells at 37°C and 5% CO. 2 Culture in an incubator until the cell confluence is ≥80%;

[0067] (3) Aspirate the culture medium, wash with PBS three times, then add 15 mL of MSC complete culture medium to each bottle and place at 37°C, 2% O 2 , 5% CO 2 Culture in an incubator for 48 hours;

[0068] [Above 2% O 2 , 5% CO 2 The atmosphere in the incubator is a nitrogen-balanced atmosphere, while the aforementioned 5% CO 2 The atmosphere in the incubator refers to an atmosphere of 21% oxygen and a balance of nitrogen, and the meaning of this expression is generally recognized by those skilled in the art]

[0069] (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:

[0070] 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;

[0071] Centrifuge at 2000 g and 4 °C for 20 min (to further remove cell debris and other impurities), and pipette the supernatant into another (high-speed) centrifuge tube;

[0072] Centrifuge at 10,000 g and 4°C for 30 min (to further remove smaller cell debris and impurities), and filter the supernatant with a 0.22 μm filter (PES filter, millipore) and place in another (ultracentrifuge) tube;

[0073] Centrifuge at 100,000 g and 4 °C for 90 min and discard the supernatant;

[0074] (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.

[0075] The centrifuge tubes and centrifuges with different centrifugal force requirements used in the above steps were purchased from Beckman Coulter.

[0076] Example 2a: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes by differential centrifugation

[0077] (1) The mesenchymal stem cells (P3 generation) obtained in Example 1 were inoculated into T75 culture flasks at a density of 5×10^5 cells, and 17.5 mL of MSC complete medium was added to each flask. The flask was placed at 37°C and 5% CO. 2 After culturing in an incubator (20 h) to allow cells to adhere, IL-1β was added to the culture medium to a concentration of 8 ng / mL and culture was continued for 24 h;

[0078] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium. Keep the cells at 37°C and 5% CO. 2 Culture in an incubator until the cell confluence is ≥80%;

[0079] (3) Aspirate the culture medium, wash with PBS three times, then add 15 mL of MSC complete culture medium to each bottle and place at 37°C, 2% O 2 , 5% CO 2 Culture in an incubator for 48 hours;

[0080] (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:

[0081] 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;

[0082] Centrifuge at 2500 g and 4 °C for 18 min (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0083] 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 (PES filter, millipore) and place in another (ultracentrifuge) tube;

[0084] Centrifuge at 120,000 g and 4 °C for 75 min and discard the supernatant;

[0085] (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.

[0086] Example 2b: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes by differential centrifugation

[0087] (1) The mesenchymal stem cells (P6 generation) obtained in Example 1 were inoculated into T75 culture flasks at a density of 5×10^5 cells, and 12.5 mL of MSC complete medium was added to each flask. The flask was placed at 37°C and 5% CO. 2 After culturing in an incubator (30 h) to allow cells to adhere, IL-1β was added to the culture medium to a concentration of 12 ng / mL and culture was continued for 24 h;

[0088] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium. Keep the cells at 37°C and 5% CO. 2 Culture in an incubator until the cell confluence is ≥80%;

[0089] (3) Aspirate the culture medium, wash with PBS three times, then add 15 mL of MSC complete culture medium to each bottle and place at 37°C, 2% O 2 , 5% CO 2 Culture in an incubator for 48 hours;

[0090] (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:

[0091] 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;

[0092] Centrifuge at 1500 g and 4 °C for 25 min (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0093] Centrifuge at 12000g and 4°C for 25 min (to further remove smaller cell debris and impurities), and filter the supernatant with a 0.22 μm filter (PES filter, millipore) and place in another (ultracentrifuge) tube;

[0094] Centrifuge at 80,000 g and 4 °C for 120 min and discard the supernatant;

[0095] (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.

[0096] Example 3: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes by differential centrifugation

[0097] (1) The mesenchymal stem cells (P4 generation) obtained in Example 1 were inoculated into T75 culture flasks at a density of 5 × 10^5 cells, and 15 mL of MSC complete medium was added to each flask. The flask was placed at 37°C and 5% CO. 2 The cells were cultured in an incubator (24 hours) to allow them to adhere to the wall, and IL-1β, sodium potassium tartrate, and lysine were added to the culture medium to concentrations of 10 ng / mL, 0.15 mg / mL, and 2.2 mg / mL, respectively, and the culture was continued for 24 hours;

[0098] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium. Keep the cells at 37°C and 5% CO. 2 Culture in an incubator until the cell confluence is ≥80%;

[0099] (3) Aspirate the culture medium, wash with PBS three times, then add 15 mL of MSC complete culture medium to each bottle and place at 37°C, 2% O 2 , 5% CO 2 Culture in an incubator for 48 hours;

[0100] [Above 2% O 2 , 5% CO 2 The atmosphere in the incubator is a nitrogen-balanced atmosphere, while the aforementioned 5% CO 2 The atmosphere in the incubator refers to an atmosphere of 21% oxygen and a balance of nitrogen, and the meaning of this expression is generally recognized by those skilled in the art]

[0101] (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:

[0102] 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;

[0103] Centrifuge at 2000 g and 4 °C for 20 min (to further remove cell debris and other impurities), and pipette the supernatant into another (high-speed) centrifuge tube;

[0104] Centrifuge at 10,000 g and 4°C for 30 min (to further remove smaller cell debris and impurities), and filter the supernatant with a 0.22 μm filter (PES filter, millipore) and place in another (ultracentrifuge) tube;

[0105] Centrifuge at 100,000 g and 4 °C for 90 min and discard the supernatant;

[0106] (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.

[0107] Example 3a: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes by differential centrifugation

[0108] (1) The mesenchymal stem cells (P3 generation) obtained in Example 1 were inoculated into T75 culture flasks at a density of 5×10^5 cells, and 17.5 mL of MSC complete medium was added to each flask. The flask was placed at 37°C and 5% CO. 2 The cells were cultured in an incubator (20 hours) to allow the cells to adhere to the wall, and IL-1β, potassium sodium tartrate, and lysine hydrochloride were added to the culture medium to concentrations of 8 ng / mL, 0.175 mg / mL, and 2 mg / mL, respectively, and the culture was continued for 24 hours;

[0109] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium. Keep the cells at 37°C and 5% CO. 2 Culture in an incubator until the cell confluence is ≥80%;

[0110] (3) Aspirate the culture medium, wash with PBS three times, then add 15 mL of MSC complete culture medium to each bottle and place at 37°C, 2% O 2 , 5% CO 2 Culture in an incubator for 48 hours;

[0111] (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:

[0112] 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;

[0113] Centrifuge at 2500 g and 4 °C for 18 min (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0114] 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 (PES filter, millipore) and place in another (ultracentrifuge) tube;

[0115] Centrifuge at 120,000 g and 4 °C for 75 min and discard the supernatant;

[0116] (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.

[0117] Example 3b: Treatment of mesenchymal stem cells with IL-1β and separation and purification of exosomes by differential centrifugation

[0118] (1) The mesenchymal stem cells (P6 generation) obtained in Example 1 were inoculated into T75 culture flasks at a density of 5×10^5 cells, and 12.5 mL of MSC complete medium was added to each flask. The flask was placed at 37°C and 5% CO. 2 The cells were cultured in an incubator (30 hours) to allow them to adhere to the wall, and IL-1β, potassium sodium tartrate, and lysine hydrochloride were added to the culture medium to concentrations of 12 ng / mL, 0.125 mg / mL, and 2.5 mg / mL, respectively, and the culture was continued for 24 hours;

[0119] (2) Aspirate the culture medium and replace with fresh MSC complete culture medium. Keep the cells at 37°C and 5% CO. 2 Culture in an incubator until the cell confluence is ≥80%;

[0120] (3) Aspirate the culture medium, wash with PBS three times, then add 15 mL of MSC complete culture medium to each bottle and place at 37°C, 2% O 2 , 5% CO 2 Culture in an incubator for 48 hours;

[0121] (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:

[0122] 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;

[0123] Centrifuge at 1500 g and 4 °C for 25 min (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0124] Centrifuge at 12000g and 4°C for 25 min (to further remove smaller cell debris and impurities), and filter the supernatant with a 0.22 μm filter (PES filter, millipore) and place in another (ultracentrifuge) tube;

[0125] Centrifuge at 80,000 g and 4 °C for 120 min and discard the supernatant;

[0126] (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.

[0127] Example 3c: Referring to Example 3, Example 3a and Example 3b respectively, the only difference is that potassium 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.

[0128] Example 3d: Referring to Example 3, Example 3a and Example 3b, 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.

[0129] Example 4: Transmission electron microscopy observation of exosome particles

[0130] 1. Take 50 μl of the isolated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and place it in a 4°C refrigerator for fixation for 1 hour;

[0131] 2. Add 20 μl of the fixed exosome suspension onto the front of the copper mesh and let it stand for 20 minutes;

[0132] 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;

[0133] 4. Add 1 drop of 2% uranyl acetate dye solution to the front of the copper mesh, dye for 1 minute, and then use filter paper to absorb the excess dye along the edge of the copper mesh;

[0134] 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.

[0135] The above method was used to detect the exosomes (suspension) obtained in each embodiment of the present invention. The number of exosomes obtained from 1×10^6 cells (2 culture flasks) in each embodiment was calculated, and the results were as follows: the number of exosome particles in Example 2, Example 2a, and Example 2b was 3.4×10^9, 4.3×10^9, and 3.1×10^9, respectively, the number of exosome particles in Example 3, Example 3a, and Example 3b was 74.4×10^9, 66.4×10^9, and 71.3×10^9, respectively, the number of exosome particles in Example 3c1, Example 3c2, and Example 3c3 was 4.7×10^9, 5.2×10^9, and 3.8×10^9, respectively, and the number of exosome particles in Example 3d1, Example 3d2, and Example 3d3 was 5.8×10^9, 5.4×10^9, and 4.6×10^9, respectively. Taking the exosomes in Example 2 as an example, the initial amount of MSC cells is 1×10^6 cells, the obtained exosome volume is 1 ml, and the exosome concentration is measured to be 3.4×10^9 particles / mL, which is equivalent to obtaining 3.4×10^9 exosomes from 1×10^6 cells. The above "3.4×10^9 exosomes" means 3.4 times 10 to the 9th power of exosomes, and other similar expressions have similar meanings.

[0136] Figure 1 The electron micrograph of the umbilical cord mesenchymal stem cell exosomes obtained in Example 3 is shown. Figure 2 The particle size and distribution of umbilical cord mesenchymal stem cell exosomes obtained in Example 3 are shown, with a peak particle size of 102.5±3.0nm and an average particle size of 116.3±0.4nm. Electron micrographs of exosomes obtained in other examples and the particle size of exosomes Figure 1 and Figure 2 There is no significant difference.

[0137] Example 5: Detection of protein expression on the surface of exosome particles by flow cytometry

[0138] 1. Mix the components of 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;

[0139] 2. Add 200 μl of magnetic bead cleaning solution to the EP tube and mix thoroughly with a pipette tip;

[0140] 3. Place the EP on the magnetic stand for 1 minute, then discard the supernatant;

[0141] 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;

[0142] 5. Place the exosome-washing solution mixture on a rotary mixer, set the speed to 10 rpm, and incubate overnight at 2-8°C;

[0143] 6. The next day, centrifuge the sample for 3 to 5 seconds to collect the precipitate;

[0144] 7. Add 300 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds;

[0145] 8. Place the sample on the magnetic rack for about 1 minute and discard the supernatant;

[0146] 9. Add 400 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds;

[0147] 10. Place the sample on the magnetic rack for about 1 minute, discard the supernatant, and resuspend with 300 μl of washing solution;

[0148] 11. Take 100 μl of each sample, add CD9-PE and CD81-FITC flow cytometry antibodies to the samples, and incubate at 4°C in the dark for 30 minutes.

[0149] 12. Place the incubated sample on the magnetic rack for about 1 minute, discard the supernatant, and add 300 μl of cleaning solution for washing;

[0150] 13. Repeat step 12 once, resuspend the selected sample with 300 μl PB and then test on the instrument.

[0151] Figure 3 The expression level of exosome membrane protein CD9 obtained in Example 3 is shown. Figure 4 The expression of membrane protein CD81 in the exosomes obtained in Example 3 is shown. The positive expression rate of Cd9 is 81.2%, and the positive expression rate of Cd81 is 96.3%. The expression of membrane protein CD9 and CD81 in the exosomes obtained in other examples is similar to Figure 3 and Figure 4 There is no significant difference.

[0152] Example 6: Detection of exosome protein content

[0153] This example uses Pierce TM The protein quantification kit (Cat. No. 23225, Thermo Scientific) was used for the test.

[0154] 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 the standard solution to the protein standard wells of a 96-well plate and add PBS to make up to 20 μL.

[0155] 2. Dilute the exosome sample appropriately and add 20 μL to the sample wells of the 96-well plate. Since the pipette has a large error when taking a small amount of sample, the point before the standard line may not be very accurate, so try to make the sample concentration point fall after 1 / 2 of the standard line.

[0156] 3. Add 200 μL of BCA working solution to each well and place at 37°C for 15-30 minutes. Use an enzyme-labeled instrument to measure the OD value at A562nm and calculate the protein concentration based on the standard curve.

[0157] Figure 5 The BCA standard curve obtained by the method of this example is shown, and 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.

[0158] The above method was used to detect the exosomes (suspension) obtained in each embodiment of the present invention. The protein content of the exosomes obtained from 1×10^6 cells in each embodiment was calculated, and the results were as follows: the protein content of Example 2, Example 2a and Example 2b was 48.6μg, 52.2μg and 46.7μg, respectively, the protein content of Example 3, Example 3a and Example 3b was 275.3μg, 257.4μg and 286.6μg, respectively, and the protein content of Example 3c1, Example 3c2 and Example 3c3 was 57.8μg, 44.7μg and 49.4μg, respectively, and the protein content of Example 3d1, Example 3d2 and Example 3d3 was 58.2μg, 54.1μg and 43.8μg, respectively.

[0159] Example 7: Elisa test of immune regulation of exosomes on TNF-α secretion level of PBMC cells

[0160] 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 exerts its effects in the body in two forms: transmembrane TNF (tmTNF) and secretory TNF (sTNF). tmTNF is distributed in the form of membrane proteins on cells that secrete TNF-α, and sTNF is produced after activation and cleavage by TACE (TNF-α-converting enzyme). There are two types of TNFα receptors (TNFRⅠ and TNFRⅡ), which exist on the surface of many cells. The binding of TNFα to TNFR usually causes cell apoptosis, inflammation, and tumorigenesis.

[0161] The exosomes secreted by the 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 and PBMC were co-cultured in a certain ratio, and then the ELISA method was used to detect the expression level of TNF-α in the cell supernatant. The inhibitory ability of exosomes on the release of TNF-α by lymphocytes was detected by analyzing the changes in the expression level of TNF-α.

[0162] 1. Use the Ficoll method to isolate mononuclear cells (PBMC) from the peripheral blood of healthy adults, resuspend PBMC in immune cell serum-free medium (Miltenyi, containing 2.5% serum replacement), adjust the density of PBMC to 4×10^5 / ml, and then add 50μl CD3 / CD28 magnetic beads (Thermofisher) to each ml of PBMC suspension; after mixing PBMC and magnetic beads, add 0.5ml PBMC to the well of a 24-well plate, and add 50μl of a suspension containing 1×10^7 exosomes to the well. Set up the experimental group wells (PBMC activated with magnetic beads and added with exosomes), the control group wells (PBMC activated with magnetic beads and without exosomes), the negative group wells (PBMC not activated with magnetic beads), and the blank group wells (containing only immune cell serum-free medium);

[0163] 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;

[0164] 3. Take out the ELISA kit (R&D Systems) and reagents for detecting TNF-α from the refrigerator and place them at room temperature (equilibrate to room temperature before use). Remove the microwell strips and place them in the microwell strip holder. Put the remaining microwell strips back into the foil bag, seal it, and return it to the refrigerator.

[0165] 4. Take out a TNF-α standard, add 0.95ml deionized water, and gently blow a few times with a pipette to dissolve PGE2 to prepare a standard stock solution with a concentration of 10000pg / ml; leave the dissolved PGE2 standard at room temperature for minutes, and shake the TNF-α standard by hand 2-3 times every 4-5 minutes; then take 7 1.5ml EP tubes and use Calibrator Diluent RD6-12 to dilute the TNF-α standard into different concentration gradients: 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.3pg / ml and 15.6pg / ml;

[0166] 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;

[0167] 6. Add samples and incubate: Add 50μl of Assay Diluent RD1F to each well of the microplate; add 50μl of culture medium to the microplate, repeat 3 wells as a control group; add 50μl of standard solutions of different concentrations to the microplate, repeat 3 wells; add 50μl of diluted samples to the microplate, repeat 3 wells; carefully cover the microplate film, and shake at 450rpm for 2 hours at room temperature;

[0168] 7. Carefully peel off the film, pour out the liquid in the microplate, turn the microplate upside down on absorbent paper and tap it several times; add 300μl 1×Wash Buffer to each well, then pour out 1×Wash Buffer, turn the microplate upside down on absorbent paper and tap it several times; repeat 3 times;

[0169] 8. Add 200 μl TNF-α Conjugate to each well, carefully cover with a new film, and incubate at room temperature for 2 hours;

[0170] 9. Carefully remove the film, pour out the liquid in the microplate, turn the microplate upside down on absorbent paper and tap it several times; add 300μl 1×Wash Buffer to each well, then pour out 1×Wash Buffer, turn the microplate upside down on absorbent paper and tap it several times; repeat 3 times;

[0171] 10. Add 200 μl of Substrate Solution to each well and place it at room temperature in the dark for 30 minutes; then add 50 μl of Stop Solution to each well;

[0172] 11. Read the OD value: Place the microplate into the microplate reader (Thermofisher, Multiscan), then set the microplate reader program to detect the absorbance at a wavelength of 450nm and perform data analysis;

[0173] 12. After subtracting the average OD value of the culture medium blank group, 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, and fit with the four-parameter method to draw the standard curve. Enter the absorbance value of the sample and calculate the TNF-α concentration of the sample.

[0174] The above method was used to measure the level of exosomes inhibiting PBMC secretion of TNF-α. The results were as follows:

[0175] The TNF-α level in the negative group was 802.2 pg / ml.

[0176] The TNF-α level in the control group was 5203.7 pg / ml.

[0177] The TNF-α level of the test groups of Example 2, Example 2a, and Example 2b was 1031-1327 pg / ml. For example, the TNF-α level of the test group of Example 2 was 1160.8 pg / ml.

[0178] The TNF-α level of the test groups of Example 3, Example 3a, and Example 3b was 1123-1386 pg / ml. For example, the TNF-α level of the test group of Example 3 was 1143.3 pg / ml.

[0179] The TNF-α levels of the test groups of Example 3c and Example 3d were in the range of 1072 to 1421 pg / ml.

[0180] Example 8: RT-PCR analysis of the inhibitory effect of exosomes on TNF-α gene expression level in PBMC

[0181] 1. Extraction of total RNA from PBMC: 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 was aspirated from a 24-well plate and placed in a 1.5 ml EP tube. Centrifuge at 300 g for 5 minutes, discard the supernatant, add 1 ml of Trizol reagent (Life Company) to the precipitate, and leave at room temperature for 5 minutes to fully lyse.

[0182] 2. Place the EP tube in a high-speed centrifuge, centrifuge at 4°C, 12,000 rpm for 5 minutes, and discard the precipitate;

[0183] 3. Add 200 μl of chloroform, shake and mix well, and leave at room temperature for 15 minutes;

[0184] 4. Place the EP tube in a high-speed centrifuge, centrifuge at 12000rpm for 15 minutes;

[0185] 5. Pipette the upper aqueous phase into another centrifuge tube; add 0.5 ml of isopropanol solution, mix well, and leave at room temperature for 10 minutes;

[0186] 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;

[0187] 7. Add 1 mL of 75% ethanol solution and gently shake the centrifuge tube to suspend the precipitate;

[0188] 8. Place the EP tube in a high-speed centrifuge, centrifuge at 4°C, 8000rpm for 5 minutes, discard the supernatant; dry at room temperature for 5-10 minutes, and resuspend the RNA with 20 μl sterile deionized water;

[0189] 9. Detect RNA concentration using nanodrop instrument;

[0190] 10. Reverse transcription of cDNA: reverse transcription of RNA into cDNA according to the instructions of Bestar qPCR kit (Cat. No.: DBI-2220);

[0191] 11. qPCR detection of TNF-α gene expression level: Real-time fluorescence quantitative PCR was performed to detect TNF-α gene expression level according to the Bestar qPCR (SyberGreen) kit (Cat. No.: DBI-2043) kit operating instructions.

[0192] Real-time fluorescence quantitative PCR detected TNF-α gene expression levels and expressed them 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:

[0193] The relative mRNA level of the control group was 114.6 times,

[0194] The relative mRNA levels of the test groups of Example 2, Example 2a, and Example 2b were 67 to 75 times. For example, the relative mRNA level of the test group of Example 2 was 73.7 times.

[0195] The relative mRNA levels of the test groups of Example 3, Example 3a, and Example 3b were 62 to 73 times. For example, the relative mRNA level of the test group of Example 3 was 67.4 times.

[0196] The relative mRNA levels of the test groups of Example 3c and Example 3d were 66 to 81 times.

[0197] 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for isolating and extracting exosomes from umbilical cord mesenchymal stem cells, the method comprising the following steps: (1) Mesenchymal stem cells of passages P3 to P6 were seeded into culture flasks at a density of (0.5-5)×10^4 cells / cm^2, and MSC complete medium was added. The cells were cultured in a 37°C, 5% CO2 incubator for 20-30 hours to allow the cells to adhere to the wall. IL-1β was then added to a concentration of 8-12 ng / mL, potassium sodium tartrate to a concentration of 0.125-0.175 mg / mL, and lysine to a concentration of 2.0-2.5 mg / mL, and the culture was continued for 20-30 hours; (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 then culture in a 37°C, 2% O2, 5% CO2 incubator for 42-56 hours; (4) Pipette the cell supernatant into a centrifuge tube and perform the following centrifugation treatments: centrifuge at 250-350 g and 4°C for 8-12 minutes, and transfer the supernatant to another centrifuge tube; centrifuge at 1500-2500 g and 4°C for 18-25 minutes, and transfer the supernatant to another centrifuge tube; centrifuge at 8000-12000 g and 4°C for 25-35 minutes, and filter the supernatant with a 0.22 μm filter membrane and transfer it to another centrifuge tube; centrifuge at 80000-120000 g and 4°C for 75-120 minutes, and discard the supernatant; (5) Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80,000-120,000 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; the exosomes express membrane protein CD9 and membrane protein CD81, and the positive expression rate of membrane protein CD9 is greater than 65%, and the positive expression rate of membrane protein CD81 is greater than 75%.

2. According to the method of claim 1, in step (1), cells are added to the culture flask at a density of (0.5~2)×10^4 cells / cm^2.

3. According to the method of claim 1, step (1) uses a T75 culture flask, inoculating 2 to 10×10^5 cells per flask, and adding 10 to 20 ml of culture medium.

4. 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.

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

6. 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.

7. According to the method of claim 1, in step (4), First, centrifuge at 250 g and 4 °C for 12 min, then at 2500 g and 4 °C for 18 min, then at 8000 g and 4 °C for 35 min, and the supernatant is filtered through a 0.22 μm filter and centrifuged at 120,000 g and 4 °C for 75 min; First, centrifuge at 350g and 4°C for 8 minutes, then centrifuge at 1500g and 4°C for 25 minutes, then centrifuge at 12000g and 4°C for 25 minutes, filter the supernatant with a 0.22 μm filter and centrifuge at 80000g and 4°C for 120 minutes; or, The mixture was first centrifuged at 300 g and 4°C for 10 min, then at 2000 g and 4°C for 20 min, and then at 10,000 g and 4°C for 30 min. The supernatant was filtered through a 0.22 μm filter and centrifuged at 100,000 g and 4°C for 90 min.

8. The method according to claim 1, wherein in step (5), the mixture is 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.

9. According to the method of claim 1, in step (5), the obtained exosome suspension is stored at -80°C.

10. According to the method of claim 1, in step (5), the exosomes obtained from 1×10^6 cells in step (1) are resuspended in 0.5-5 ml of sterile PBS.

11. According to the method of claim 1, in step (1), the concentrations of potassium sodium tartrate and lysine added are 0.15 mg / mL and 2.2 mg / mL respectively.

12. According to the method of claim 1, in step (5), the average particle size of the obtained exosomes is 50-200 nm.

13. According to the method of claim 1, in step (5), the average particle size of the obtained exosomes is 75-150 nm.

14. According to the method of claim 7, the positive expression rate of exosome membrane protein CD9 is greater than 70%, and the positive expression rate of membrane protein CD81 is greater than 80%.

15. The method according to claim 1, wherein the umbilical cord mesenchymal stem cells are prepared by a method comprising the following steps: (a) Clean the surface of the placenta with tissue cleaning solution to remove surface blood, cut the umbilical cord and remove the internal blood vessels; (b) Remove Wharton's jelly from the umbilical cord, clean it, cut it into small pieces of 2-3 mm^3, and transfer it to a T75 culture flask; (c) Slowly add MSC complete medium to the culture bottle, gently shake the culture bottle to spread the umbilical cord tissue block in the culture bottle, and culture it in a 37°C, 5% CO2 incubator. Replenish the medium every 2-3 days, and after 2-3 times of replenishment, remove the tissue block and replace the MSC complete medium in full to continue culturing until the cells grow to a confluency of 70%, and then digest the obtained cells; (d) The culture medium in the culture flask was discarded, PBS was aspirated to rinse the culture flask to remove the residual culture medium, 3 mL of recombinant trypsin solution was added for digestion, the culture flask was tapped to make the cells fall off to the bottom of the culture flask, MSC complete culture medium was added to neutralize trypsin to terminate the digestion process, the cells were blown off with a pipette, the cell suspension was collected in a centrifuge tube, centrifuged, and the supernatant was discarded to obtain a cell pellet recorded as cells of the P0 generation; (e) The P0 cell pellet was resuspended in MSC complete medium, the cells were counted, and then inoculated into a culture flask, supplemented with the above MSC complete medium, and cultured in a 37°C, 5% CO2 incubator until the cell confluence reached 80%, the medium in the culture flask was discarded, the culture flask was rinsed with PBS to remove the residual medium, and a recombinant trypsin solution was added for digestion. The culture flask was tapped to make the cells fall off to the bottom of the culture flask, and MSC complete medium was added to neutralize the trypsin to terminate the digestion process. The cells were blown off with a pipette, and the cell suspension was collected in a centrifuge tube, centrifuged, and the supernatant was discarded to obtain a cell pellet recorded as P1 cells; (f) Repeat step (e) to perform subculture of MSC cells until the cell generations are P3 to P8.

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