Preparation of exosomes from cultured adipose-derived mesenchymal stem cells

Abstract

This invention relates to the preparation of exosomes from cultured adipose-derived mesenchymal stem cells. On one hand, this invention relates to exosomes isolated and extracted from adipose-derived mesenchymal stem cells, having an average particle size of 50–200 nm, a positive expression rate of membrane protein CD9 greater than 70%, and a positive expression rate of membrane protein CD81 greater than 80%. These exosomes are prepared by treating adipose-derived mesenchymal stem cells with IL-1β, followed by cell culture in a hypoxic environment, and then by differential centrifugation of the culture medium. This invention also provides a method for preparing these exosomes and their medical applications. The exosomes of this invention exhibit excellent bioregulatory properties and biological activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for obtaining exosomes from cultured mesenchymal stem cells. Specifically, it relates to a method for inducing cultured mesenchymal stem cells to secrete exosomes under inflammatory factors and a hypoxic environment, followed by extraction. The exosomes obtained by this method can significantly enhance the anti-inflammatory activity of the cells. The mesenchymal stem cells of this invention are obtained from fat isolation and passage culture. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of non-hematopoietic pluripotent stem cells derived from the mesoderm. It has been confirmed that in addition to their self-renewal, self-replication, and multi-lineage differentiation potential, MSCs also have strong anti-inflammatory and inhibitory abilities against various 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 various 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.). (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, 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 (MSCs) have low immunogenicity. MSC-based cell therapy has been successfully applied to the treatment of cardiovascular diseases, bone and cartilage defects, diabetes, and other conditions. MSCs release various cytokines and growth factors through paracrine and autocrine processes. These secreted bioactive factors can inhibit fibrosis and apoptosis, enhance angiogenesis, and participate in tissue repair and regeneration.

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

[0005] Exosomes are membrane-bound microvesicles secreted by cells, with a diameter of approximately 30–200 nm and a density ranging from 1.13 to 1.19 g / ml. Exosomes can carry a variety of proteins, mRNAs, and miRNAs similar to those of their source cells, and participate in processes such as immune regulation, cell communication, cell migration, and angiogenesis (see: Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles. 2015; 4:27066.). Lai et al. found that exosomes derived from MSCs can reduce myocardial ischemia-reperfusion injury and confirmed that exosome miRNAs 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 neural axons 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 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 further research, they have been found to perform functions such as transporting proteins and nucleic acids, specifically targeting receptor cells, exchanging proteins and lipids, triggering downstream signaling events, and participating in intercellular communication, thus gaining increasing attention. Exosomes carry two types of proteins: non-specific and cell-specific. The former, likely related to the biogenesis and biological functions of exosomes, mainly includes cytosolic proteins, proteins involved in intracellular signal transduction, various metabolic enzymes, heat shock proteins, and tetraspan proteins. The other type consists of specific proteins, which are only present in exosomes secreted by certain specific cells. These cell-derived exosomes are closely related to their biological functions; for example, molecularly derived exosomes contain MHC class II molecules. Therefore, exosomes from different cell sources carry different signaling molecules and perform different functions. For example, exosomes secreted by tumor cells can mediate angiogenesis, tumor cell proliferation, and immune escape, while dendritic cell-derived exosomes can elicit an effective anti-tumor immune response. Existing studies have found that exosomes contain cell-derived protein rRNA and microRNA, and that exosomes can cross biological barriers to deliver functional nucleic acid molecules between cells, thereby performing various biological functions. Therefore, exosomes are expected to become a novel drug delivery route and gene therapy vector.

[0007] Exosomes are nanoscale lipid-encapsulated structures that encapsulate proteins, mRNA, and microRNA. They are naturally present in bodily fluids, including blood, saliva, urine, and breast milk. Exosomes are membrane-bound vesicles secreted by living cells, originating from late-stage endosomes (also known as multivesicular bodies). Almost all cells, including tumor cells, can produce and release exosomes. Released from cellular secretion, exosomes spread through bodily fluids such as blood and can eventually be phagocytosed by other cells, serving as important mediators of intercellular communication. Exosomes secreted by both host cells and tumor cells participate in cell growth, proliferation, metabolism, and regulation. Immune cells and tumor cells can also exchange information through exosomes. This communication plays a dual role in regulating tumor immunity: exosomes can both induce anti-tumor responses by inhibiting immune cells (DCs, NK cells, CD4+, CD8+ T cells, etc.) and induce or regulate immunosuppression in cell populations (MDSCs, Tregs, Bregs).

[0008] Current research has found that exosomes secreted by mesenchymal stem cells carry a variety of effective cytokines, proteins, and small nucleic acid molecules, which can effectively mediate cell proliferation, apoptosis, and functional regulation. Studies have shown that exosomes contain vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, tumor necrosis factor, and tumor growth factor, which can inhibit apoptosis and fibrosis, promote angiogenesis and mitosis, and mediate immune responses. Experiments have demonstrated that insulin-like growth factor and vascular endothelial growth factor carried by exosomes secreted by mesenchymal stem cells are key factors in the treatment of acute kidney injury. In terms of immunology, the lipid membrane surface of exosomes secreted by mesenchymal stem cells expresses various membrane proteins, such as coagulation factors, tumor necrosis factor, MHC I / II molecules, and CCR5 chemokine receptors. These lipid membrane surface proteins play an important role in resisting inflammation.

[0009] Currently, there are various methods and approaches for exosome extraction. Existing methods mainly employ ultracentrifugation or expensive column chromatography kits. However, ultracentrifugation results in inconsistent exosome quality, inconsistent yields, lengthy centrifugation times, and sometimes even failure to separate exosomes due to centrifugation time or speed, wasting time and money. Column chromatography kits are typically only suitable for obtaining small quantities of exosomes and are also expensive.

[0010] There are some existing reports on methods for isolating and extracting exosomes. 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 treating 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 application of exosomes secreted from human blood or mesenchymal stem cells. However, existing technologies that use mesenchymal stem cell culture supernatants from various sources to extract exosomes all suffer from drawbacks such as low yield and complex processes.

[0011] A type of stem cell with multipotent differentiation potential has been isolated from adipose tissue and successfully differentiated into adipocytes, osteoblasts, chondrocytes, and myocytes in a specialized in vitro culture system. These differentiateable stem cells are recognized as adipose-derived mesenchymal cells, or adipose-derived stem cells (ADSCs). ADSCs are a type of stem cell with multipotent differentiation potential isolated from adipose tissue in recent years. They possess the characteristic of differentiating into multiple germ layers, including the ability to differentiate into mesoderm, endoderm, and ectoderm cells, and can secrete many potentially beneficial growth factors and cytokines. Cell-based therapies to promote wound healing are rapidly developing. Adipose-derived stem cells are a group of pluripotent mesenchymal stem cells that can differentiate into other cell lines. A large number of adipose-derived cells exist in the non-adipose (mesenchymal) fragments of adipose tissue and are easily isolated and obtained.

[0012] Adipose-derived stem cells (ADSCs) are recommended as an adjunctive treatment for various tissue defects. Angiogenesis is a crucial step in wound healing; the formation of new blood vessels is essential for supporting granulation tissue formation and the survival and expansion of keratinocytes to heal wounds. Therefore, it is believed that ADSCs promote wound healing by rapidly inducing angiogenesis. Furthermore, ADSCs have also shown to promote wound healing through cell differentiation and the secretion of long-term factors that promote collagen synthesis and dermal fibroblast migration.

[0013] Adipose-derived stem cells (ADSCs) have been combined with extracellular matrix scaffold components for use in animal skin and soft tissue wounds, revealing that ADSCs promote scaffold vascularization. Studies have shown that the wound-healing-promoting effect of ADSCs is related to their ability to promote epithelialization and granulation tissue formation, and ADSCs also possess potential epidermal cell differentiation capabilities. Further research suggests that the distant differentiation capacity of ADSCs is a response of pluripotent ADSCs to the wound microenvironment. ADSCs contribute to local vascular remodeling through direct differentiation into vascular endothelial cells and the secretion of angiogenesis factors, both of which benefit vascular regeneration. In other words, ADSCs promote wound healing through cell differentiation and angiogenesis. Furthermore, ADSCs have been combined with platelet-derived growth factor (PDGF), a well-known factor involved in normal wound healing, for wound treatment. Research indicates a synergistic effect, potentially promoting wound healing by increasing PGF levels.

[0014] In theory, cell therapy holds great promise because pluripotent stem cells or progenitor cells promote wound healing by regenerating missing tissue and continuously providing numerous beneficial factors. Nevertheless, the mechanisms by which adipose-derived stem cells contribute to wound healing still require further investigation. The remodeling of local blood vessels by adipose-derived stem cells includes direct differentiation into vascular endothelial cells and the secretion of angiogenesis factors, both of which are beneficial for vascular regeneration.

[0015] The application prospects of adipose-derived mesenchymal stem cells (ADSCs) have been validated through their use in various animal models of diseases. They have been used to intervene in the treatment of diabetes, liver damage repair, bladder reconstruction, type 2 diabetic eczema, myocardial infarction, cerebral infarction, cognitive impairment, stroke, intervertebral disc repair, glioblastoma treatment, bone defects, post-traumatic angiogenesis, rheumatoid arthritis, cleft lip and palate, heart failure, colitis, and urinary incontinence. Some researchers have transformed ADSCs into cardiomyocytes, reprogramming mature stem cells and improving the treatment of heart disease. Others have injected ADSCs, combined with fibrin glue, into the left ventricular wall of rats with myocardial infarction; ADSCs show great promise for clinical tissue engineering treatment of myocardial infarction. Furthermore, ADSCs, used as seed cells in a mesh scaffold, successfully repaired skull injuries in dogs, providing a potential clinical treatment for skull injuries. Studies have shown that injecting adipose-derived mesenchymal stem cells (ADMSCs) from the abdominal fat of heart patients into their hearts reduced cardiac damage and increased blood flow. Six months after the injection, the patients' hearts showed improved oxygenation, and the left ventricle pumped 3.5 times more blood. Spelt imaging demonstrated a 5.7% increase in cardiac pumping capacity in patients receiving ADMSCs, and MRI scans showed a decrease in the average myocardial scar area from 31.6% to 15.4%. These findings were presented at the 2010 American Heart Association Scientific Sessions. During tissue repair, ADMSCs can regulate growth hormones and cytokines in surrounding tissues, preventing apoptosis. Experiments have shown that ADMSCs can secrete various skin growth factors, such as basic fibroblast growth factor (bFGF), to promote fibroblast proliferation and possess anti-photoaging, antioxidant, anti-wrinkle, and anti-ultraviolet radiation effects. Research using ADMSCs to treat complex anal ulcers has entered Phase II clinical trials. Furthermore, human adipose-derived mesenchymal stem cells have been transplanted into the breasts of patients with breast atrophy, uneven size, or those requiring breast augmentation. The resulting breasts are symmetrical and balanced in size. Mammograms show that the repaired breasts are naturally soft, completely free of calcification, cysts, and obvious injection scars. Recent clinical studies have shown that intramuscular injection of adipose-derived mesenchymal stem cells also has some therapeutic effect on diabetic foot and arteriosclerosis obliterans. Six months after injection, patients experienced relief of resting pain, a significant increase in painless walking distance, and no complications were observed. Trials have shown that adipose-derived mesenchymal stem cells induced into islet-like cells and reinfused into diabetic patients, with a 23-month follow-up, resulted in a decrease in exogenous insulin usage, an increase in average weight, and no discomfort or side effects.

[0016] Scholars have discovered that adipose tissue participates in wound repair. When adipocytes are stimulated by trauma, gene expression undergoes stress response, leading to increased secretion of genes involved in the inflammatory matrix, including TNF-α, IL-1, IL-6, intercellular adhesion factors, chemokines, acute response proteins, and receptors. These factors regulate the intrinsic therapeutic mechanisms by which adipocytes participate in repair, and this regulatory system warrants further consideration. The research findings enrich the theoretical foundation for wound repair. In clinical applications, fat transfusion therapy can achieve ideal therapeutic effects by accelerating wound repair in deep skin lesions affecting the subdermal fat layer. International researchers have also used tissue engineering techniques to prepare artificial skin containing adipocytes, collagen, and elastin. Clinical applications have shown that this reduces wound contraction caused by tissue repair of full-thickness skin defects, suggesting the presence of repair-functional factors and cells within adipose tissue.

[0017] The strong and rapid adhesion of adipose-derived mesenchymal stem cells to the extracellular matrix has enabled the development of specific methods for screening and culturing human adipose-derived mesenchymal stem cells both domestically and internationally. Furthermore, the biological properties of stem cells allow for enhanced function, avoidance of potentially harmful genes, and control over differentiation time and different differentiation directions. These advantages provide irreplaceable guidance in basic research, clinical treatment, stem cell therapy research, skin regeneration and repair, and the construction of highly tissue-engineered skin.

[0018] Adipose-derived mesenchymal stem cells (ADSCs), as mesenchymal stem cells derived from fat, are easy to obtain, with an extraction efficiency 40 times higher than that of bone marrow mesenchymal stem cells, and they proliferate faster under in vitro culture conditions. They possess multi-lineage differentiation capabilities, capable of differentiating into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, and even nerve cells. Current Phase I and Phase II clinical trials have demonstrated that ADSCs are safe and effective in repairing injuries to various organs, including the heart, rectum, and breast. Furthermore, studies have confirmed that ADSCs promote skin regeneration and repair.

[0019] ADSCs exhibit inherent advantages in several aspects. Belonging to the monocyte family, ADSCs are a type of adult stem cell. Derived from processed adipose tissue, they grow in cell culture dishes using adherent and adhesive methods and possess diverse differentiation capabilities. They can differentiate not only into adipocytes but also into myocytes, chondrocytes, nerve cells, vascular endothelial cells, osteocytes, etc., thus finding widespread application in tissue engineering. For example, ADSCs can self-replicate and differentiate into adipocytes, playing a role in tissue regeneration; ADSCs can differentiate into vascular endothelial cells or peripheral cells; ADSCs can secrete angiogenesis factors under hypoxic or other conditions; when ADSCs are transplanted together with adipocytes, they can differentiate into endothelial cells, preventing fibrosis and fat necrosis, and improving fat survival rate; recent studies have shown that ADSCs can express various cytokines such as VEGF, IGF, TGF-β1, bFGF, and EGF, and also prevent apoptosis and maintain adipocyte survival rate by expressing angiogenesis factors such as VEGF and IGF-1.

[0020] Numerous methods for culturing adipose-derived mesenchymal stem cells have been disclosed in the prior art. For example, CN106479970A (201611050980.1) discloses a method for large-scale culture of human adipose-derived mesenchymal stem cells; CN104762260A (201510197879.8) relates to a method for preparing adipose-derived mesenchymal stem cells; CN104974984A (application number 201510173453.9) discloses a method for expanding and culturing mesenchymal stem cells derived from adipose tissue; CN106 Application No. 520686A (201610887111.8) discloses a method for culturing adipose-derived mesenchymal stem cells; Application No. 201010580537.1 (CN101984049A) discloses a method for isolating mesenchymal stem cells from adipose tissue; Application No. 201110310461.5 (CN102329783A) discloses a method for isolating adipose-derived mesenchymal stem cells from the tumescent fluid in a surgical liposuction procedure.

[0021] In addition, the inventors of this application also provided an effective method for isolating and culturing primary adipose-derived mesenchymal stem cells in Chinese patent ZL2020113812050.

[0022] However, the art still anticipates new methods for isolating and extracting exosomes, particularly exolizing them through mesenchymal stem cell secretion in a simple, readily available, and high-yield manner. Furthermore, the art also anticipates providing a method for treating inflammatory diseases, such as a method for treating inflammatory diseases using exosomes. In particular, the art particularly anticipates providing a method for treating inflammatory diseases using exosomes isolated from human adipose-derived mesenchymal stem cells. Summary of the Invention

[0023] The object of this invention is to provide a novel method for isolating and extracting exosomes, particularly a method that enables the simple, readily available, and high-yield extraction of exosomes from mesenchymal stem cell secretion. It has been unexpectedly discovered that the method of this invention can advantageously achieve the above-mentioned object. This invention is based on this discovery.

[0024] The mesenchymal stem cells involved in this invention are derived from adipose tissue.

[0025] Therefore, the first aspect of the present invention provides exosomes isolated and extracted using adipose-derived mesenchymal stem cells, having an average particle size of 50 to 200 nm, for example, having an average particle size of 75 to 150 nm.

[0026] According to a first aspect of the invention, the exosomes express membrane proteins CD9 and CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0027] According to the exosomes of the first aspect of the present invention, the adipose-derived mesenchymal stem cells are prepared by a method comprising the following steps:

[0028] (a) Process fat samples transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet;

[0029] (b) Centrifuge the fat sample, remove the upper adipose tissue, wash it with D-Hanks solution, centrifuge it again, remove the adipose tissue, add 1% type II collagenase and shake to digest.

[0030] (c) After digestion, dilute with an equal volume of D-hanks solution, centrifuge, and collect the cell pellet at the bottom for the next operation;

[0031] (d) Take the cell pellet obtained in step (c), resuspend it in primary supplemental medium, take samples for counting, and seed it into culture flasks according to the specified cell amount; place it in a CO2 incubator for culture; the primary supplemental medium is prepared with DMEM-F12 medium as the matrix and the following are added: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose;

[0032] (e) After culturing for 3 days, the culture medium was completely replaced once. When the cell confluence reached more than 80%, the old culture medium was removed, the cells were washed with D-hanks solution, recombinant trypsin solution was added to digest the cells to cause them to detach, D-hanks solution was added to dilute, centrifuged, and the cell pellet was resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells.

[0033] (f) The primary adipose mesenchymal stem cells are purified, cultured and passaged in steps (d) to (e) to obtain P1 generation cells; and so on, to obtain P2 to P8 generation mesenchymal stem cells.

[0034] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, in step (b), both centrifugations are performed at 100xg for 5 min; and / or, 2 volumes of 1% type II collagenase are added and the cells are shaken and digested for 30 min; and / or, in step (c), centrifugation is performed at 100xg for 5 min; and / or, in step (d), seeding the cells into the culture flask according to the specified amount means seeding the cells at a rate of 1–5 × 10⁶ cells / mL. 4 / cm 2 Inoculate into T75 flasks; and / or, in step (d), the conditions for incubation in a CO2 incubator are: 5% CO2, 37°C, saturated humidity; and / or, in step (e), add 2 ml of recombinant trypsin solution to each flask to digest the cells for 2 min; and / or, in step (e), centrifuge at 100 x g for 10 min; and / or, in step (e), add 10 ml of D-hanks solution to each flask for dilution.

[0035] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, the D-Hanks solution is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4·12H2O, 0.35g NaHCO3, and water to 1000ml.

[0036] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, the DMEM-F12 culture medium composition is as follows: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, and L-phenylalanine 35 mg. 48 mg, 1,4-Butanediamine dihydrochloride 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, vitamin H 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous dihydrogen phosphate Sodium 54.35 mg, L-Aspartic acid 6.65 mg, Folic acid 2.65 mg, Disodium hydrogen phosphate 71.02 mg, L-cysteine ​​hydrochloride 17.56 mg, I-inositol 12.6 mg, Zinc sulfate heptahydrate 0.432 mg, L-glutamate 7.35 mg, Nicotinamide 2.02 mg, L-arginine hydrochloride 147.5 mg, L-proline 17.25 mg, Pyridoxal hydrochloride 2 mg, L-cysteine ​​hydrochloride 31.29 mg, L-tryptophan 9.02 mg mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water added to 1000mL.

[0037] According to the first aspect of the present invention, the exosomes are prepared using adipose-derived mesenchymal stem cells by a method comprising the following steps:

[0038] (1) Seed mesenchymal stem cells into culture flasks, add MSC complete medium, and culture in a 37°C, 5% CO2 incubator to allow the cells to adhere to the culture wall. Then add IL-1β to the culture medium to a concentration of 8-12 ng / mL and continue culturing.

[0039] (2) Discard 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%.

[0040] (3) Discard the culture medium, wash with PBS, then add MSC complete culture medium, and incubate in a 37℃, 2% O2, 5% CO2 incubator for 42-56 hours;

[0041] (4) Transfer the cell supernatant into a centrifuge tube and centrifuge as follows:

[0042] Centrifuge at 250–350 g and 4 °C for 8–12 minutes, and transfer the supernatant to another centrifuge tube;

[0043] Centrifuge at 1500–2500g and 4℃ for 18–25 minutes, and transfer the supernatant to another centrifuge tube;

[0044] Centrifuge at 8000–12000g, 4℃ for 25–35 minutes, filter the supernatant through a 0.22μm filter membrane and place it in another centrifuge tube;

[0045] Centrifuge at 80,000–120,000 g at 4°C for 75–120 minutes, and discard the supernatant;

[0046] (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, add sterile PBS to resuspend the exosome, and obtain the exosome in suspension form.

[0047] According to the exosomes 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 P7.

[0048] According to the exosomes of the first aspect of the present invention, in step (1), cells are added to the culture flask at a density of (0.5 to 5) × 10^4 cells / cm^2, for example, at a density of (0.5 to 2) × 10^4 cells / cm^2. In one embodiment, step (1) uses T75 culture flasks, and each flask is seeded with (2 to 10) × 10^5 cells, for example, 6 × 10^5 cells, and 10 to 20 ml of culture medium is added.

[0049] According to the exosomes of the first aspect of the present invention, in step (1), the cells are cultured for 20 to 30 hours, for example, 24 hours, to allow the cells to adhere.

[0050] According to the exosomes of the first aspect of the present invention, in step (1), after adding IL-1β, the culture is continued for 20 to 30 hours, for example, 24 hours.

[0051] According to the exosomes of the first aspect of the present invention, in step (3), the exosomes are cultured for 48 hours in an incubator at 37°C, 2% O2, and 5% CO2.

[0052] According to the exosomes of the first aspect of the present invention, in step (4), the exosomes are first centrifuged at 250g and 4°C for 12 minutes, then centrifuged at 2500g and 4°C for 18 minutes, then centrifuged at 8000g and 4°C for 35 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 120000g and 4°C for 75 minutes.

[0053] According to the exosomes of the first aspect of the present invention, in step (4), the exosomes are first centrifuged at 350g and 4°C for 8 minutes, then centrifuged at 1500g and 4°C for 25 minutes, then centrifuged at 12000g and 4°C for 25 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0054] According to the exosomes of the first aspect of the present invention, in step (4), the exosomes are first centrifuged at 300g and 4°C for 10 minutes, then centrifuged at 2000g and 4°C for 20 minutes, then centrifuged at 10000g and 4°C for 30 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 100000g and 4°C for 90 minutes.

[0055] According to the exosomes of the first aspect of the present invention, in step (5), the centrifugation is performed at 100,000g and 4°C for 90 minutes, or at 80,000g and 4°C for 120 minutes, or at 120,000g and 4°C for 75 minutes.

[0056] According to the exosomes of the first aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0057] According to the exosomes of the first aspect of the present invention, in step (5), the exosomes obtained from 1.2 × 10^6 cells in step (1) are resuspended in 0.5 to 5 ml of sterile PBS, for example, 0.5 to 2 ml of sterile PBS, for example, 1 ml of sterile PBS.

[0058] According to the exosomes of the first aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture medium at the same time as IL-1β, and the concentrations of the two are 0.1-0.2 mg / mL, for example 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, for example 0.15 mg / mL and 2.2 mg / mL, respectively.

[0059] Furthermore, a second aspect of the present invention provides a method for isolating and extracting exosomes using adipose-derived mesenchymal stem cells, the method comprising the following steps:

[0060] (1) Seed mesenchymal stem cells into culture flasks, add MSC complete medium, and culture in a 37°C, 5% CO2 incubator to allow the cells to adhere to the culture wall. Then add IL-1β to the culture medium to a concentration of 8-12 ng / mL and continue culturing.

[0061] (2) Discard 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%.

[0062] (3) Discard the culture medium, wash with PBS, then add MSC complete culture medium, and incubate in a 37℃, 2% O2, 5% CO2 incubator for 42-56 hours;

[0063] (4) Transfer the cell supernatant into a centrifuge tube and centrifuge as follows:

[0064] Centrifuge at 250–350 g and 4 °C for 8–12 minutes, and transfer the supernatant to another centrifuge tube;

[0065] Centrifuge at 1500–2500g and 4℃ for 18–25 minutes, and transfer the supernatant to another centrifuge tube;

[0066] Centrifuge at 8000–12000g, 4℃ for 25–35 minutes, filter the supernatant through a 0.22μm filter membrane and place it in another centrifuge tube;

[0067] Centrifuge at 80,000–120,000 g at 4°C for 75–120 minutes, and discard the supernatant;

[0068] (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, add sterile PBS to resuspend the exosome, and obtain the exosome in suspension form.

[0069] According to the method of the second 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 P7.

[0070] 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 to 5) × 10^4 cells / cm^2, for example, at a density of (0.5 to 2) × 10^4 cells / cm^2. In one embodiment, step (1) uses T75 culture flasks, and each flask is seeded with (2 to 10) × 10^5 cells, for example, 6 × 10^5 cells, and 10 to 20 ml of culture medium is added.

[0071] According to the method of the second aspect of the present invention, in step (1), the cells are cultured for 20 to 30 hours, for example, 24 hours, to allow the cells to adhere to the wall.

[0072] According to the method of the second aspect of the present invention, in step (1), after adding IL-1β, the culture is continued for 20 to 30 hours, for example, 24 hours.

[0073] According to the method of the second aspect of the present invention, in step (3), the culture is carried out for 48 hours in an incubator at 37°C, 2% O2, and 5% CO2;

[0074] According to the method of the second aspect of the present invention, in step (4), the mixture is first centrifuged at 250g and 4°C for 12 minutes, then centrifuged at 2500g and 4°C for 18 minutes, then centrifuged at 8000g and 4°C for 35 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 120000g and 4°C for 75 minutes.

[0075] According to the method of the second aspect of the present invention, in step (4), the mixture is first centrifuged at 350g and 4°C for 8 minutes, then centrifuged at 1500g and 4°C for 25 minutes, then centrifuged at 12000g and 4°C for 25 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0076] According to the method of the second aspect of the present invention, in step (4), the mixture is first centrifuged at 300g and 4°C for 10 minutes, then centrifuged at 2000g and 4°C for 20 minutes, then centrifuged at 10000g and 4°C for 30 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 100000g and 4°C for 90 minutes.

[0077] According to the method of the second aspect of the present invention, in step (5), centrifugation is performed at 100,000g and 4°C for 90 minutes, or at 80,000g and 4°C for 120 minutes, or at 120,000g and 4°C for 75 minutes.

[0078] According to the method of the second aspect of the present invention, in step (5), the obtained exosome suspension is stored at -80°C.

[0079] According to the method of the second aspect of the present invention, in step (5), the exosomes obtained from 1.2 × 10^6 cells in step (1) are resuspended in 0.5 to 5 ml of sterile PBS, for example, 0.5 to 2 ml of sterile PBS, for example, 1 ml of sterile PBS.

[0080] According to the method of the second aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture medium at the same time as IL-1β are added, with concentrations of 0.1-0.2 mg / mL, for example 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, for example 0.15 mg / mL and 2.2 mg / mL, respectively.

[0081] According to the method of the second aspect of the present invention, the average particle size of the obtained exosomes is 50 to 200 nm, for example, 75 to 150 nm.

[0082] According to the method of a second aspect of the present invention, the resulting exosomes express membrane proteins CD9 and CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0083] According to a second aspect of the present invention, the adipose-derived mesenchymal stem cells are prepared by a method comprising the following steps:

[0084] (a) Process fat samples transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet;

[0085] (b) Centrifuge the fat sample, remove the upper adipose tissue, wash it with D-Hanks solution, centrifuge it again, remove the adipose tissue, add 1% type II collagenase and shake to digest.

[0086] (c) After digestion, dilute with an equal volume of D-hanks solution, centrifuge, and collect the cell pellet at the bottom for the next operation;

[0087] (d) Take the cell pellet obtained in step (c), resuspend it in primary supplemental medium, take samples for counting, and seed it into culture flasks according to the specified cell amount; place it in a CO2 incubator for culture; the primary supplemental medium is prepared with DMEM-F12 medium as the matrix and the following are added: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose;

[0088] (e) After culturing for 3 days, the culture medium was completely replaced once. When the cell confluence reached more than 80%, the old culture medium was removed, the cells were washed with D-hanks solution, recombinant trypsin solution was added to digest the cells to cause them to detach, D-hanks solution was added to dilute, centrifuged, and the cell pellet was resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells.

[0089] (f) The primary adipose mesenchymal stem cells are purified, cultured and passaged in steps (d) to (e) to obtain P1 generation cells; and so on, to obtain P2 to P8 generation mesenchymal stem cells.

[0090] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, in step (b), both centrifugations are performed at 100xg for 5 min; and / or, 2 volumes of 1% type II collagenase are added and the cells are shaken and digested for 30 min; and / or, in step (c), centrifugation is performed at 100xg for 5 min; and / or, in step (d), seeding the cells into the culture flask according to the specified amount means seeding the cells at a rate of 1–5 × 10⁶ cells / mL. 4 / cm 2 Inoculate into T75 flasks; and / or, in step (d), the conditions for incubation in a CO2 incubator are: 5% CO2, 37°C, saturated humidity; and / or, in step (e), add 2 ml of recombinant trypsin solution to each flask to digest the cells for 2 min; and / or, in step (e), centrifuge at 100 x g for 10 min; and / or, in step (e), add 10 ml of D-hanks solution to each flask for dilution.

[0091] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, the D-Hanks solution is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4·12H2O, 0.35g NaHCO3, and water to 1000ml.

[0092] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, the DMEM-F12 culture medium composition is as follows: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, and L-phenylalanine 35 mg. 48 mg, 1,4-Butanediamine dihydrochloride 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, vitamin H 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous dihydrogen phosphate Sodium 54.35 mg, L-Aspartic acid 6.65 mg, Folic acid 2.65 mg, Disodium hydrogen phosphate 71.02 mg, L-cysteine ​​hydrochloride 17.56 mg, I-inositol 12.6 mg, Zinc sulfate heptahydrate 0.432 mg, L-glutamate 7.35 mg, Nicotinamide 2.02 mg, L-arginine hydrochloride 147.5 mg, L-proline 17.25 mg, Pyridoxal hydrochloride 2 mg, L-cysteine ​​hydrochloride 31.29 mg, L-tryptophan 9.02 mg mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water added to 1000mL.

[0093] Furthermore, a third aspect of the present invention provides the use of exosomes isolated from adipose-derived mesenchymal stem cells in the preparation of medicaments for treating inflammatory diseases, said exosomes having an average particle size of 50–200 nm, for example having an average particle size of 75–150 nm.

[0094] According to a third aspect of the invention, the exosomes express membrane proteins CD9 and CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0095] According to a third aspect of the invention, the exosomes are prepared using adipose-derived mesenchymal stem cells by a method comprising the following steps:

[0096] (1) Mesenchymal stem cells were seeded into culture flasks, MSC complete medium was added, and the flasks were incubated at 37°C in a 5% CO2 incubator to allow the cells to adhere to the culture vessel. Then, IL-1β was added to the culture medium to a concentration of 8–12 ng / mL, and the culture was continued. The adipose-derived mesenchymal stem cells were prepared by a method including the following steps:

[0097] (a) Process fat samples transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet;

[0098] (b) Centrifuge the fat sample, remove the upper adipose tissue, wash it with D-Hanks solution, centrifuge it again, remove the adipose tissue, add 1% type II collagenase and shake to digest.

[0099] (c) After digestion, dilute with an equal volume of D-hanks solution, centrifuge, and collect the cell pellet at the bottom for the next operation;

[0100] (d) Take the cell pellet obtained in step (c), resuspend it in primary supplemental medium, take samples for counting, and seed it into culture flasks according to the specified cell amount; place it in a CO2 incubator for culture; the primary supplemental medium is prepared with DMEM-F12 medium as the matrix and the following are added: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose;

[0101] (e) After culturing for 3 days, the culture medium was completely replaced once. When the cell confluence reached more than 80%, the old culture medium was removed, the cells were washed with D-hanks solution, recombinant trypsin solution was added to digest the cells to cause them to detach, D-hanks solution was added to dilute, centrifuged, and the cell pellet was resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells.

[0102] (f) The primary adipose mesenchymal stem cells are purified, cultured and passaged in steps (d) to (e) to obtain P1 generation cells; and so on, to obtain P2 to P8 generation mesenchymal stem cells.

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

[0104] (3) Discard the culture medium, wash with PBS, then add MSC complete culture medium, and incubate in a 37℃, 2% O2, 5% CO2 incubator for 42-56 hours;

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

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

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

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

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

[0110] (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, add sterile PBS to resuspend the exosome, and obtain the exosome in suspension form.

[0111] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, in step (b), both centrifugations are performed at 100xg for 5 min; and / or, 2 volumes of 1% type II collagenase are added and the cells are shaken and digested for 30 min; and / or, in step (c), centrifugation is performed at 100xg for 5 min; and / or, in step (d), seeding the cells into the culture flask according to the specified amount means seeding the cells at a rate of 1–5 × 10⁶ cells / mL. 4 / cm 2Inoculate into T75 flasks; and / or, in step (d), the conditions for incubation in a CO2 incubator are: 5% CO2, 37°C, saturated humidity; and / or, in step (e), add 2 ml of recombinant trypsin solution to each flask to digest the cells for 2 min; and / or, in step (e), centrifuge at 100 x g for 10 min; and / or, in step (e), add 10 ml of D-hanks solution to each flask for dilution.

[0112] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, the D-Hanks solution is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4·12H2O, 0.35g NaHCO3, and water to 1000ml.

[0113] In the above-mentioned preparation of adipose-derived mesenchymal stem cells, the DMEM-F12 culture medium composition is as follows: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, and L-phenylalanine 35 mg. 48 mg, 1,4-Butanediamine dihydrochloride 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, vitamin H 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous dihydrogen phosphate Sodium 54.35 mg, L-Aspartic acid 6.65 mg, Folic acid 2.65 mg, Disodium hydrogen phosphate 71.02 mg, L-cysteine ​​hydrochloride 17.56 mg, I-inositol 12.6 mg, Zinc sulfate heptahydrate 0.432 mg, L-glutamate 7.35 mg, Nicotinamide 2.02 mg, L-arginine hydrochloride 147.5 mg, L-proline 17.25 mg, Pyridoxal hydrochloride 2 mg, L-cysteine ​​hydrochloride 31.29 mg, L-tryptophan 9.02 mg mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water added to 1000mL.

[0114] According to the third aspect of the present invention, in the above method for preparing adipose-derived mesenchymal stem cells, the basic balanced salt solution is prepared by dissolving 0.4 g of KCl, 0.06 g of KH2PO4, 0.132 g of Na2HPO4·12H2O, 8 g of NaCl, 0.35 g of NaHCO3, 1.0 g of D-glucose, 0.10 g of streptomycin, and 0.06 g of penicillin in water and making up to 1 L of solution.

[0115] According to the third aspect of the present invention, in the above method for preparing amniotic mesenchymal stem cells, the mixed enzyme digestion solution contains: 0.1 mg / ml of type I collagenase, 0.3 mg / ml of type II collagenase, 0.1 mg / ml of hyaluronidase and 0.05 mg / ml of neutral protease.

[0116] According to the third aspect of the present invention, in step (e) of the above method for preparing amniotic mesenchymal stem cells, the trypsin digest is a digest comprising 0.25% trypsin and 0.02% EDTA.

[0117] According to the third aspect of the invention, in step (1), the mesenchymal stem cells are cells of generation P2 to P8, for example, cells of generation P3 to P7.

[0118] According to the third aspect of the invention, in step (1), cells are added to the culture flask at a density of (0.5–5) × 10⁴ cells / cm², for example, at a density of (0.5–2) × 10⁴ cells / cm². In one embodiment, step (1) uses T75 culture flasks, with each flask seeded with (2–10) × 10⁵ cells, for example, 6 × 10⁵ cells, and 10–20 ml of culture medium is added.

[0119] According to the third aspect of the invention, in step (1), the cells are cultured for 20 to 30 hours, for example, 24 hours, to allow them to adhere to the wall.

[0120] According to the third aspect of the invention, in step (1), after adding IL-1β, the culture is continued for 20 to 30 hours, for example, 24 hours.

[0121] According to the third aspect of the invention, in step (3), the culture is carried out for 48 hours in an incubator at 37°C, 2% O2, and 5% CO2.

[0122] According to the third aspect of the invention, in step (4), the liquid is first centrifuged at 250g and 4°C for 12 minutes, then at 2500g and 4°C for 18 minutes, then at 8000g and 4°C for 35 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 120000g and 4°C for 75 minutes.

[0123] According to the third aspect of the invention, in step (4), the liquid is first centrifuged at 350g and 4°C for 8 minutes, then centrifuged at 1500g and 4°C for 25 minutes, then centrifuged at 12000g and 4°C for 25 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 80000g and 4°C for 120 minutes.

[0124] According to the third aspect of the invention, in step (4), the liquid is first centrifuged at 300g and 4°C for 10 minutes, then centrifuged at 2000g and 4°C for 20 minutes, then centrifuged at 10000g and 4°C for 30 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 100000g and 4°C for 90 minutes.

[0125] According to the third aspect of the invention, in step (5), the centrifugation is performed at 100,000g and 4°C for 90 minutes, or at 80,000g and 4°C for 120 minutes, or at 120,000g and 4°C for 75 minutes.

[0126] According to the third aspect of the invention, in step (5), the resulting exosome suspension is stored at -80°C.

[0127] According to the third aspect of the invention, in step (5), the exosomes obtained from 1.2 × 10^6 cells in step (1) are resuspended in 0.5 to 5 ml of sterile PBS, for example, 0.5 to 2 ml of sterile PBS, for example, 1 ml of sterile PBS.

[0128] According to the third aspect of the invention, in step (1), sodium tartrate and lysine are added to the culture medium at the same time as IL-1β, with concentrations of 0.1-0.2 mg / mL, for example 0.125-0.175 mg / mL and 2.0-2.5 mg / mL, for example 0.15 mg / mL and 2.2 mg / mL, respectively.

[0129] Furthermore, a fourth aspect of the present invention provides a method for regulating the secretion of TNF-α by peripheral blood mononuclear cells (PBMCs) using exosomes, the method comprising exposing the PBMCs to the exosomes; wherein the exosomes have an average particle size of 50–200 nm, for example, an average particle size of 75–150 nm, and the exosomes express membrane proteins CD9 and CD81. In one embodiment, the positive expression rate of exosomal membrane protein CD9 is greater than 70%, for example, greater than 75%. In one embodiment, the positive expression rate of exosomal membrane protein CD81 is greater than 80%, for example, greater than 85%.

[0130] According to a fourth aspect of the present invention, the exosomes are prepared using adipose-derived mesenchymal stem cells by a method comprising the following steps:

[0131] (1) Mesenchymal stem cells were seeded into culture flasks, MSC complete medium was added, and the flasks were incubated at 37°C in a 5% CO2 incubator to allow the cells to adhere to the culture vessel. Then, IL-1β was added to the culture medium to a concentration of 8–12 ng / mL, and the culture was continued. The adipose-derived mesenchymal stem cells were prepared by a method including the following steps:

[0132] (a) Process fat samples transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet;

[0133] (b) Centrifuge the fat sample, remove the upper adipose tissue, wash it with D-Hanks solution, centrifuge it again, remove the adipose tissue, add 1% type II collagenase and shake to digest.

[0134] (c) After digestion, dilute with an equal volume of D-hanks solution, centrifuge, and collect the cell pellet at the bottom for the next operation;

[0135] (d) Take the cell pellet obtained in step (c), resuspend it in primary supplemental medium, take samples for counting, and seed it into culture flasks according to the specified cell amount; place it in a CO2 incubator for culture; the primary supplemental medium is prepared with DMEM-F12 medium as the matrix and the following are added: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose;

[0136] (e) After culturing for 3 days, the culture medium was completely replaced once. When the cell confluence reached more than 80%, the old culture medium was removed, the cells were washed with D-hanks solution, recombinant trypsin solution was added to digest the cells to cause them to detach, D-hanks solution was added to dilute, centrifuged, and the cell pellet was resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells.

[0137] (f) The primary adipose mesenchymal stem cells are purified, cultured and passaged in steps (d) to (e) to obtain P1 generation cells; and so on, to obtain P2 to P8 generation mesenchymal stem cells.

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

[0139] (3) Discard the culture medium, wash with PBS, then add MSC complete culture medium, and incubate in a 37℃, 2% O2, 5% CO2 incubator for 42-56 hours;

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

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

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

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

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

[0145] (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, add sterile PBS to resuspend the exosome, and obtain the exosome in suspension form.

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

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

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

[0149] According to the method of the fourth aspect of the present invention, in step (1), the mesenchymal stem cells are cells of generation P2 to P8, for example, cells of generation P3 to P7.

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

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

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

[0153] According to the method of the fourth aspect of the present invention, in step (3), the culture is carried out for 48 hours in an incubator at 37°C, 2% O2, and 5% CO2;

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

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

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

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

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

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

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

[0161] Although the specific steps described in the various operational steps of the present invention may differ in some details or language from the steps described in the preparation examples in the detailed embodiments section below, those skilled in the art can fully summarize the above-described method steps based on the detailed disclosure of the entire present invention.

[0162] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not contradict each other. Furthermore, any technical feature in any embodiment of any aspect of the present invention can be applied to the same technical feature in other embodiments, as long as they do not contradict each other. The present invention will now be further described.

[0163] All references cited in this invention are incorporated herein by reference in their entirety, and in the event of any inconsistency between the meanings expressed in these references and those expressed herein, the meanings expressed herein shall prevail. Furthermore, the various terms and phrases used in this invention have their general meanings known to those skilled in the art; however, this invention still seeks to provide a more detailed explanation and interpretation of these terms and phrases, and in the event of any inconsistency between the mentioned terms and their known meanings and those expressed herein, the meanings expressed herein shall prevail.

[0164] Although MSC-derived exosomes possess numerous advantages, such as promoting angiogenesis, cell proliferation, growth, and migration, the vast majority of MSC-derived exosomes cultured using conventional methods do not exhibit significant effects in mediating inflammation regulation. Therefore, this invention designs a method to induce MSC culture and isolate purified exosomes secreted by MSCs using a hypoxia partial pressure combined with inflammatory factor stimulation. These exosomes demonstrate excellent inflammation-regulating effects in vitro, providing a novel approach for exosome-based treatment of inflammatory diseases. Attached Figure Description

[0165] Figure 1 Electron micrographs of exosomes from adipose-derived mesenchymal stem cells obtained in Example 2 are shown.

[0166] Figure 2 Electron micrographs of exosomes from adipose-derived mesenchymal stem cells obtained in Example 3 are shown.

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

[0168] Figure 4 The particle size distribution curve of exosomes from Example 3 is shown.

[0169] Figure 5 The expression level of the exosomal membrane protein CD9 obtained in Example 3 is shown.

[0170] Figure 6 The expression level of the exosomal membrane protein CD81 obtained in Example 3 is shown. Detailed Implementation

[0171] The invention can be further described through the following embodiments; however, the scope of the invention is not limited to the embodiments described below. Those skilled in the art will understand that various changes and modifications can be made to the invention without departing from its spirit and scope. The invention provides a general and / or specific description of the materials and methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of the invention are well known in the art, the invention is still described herein in as much detail as possible.

[0172] Unless otherwise stated, some of the reagents used in this invention are conventional in the art or readily available from commercial sources. For example, serum-free culture medium (Gibco) was purchased from Thermo Fisher Scientific; human platelet lysate was purchased from Precicion BioMedicals; phosphate-buffered saline (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 washing buffer (PRS-TCR-1) was purchased from Precicion. The complete MSC culture medium described herein is a serum-free culture medium containing 2% human platelet lysate.

[0173] Example 1: Primary and passaged culture of adipose-derived mesenchymal stem cells

[0174] Methods for obtaining primary and passaged mesenchymal stem cells through adipose tissue have been reported in numerous publications. The mesenchymal stem cells used in the preparation of exosomes in this invention can be obtained using these methods. Although the key to the technology of this invention is not in this invention, by way of example, this invention is still willing to describe a method for preparing mesenchymal stem cells herein.

[0175] This embodiment refers to the method described in Chinese Patent No. ZL2020113812050 (Example 1a) of the inventive team of this application to prepare adipose mesenchymal stem cells. The materials and sources used are also referred to in that patent document, the entire contents of which are incorporated herein by reference.

[0176] (a) Processing volunteer-donated fat (sample M) transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet;

[0177] (b) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min.

[0178] (c) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation;

[0179] (d) Collect the cell pellet, resuspend it in primary culture medium, take samples for counting, and perform 2×10⁶ cell counts. 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37°C, saturated humidity);

[0180] (e) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle to dilute the cells. The cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation).

[0181] (f) The P0 generation cells are purified, cultured and passaged in steps (d) to (e) to obtain the P1 generation cells; and so on, to obtain the P2 to P8 generation mesenchymal stem cells.

[0182] The D-Hanks solution used in Example 1 has the following composition and preparation method: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4·12H2O, 0.35g NaHCO3, and water to 1000ml; Preparation: Dissolve each material in 1000ml, filter and sterilize through a 0.22μm microporous membrane to obtain the solution.

[0183] The primary supplemental culture medium used in Example 1 was prepared using DMEM-F12 medium as the matrix and contained: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose.

[0184] The DMEM-F12 culture medium used in Example 1 of this experiment has the following composition: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, L-phenylalanine 35.48 mg, and 1,4-butanediamine dihydrochloride. Salt 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, vitamin H 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous sodium dihydrogen phosphate 54.35 mg, L-aspartic acid 6.65 mg Folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine ​​hydrochloride 17.56mg, I-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamate 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cysteine ​​hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L -Tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water added to 1000mL; Preparation: Dissolve all materials in 1000mL, filter through a 0.22μm microporous membrane for sterilization, and the product is ready.

[0185] As described in ZL2020113812050, the human adipose-derived mesenchymal stem cells obtained by the above method exhibit excellent properties in terms of cell morphology, cell phenotype, and differentiation potential, such as excellent ability to induce differentiation into osteoblasts, chondrocytes, and adipocytes.

[0186] Example 2: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0187] (1) The mesenchymal stem cells (P5 generation) obtained in Example 1 were seeded at a density of 6 × 10^5 cells into T75 culture flasks, with 15 mL of MSC complete culture medium added to each flask. The flasks were then incubated at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere. IL-1β was then added to the culture medium to a concentration of 10 ng / mL, and the culture was continued for another 24 hours. [Those skilled in the art will understand that other sizes of culture flasks, such as T25 or T75 flasks, can be used. The number of cells seeded and / or the amount of culture medium added can be changed depending on the culture area. For example, using the above concentration and adding approximately 15 mL of culture medium during MSC culture is a routine procedure in the art.]

[0188] (2) Discard 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%.

[0189] (3) Discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then incubate in a 37℃, 2% O2, 5% CO2 incubator for 48 hours.

[0190] [The atmosphere in the aforementioned 2% O2 and 5% CO2 incubators refers to a nitrogen-balanced atmosphere, while the atmosphere in the aforementioned 5% CO2 incubator refers to an atmosphere balanced with 21% oxygen and the remainder nitrogen. The meaning of such expressions is generally recognized by those skilled in the art.]

[0191] (4) Transfer 30 ml of cell supernatant (from two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0192] Centrifuge at 300g, 4℃ for 10 minutes (to remove dead cells and larger cell debris), and transfer the supernatant to another (50ml) centrifuge tube;

[0193] Centrifuge at 2000g, 4℃ for 20 minutes (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0194] Centrifuge at 10000g, 4℃ for 30 minutes (to further remove smaller cell debris and impurities), filter the supernatant through a 0.22μm filter membrane (PES filter membrane, millipore) and place it in another (ultra-speed) centrifuge tube;

[0195] Centrifuge at 100,000g, 4℃ for 90 minutes, and discard the supernatant;

[0196] (5) Add 20 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate. Centrifuge at 100000 g and 4 °C for 90 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosome, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance testing.

[0197] The centrifuge tubes and centrifuges used in the above steps with different centrifugal force requirements were all purchased from Beckman Coulter.

[0198] Example 2a: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0199] (1) The mesenchymal stem cells (P3 generation) obtained in Example 1 were seeded into T75 culture flasks at a rate of 6×10^5 cells. 17 mL of MSC complete culture medium was added to each flask. The flasks were then placed in a 37°C, 5% CO2 incubator for 20 hours to allow the cells to adhere. IL-1β was then added to the culture medium to a concentration of 8 ng / mL and cultured for another 24 hours.

[0200] (2) Discard 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%.

[0201] (3) Discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then incubate in a 37℃, 2% O2, 5% CO2 incubator for 48 hours.

[0202] (4) Transfer 30 ml of cell supernatant (from two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0203] Centrifuge at 250g, 4℃ for 12 minutes (to remove dead cells and larger cell debris), and transfer the supernatant to another (50ml) centrifuge tube;

[0204] Centrifuge at 2500g, 4℃ for 18 minutes (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0205] Centrifuge at 8000g, 4℃ for 35 minutes (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22μm filter membrane (PES membrane, millipore) and place it in another (ultra-speed) centrifuge tube;

[0206] Centrifuge at 120,000g, 4℃ for 75 minutes, and discard the supernatant;

[0207] (5) Add 15 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80000 g and 4 °C for 120 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosome, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance testing.

[0208] Example 2b: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0209] (1) The mesenchymal stem cells (P7 generation) obtained in Example 1 were seeded into T75 culture flasks at a rate of 6×10^5 cells. 13 mL of MSC complete culture medium was added to each flask. The flasks were then placed in a 37°C, 5% CO2 incubator for 30 hours to allow the cells to adhere to the culture wall. IL-1β was then added to the culture medium to a concentration of 12 ng / mL and cultured for another 24 hours.

[0210] (2) Discard 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%.

[0211] (3) Discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then incubate in a 37℃, 2% O2, 5% CO2 incubator for 48 hours.

[0212] (4) Transfer 30 ml of cell supernatant (from two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0213] Centrifuge at 350g, 4℃ for 8 minutes (to remove dead cells and larger cell debris), and transfer the supernatant to another (50ml) centrifuge tube;

[0214] Centrifuge at 1500g, 4℃ for 25 minutes (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0215] Centrifuge at 12000g, 4℃ for 25 minutes (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22μm filter membrane (PES filter membrane, millipore) and place it in another (ultra-speed) centrifuge tube;

[0216] Centrifuge at 80000g, 4℃ for 120 minutes, and discard the supernatant;

[0217] (5) Add 25 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 120000 g and 4 °C for 75 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosome, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance testing.

[0218] Example 3: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0219] (1) The mesenchymal stem cells (P5 generation) obtained in Example 1 were seeded into T75 culture flasks at a rate of 6×10^5 cells per flask. 15 mL of MSC complete culture medium was added to each flask, and the flasks were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere. Then, IL-1β, sodium tartrate, and lysine were added to the culture medium to a concentration of 10 ng / mL, 0.15 mg / mL, and 2.2 mg / mL, respectively, and the flasks were cultured for another 24 hours.

[0220] (2) Discard 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%.

[0221] (3) Discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then incubate in a 37℃, 2% O2, 5% CO2 incubator for 48 hours.

[0222] [The atmosphere in the aforementioned 2% O2 and 5% CO2 incubators refers to a nitrogen-balanced atmosphere, while the atmosphere in the aforementioned 5% CO2 incubator refers to an atmosphere balanced with 21% oxygen and the remainder nitrogen. The meaning of such expressions is generally recognized by those skilled in the art.]

[0223] (4) Transfer 30 ml of cell supernatant (from two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0224] Centrifuge at 300g, 4℃ for 10 minutes (to remove dead cells and larger cell debris), and transfer the supernatant to another (50ml) centrifuge tube;

[0225] Centrifuge at 2000g, 4℃ for 20 minutes (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0226] Centrifuge at 10000g, 4℃ for 30 minutes (to further remove smaller cell debris and impurities), filter the supernatant through a 0.22μm filter membrane (PES filter membrane, millipore) and place it in another (ultra-speed) centrifuge tube;

[0227] Centrifuge at 100,000g, 4℃ for 90 minutes, and discard the supernatant;

[0228] (5) Add 20 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate. Centrifuge at 100000 g and 4 °C for 90 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosome, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance testing.

[0229] Example 3a: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0230] (1) The mesenchymal stem cells (P3 generation) obtained in Example 1 were seeded into T75 culture flasks at a rate of 6×10^5 cells. 17 mL of MSC complete culture medium was added to each flask. The flasks were then incubated at 37°C and 5% CO2 for 20 hours to allow the cells to adhere to the culture wall. IL-1β, sodium tartrate, and lysine hydrochloride were then added to the culture medium to a concentration of 8 ng / mL, 0.175 mg / mL, and 2 mg / mL, respectively. The culture was continued for 24 hours.

[0231] (2) Discard 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%.

[0232] (3) Discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then incubate in a 37℃, 2% O2, 5% CO2 incubator for 48 hours.

[0233] (4) Transfer 30 ml of cell supernatant (from two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0234] Centrifuge at 250g, 4℃ for 12 minutes (to remove dead cells and larger cell debris), and transfer the supernatant to another (50ml) centrifuge tube;

[0235] Centrifuge at 2500g, 4℃ for 18 minutes (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0236] Centrifuge at 8000g, 4℃ for 35 minutes (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22μm filter membrane (PES membrane, millipore) and place it in another (ultra-speed) centrifuge tube;

[0237] Centrifuge at 120,000g, 4℃ for 75 minutes, and discard the supernatant;

[0238] (5) Add 15 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 80000 g and 4 °C for 120 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosome, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance testing.

[0239] Example 3b: Treatment of mesenchymal stem cells with IL-1β and purification of exosomes by differential centrifugation

[0240] (1) The mesenchymal stem cells (P7 generation) obtained in Example 1 were seeded into T75 culture flasks at a rate of 6×10^5 cells per flask. 13 mL of MSC complete culture medium was added to each flask. The flasks were then incubated at 37°C and 5% CO2 for 30 hours to allow the cells to adhere to the culture wall. IL-1β, sodium tartrate, and lysine hydrochloride were then added to the culture medium to a concentration of 12 ng / mL, 0.125 mg / mL, and 2.5 mg / mL, respectively. The flasks were then incubated for another 24 hours.

[0241] (2) Discard 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%.

[0242] (3) Discard the culture medium, wash 3 times with PBS, then add 15 mL of MSC complete culture medium to each bottle, and then incubate in a 37℃, 2% O2, 5% CO2 incubator for 48 hours.

[0243] (4) Transfer 30 ml of cell supernatant (from two culture flasks) into a 50 ml centrifuge tube and centrifuge as follows:

[0244] Centrifuge at 350g, 4℃ for 8 minutes (to remove dead cells and larger cell debris), and transfer the supernatant to another (50ml) centrifuge tube;

[0245] Centrifuge at 1500g, 4℃ for 25 minutes (to further remove cell debris and other impurities), and transfer the supernatant to another (high-speed) centrifuge tube;

[0246] Centrifuge at 12000g, 4℃ for 25 minutes (to further remove smaller cell debris and impurities), and filter the supernatant through a 0.22μm filter membrane (PES filter membrane, millipore) and place it in another (ultra-speed) centrifuge tube;

[0247] Centrifuge at 80000g, 4℃ for 120 minutes, and discard the supernatant;

[0248] (5) Add 25 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 120000 g and 4 °C for 75 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosome, and obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance testing.

[0249] Example 3c: Referring to Examples 3, 3a and 3b respectively, the only difference is that sodium tartrate is not added in step (1), resulting in three batches of exosomes, which can be recorded as Example 3c1, Example 3c2 and Example 3c3 respectively.

[0250] Example 3d: Refer to Examples 3, 3a and 3b respectively, except that lysine hydrochloride is not added in step (1), and three batches of exosomes are obtained, which can be recorded as Example 3d1, Example 3d2 and Example 3d3 respectively.

[0251] Example 4: Transmission electron microscopy observation of exosome particles

[0252] 1. Take 50 μl of the separated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and fix in a 4℃ refrigerator for 1 hour;

[0253] 2. Add 20 μl of the fixed exosome suspension to the front side of the copper mesh and let it stand for 20 min;

[0254] 3. Carefully absorb the excess solution with absorbent filter paper; then rinse the copper mesh with ultrapure water 5 times, 30 seconds each time, and blot dry with filter paper.

[0255] 4. Add 1 drop of 2% uranium acetate staining solution to the front of the copper mesh, stain for 1 minute, and then use filter paper to blot away the excess staining solution along the edge of the copper mesh.

[0256] 5. Place the copper mesh in room temperature air to dry naturally. After it is completely dry, observe it on the machine and calculate and statistically analyze the particle size and distribution of exosomes.

[0257] The exosomes (suspensions) obtained in the various embodiments of the present invention were tested using the above methods. The number of exosomes obtained from 1.2 × 10^6 cells (2 culture flasks) in each example was calculated. The results were as follows: the number of exosome particles in Example 2, Example 2a, and Example 2b were 9.4 × 10^9, 8.7 × 10^9, and 11.4 × 10^9, respectively; the number of exosome particles in Example 3, Example 3a, and Example 3b were 124.3 × 10^9, 133.2 × 10^9, and 109.8 × 10^9, respectively; the number of exosome particles in Example 3c1, Example 3c2, and Example 3c3 were 12.2 × 10^9, 9.7 × 10^9, and 10.3 × 10^9, respectively; and the number of exosome particles in Example 3d1, Example 3d2, and Example 3d3 were 9.2 × 10^9, 9.8 × 10^9, and 11.6 × 10^9, respectively. Taking the exosomes in Example 2 as an example, the initial number of MSC cells was 1.2 × 10^6 cells, and the volume of exosomes obtained was 1 ml. The measured concentration of exosomes was 9.4 × 10^9 particles / mL, which is equivalent to obtaining 9.4 × 10^9 exosomes from 1.2 × 10^6 cells. The phrase "9.4 × 10^9 exosomes" above refers to 9.4 multiplied by 10 to the power of 9 exosomes; other similar expressions have similar meanings.

[0258] Figure 1 and Figure 2Electron micrographs of exosomes from adipose-derived mesenchymal stem cells obtained in Examples 2 and 3 are shown. The average particle size of all exosomes obtained in Examples 2, 2a, 2b, 3, 3a, 3b, 3c, and 3d was measured to be in the range of 102–144 nm. For example, the peak particle size of the exosomes in Example 3 was 113.7 ± 4.4 nm, and the average particle size was 131.7 ± 1.6 nm. Figure 3 A scatter plot of the particle size distribution of exosomes from Example 3 is shown. Figure 4 The particle size distribution curve of exosomes from Example 3 is shown.

[0259] Example 5: Flow cytometry detection of exosome surface protein expression

[0260] 1. Mix the components of Thermofisher CD63 magnetic beads (product number 10606D) by inverting for 10 min; pipette 20 μl of the magnetic bead suspension into a 1.5 mL round-bottom EP tube;

[0261] 2. Add 200 μl of magnetic bead cleaning solution to the EP tube and mix thoroughly with the pipette tip;

[0262] 3. Place the EP on the magnetic rack for 1 minute; then aspirate the supernatant.

[0263] 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.

[0264] 5. Place the exosome-washing solution mixture on a rotary mixer, set the speed to 10 rpm, and incubate overnight at 2-8°C.

[0265] 6. The next day, the sample was rapidly centrifuged for 3-5 seconds to collect the precipitate;

[0266] 7. Add 300 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds;

[0267] 8. Place the sample on the magnetic rack for about 1 minute, then discard the supernatant;

[0268] 9. Add 400 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds;

[0269] 10. Place the sample on a magnetic rack for about 1 minute, discard the supernatant, and resuspend it with 300 μl of washing solution;

[0270] 11. Take 100 μl of each sample, add CD9-PE and CD81-FITC flow cytometry antibodies to the samples respectively, and incubate at 4°C in the dark for 30 min.

[0271] 12. Place the incubated sample on a magnetic rack for about 1 minute, discard the supernatant, and add 300 μl of washing solution to wash;

[0272] 13. After repeating step 12 once, resuspend the selected sample with 300 μl PB and then perform the detection.

[0273] Figure 5 The expression level of the exosome membrane protein CD9 obtained in Example 3 is shown. Figure 6 The expression levels of the exosomal membrane protein CD81 obtained in Example 3 are shown. The positive expression rate of CD9 was 78.3%, and the positive expression rate of CD81 was 95.4%. The expression levels of the exosomal membrane proteins CD9 and CD81 obtained in other examples are similar to those in Example 3. Figure 5 and Figure 6 No significant difference.

[0274] Example 6: Detection of exosome protein content

[0275] This example uses the Pierce™ Protein Quantitative Kit (catalog number: 23225, Thermo Scientific) for testing.

[0276] 1. Take 10 μl of 5 mg / mL BCA (2,2-biquinoline-4,4-dicarboxylate disodium salt) standard, dilute with PBS to a final concentration of 0.5 mg / mL, and use this as the BCA standard solution. Add 0, 2, 4, 6, 8, 12, 16, and 20 μL of this standard solution to the protein standard wells of a 96-well plate, and add PBS to bring the total to 20 μL.

[0277] 2. Dilute the exosome sample appropriately and add 20 μL to the wells of a 96-well plate. Since pipettes have a larger margin of error when taking small amounts of sample, the points before the standard line may not be very accurate. Therefore, try to ensure the sample concentration point falls within half of the standard line.

[0278] 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 A562 nm using a microplate reader and calculate the protein concentration based on the standard curve.

[0279] 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.

[0280] The exosomes (suspensions) obtained in each embodiment of the present invention were detected using the above methods. The protein content of exosomes obtained from 1.2 × 10^6 cells in each embodiment was calculated. The results were as follows: the protein content of Examples 2, 2a, and 2b was 66.4 μg, 56.2 μg, and 71.7 μg, respectively; the protein content of Examples 3, 3a, and 3b was 276.8 μg, 303.7 μg, and 297.4 μg, respectively; and the protein content of the six exosome samples in Examples 3c and 3d was in the range of 61–74 μg.

[0281] Example 7: An ELISA assay to detect the immunomodulatory effect of exosomes on TNF-α secretion by PBMC cells

[0282] Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine involved in normal inflammatory and immune responses. It is primarily produced by activated monocytes and macrophages and functions in vivo in two forms: transmembrane TNF (tmTNF) and secreted TNF (sTNF). tmTNF is distributed as a membrane protein on cells that secrete TNF-α, and after activation and cleavage by the TACE (TNF-α-converting enzyme), it is converted into sTNF. There are two types of TNFα receptors (TNFRⅠ and TNFRⅡ), present on the surface of various cells. The binding of TNFα to TNFR often induces apoptosis, inflammation, and tumorigenesis.

[0283] The exosomes secreted by mesenchymal stem cells in the method of this invention can inhibit the release of TNF-α from lymphocytes. In this experiment, exosomes extracted and purified from mesenchymal stem cells and PBMCs were co-cultured in a certain ratio. Then, the expression level of TNF-α in the cell supernatant was detected by ELISA. The inhibitory ability of exosomes on the release of TNF-α from lymphocytes was detected by analyzing the changes in the expression level of TNF-α.

[0284] 1. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy adults using the Ficoll method. PBMCs were resuspended in serum-free immunocellular medium (Miltenyi, containing 2.5% serum substitute) to adjust the PBMC density to 4 × 10^5 / ml. Then, 50 μl of CD3 / CD28 magnetic beads (Thermofisher) were added to each ml of PBMC suspension. After mixing the PBMCs and magnetic beads, 0.5 ml of PBMCs was added to the wells of a 24-well plate, and 50 μl of a suspension containing 1 × 10^7 exosomes was added to each well. Experimental wells (PBMCs activated with magnetic beads and with added exosomes), control wells (PBMCs activated with magnetic beads but without added exosomes), negative wells (PBMCs not activated with magnetic beads), and blank wells (containing only serum-free immunocellular medium).

[0285] 2. After co-culturing for 5 days, collect the supernatant of the co-culture medium; then centrifuge at 2000 rpm for 5 minutes; collect the supernatant.

[0286] 3. Remove the TNF-α detection ELISA kit (R&D Systems) and reagents from the refrigerator and 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 in the refrigerator.

[0287] 4. Take one TNF-α standard, add 0.95 ml of deionized water, and gently pipette to dissolve PGE2, preparing a standard stock solution with a concentration of 10000 pg / ml. Incubate the dissolved PGE2 standard at room temperature for 1 minute, and gently shake the TNF-α standard 2-3 times every 4-5 minutes. Then, take seven 1.5 ml EP tubes and dilute the TNF-α standard into different concentration gradients using 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.

[0288] 5. Sample dilution: For each sample to be tested, take an appropriate amount of the sample and dilute it with Calibrator Diluent RD6-12 to an exosome protein concentration of 5 μg / ml;

[0289] 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 each well, repeating 3 times as a control group; add 50 μl of standard solutions of different concentrations to each well, repeating 3 times each; add 50 μl of diluted sample to each well, repeating 3 times each; carefully cover the microplate with the membrane and shake at 450 rpm at room temperature for 2 hours.

[0290] 7. Carefully peel off the film, pour out the liquid inside the microplate, and invert the microplate onto absorbent paper and tap it a few times; add 300μl of 1×Wash Buffer to each well, then pour out the 1×Wash Buffer, and invert the microplate onto absorbent paper and tap it a few times; repeat 3 times.

[0291] 8. Add 200 μl of TNF-α Conjugate to each well, carefully cover with a new membrane, and incubate at room temperature for 2 hours;

[0292] 9. Carefully peel off the film, pour out the liquid inside the microplate, and invert the microplate onto absorbent paper and tap it a few times; add 300μl of 1×Wash Buffer to each well, then pour out the 1×Wash Buffer, and invert the microplate onto absorbent paper and tap it a few times; repeat 3 times.

[0293] 10. Add 200 μl of Substrate Solution to each well and let it stand at room temperature in the dark for 30 minutes; then add 50 μl of Stop Solution to each well.

[0294] 11. Read OD value: Place the microplate into the microplate reader (Thermofisher, Multiscan), then set the microplate reader program, detect the absorbance at a wavelength of 450nm, and perform data analysis;

[0295] 12. After subtracting the average OD value of the blank group from the results, the Origin software was used to plot the standard curve. The theoretical concentration at each point of the standard curve was used as the X-axis and the absorbance value as the Y-axis. Logarithmic transformation was performed on both sides, and the four-parameter method was used for fitting. The standard curve was plotted, the absorbance value of the sample was input, and the TNF-α concentration value of the sample was calculated.

[0296] The above method was used to determine the level of TNF-α secretion by PBMCs inhibited by exosomes. Results:

[0297] The TNF-α level in the negative group was 802.2 pg / ml.

[0298] The TNF-α level in the control group was 5203.7 pg / ml.

[0299] In Examples 2, 2a, and 2b, the TNF-α levels in the experimental groups ranged from 1044 to 1307 pg / ml. For example, in Example 2, the TNF-α level in the experimental group was 1143.2 pg / ml.

[0300] The TNF-α levels in the experimental groups of Examples 3, 3a, and 3b ranged from 1103 to 1367 pg / ml. For example, the TNF-α level in the experimental group of Example 3 was 1241.4 pg / ml.

[0301] The TNF-α levels in the experimental groups of Examples 3c and 3d were in the range of 1173–1407 pg / ml.

[0302] Example 8: RT-PCR analysis of the inhibitory effect of exosomes on TNF-α gene expression in PBMCs

[0303] 1. Extraction of total RNA from PBMCs: 1 ml of PBMCs from each group (experimental group, control group, and negative group) after co-culturing 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. After centrifugation at 300 g for 5 minutes, the supernatant was discarded, and 1 ml of Trizol reagent (Life) was added to the precipitate. The precipitate was incubated at room temperature for 5 minutes to allow for complete lysis.

[0304] 2. Place the EP tube into a high-speed centrifuge, centrifuge at 4°C and 12,000 rpm for 5 minutes, then discard the precipitate;

[0305] 3. Add 200 μl of chloroform, shake to mix, and let stand at room temperature for 15 minutes;

[0306] 4. Place the EP tube in a high-speed centrifuge and centrifuge at 12,000 rpm for 15 minutes;

[0307] 5. Transfer the upper aqueous phase to another centrifuge tube; add 0.5 ml of isopropanol solution, mix well, and let stand at room temperature for 10 minutes;

[0308] 6. Place the EP tube in a high-speed centrifuge and centrifuge at 4°C and 12,000 rpm for 10 minutes. Discard the supernatant, and the RNA precipitate will remain at the bottom of the tube.

[0309] 7. Add 1 mL of 75% ethanol solution and gently shake the centrifuge tube to suspend the precipitate;

[0310] 8. Place the EP tube in a high-speed centrifuge at 4°C and 8000 rpm for 5 minutes, then discard the supernatant; air dry at room temperature for 5-10 minutes, and resuspend the RNA in 20 μl of sterile deionized water.

[0311] 9. Use a nanodrop instrument to detect RNA concentration;

[0312] 10. Reverse transcription of cDNA: RNA was reverse transcribed into cDNA according to the Bestar qPCR kit (catalog number: DBI-2220) instructions;

[0313] 11. qPCR detection of TNF-α gene expression level: Real-time quantitative PCR was performed to detect TNF-α gene expression level according to the Bestar qPCR (SyberGreen) kit (catalog number: DBI-2043) instructions.

[0314] TNF-α gene expression levels were detected by real-time quantitative PCR and characterized by relative mRNA levels (folds). The relative mRNA level of the negative group was set at 1. The relative mRNA levels of the other groups were calculated. Results:

[0315] The relative mRNA level in the control group was 114.6 times higher.

[0316] The relative mRNA levels in the experimental groups of Examples 2, 2a, and 2b were 55–67 times higher. For example, the relative mRNA level in the experimental group of Example 2 was 63.1 times higher.

[0317] The relative mRNA levels in the experimental groups of Examples 3, 3a, and 3b were 61–70 times higher. For example, the relative mRNA level in the experimental group of Example 3 was 68.4 times higher.

[0318] The relative mRNA levels in the experimental groups of Examples 3c and 3d were 64–72 times.

[0319] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for isolating and extracting exosomes using adipose-derived mesenchymal stem cells, comprising the following steps: (1) Seed mesenchymal stem cells into culture flasks, add MSC complete medium, and culture in a 37℃, 5% CO2 incubator to allow the cells to adhere to the culture wall. Then add IL-1β, sodium tartrate, and lysine hydrochloride to the culture medium to concentrations of 8~12ng / mL, 0.125~0.175mg / mL, and 2.0~2.5mg / mL, respectively, and continue culturing. (2) Discard the culture medium and replace it with fresh MSC complete culture medium, and continue to culture the cells in a 37℃, 5% CO2 incubator until the cell confluence is ≥80%; (3) Discard the culture medium, wash with PBS, then add MSC complete culture medium, and incubate in a 37℃, 2% O2, 5% CO2 incubator for 42-56 hours; (4) Transfer the cell supernatant into a centrifuge tube and centrifuge as follows: Centrifuge at 250-350g and 4℃ for 8-12 minutes, and transfer the supernatant to another centrifuge tube; Centrifuge at 1500-2500g, 4℃ for 18-25 minutes, and transfer the supernatant to another centrifuge tube; Centrifuge at 8000~12000g, 4℃ for 25~35 minutes, filter the supernatant through a 0.22μm filter membrane and place it in another centrifuge tube; Centrifuge at 80,000-120,000 g at 4°C for 75-120 minutes, and discard the supernatant; (5) Add sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 80,000-120,000 g and 4°C for 75-120 minutes, discard the supernatant, add sterile PBS to resuspend the exosomes again, and obtain exosomes in suspension form. in, The adipose-derived mesenchymal stem cells were prepared by a method comprising the following steps: (a) Processing fat samples transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (b) Centrifuge the fat sample, remove the upper adipose tissue, wash it with D-Hanks solution, centrifuge again, remove the adipose tissue, add 1% type II collagenase and shake to digest; in this step, both centrifugations are performed at 100g for 5 min, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (c) After digestion, dilute with an equal volume of D-hanks solution, centrifuge, and collect the cell pellet at the bottom for the next operation; (d) Take the cell pellet obtained in step (c), resuspend it in primary supplemental medium, take samples for counting, and seed it into culture flasks according to the specified cell amount; place it in a CO2 incubator for culture; the primary supplemental medium is prepared with DMEM-F12 medium as the matrix and the following are added: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose; (e) After culturing for 3 days, the culture medium was completely replaced once. When the cell confluence reached more than 80%, the old culture medium was removed, and the cells were washed with D-Hanks solution. 2 ml of recombinant trypsin solution was added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution was added to each bottle to dilute the cells. The cells were centrifuged at 100g for 10 min. The cell pellet was resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells. (f) The primary adipose mesenchymal stem cells are purified, cultured and passaged in steps (d) to (e) to obtain P1 generation cells; and so on, to obtain P2 to P8 generation mesenchymal stem cells.

2. According to the method of claim 1, in step (1), the mesenchymal stem cells are cells of generation P3 to P7.

3. According to the method of claim 1, the resulting exosomes have an average particle size of 50-200 nm, a positive expression rate of membrane protein CD9 greater than 70%, and a positive expression rate of membrane protein CD81 greater than 80%.

4. According to the method of claim 1, in step (c), the centrifugation is performed at 100g for 5 minutes.

5. According to the method of claim 1, in step (d), seeding cells into the culture flask according to the specified cell amount means seeding cells at a amount of 1~5×10⁶ cells / year. 4 / cm 2 Inoculate into T75 bottles; incubate in a CO2 incubator under the following conditions: 5% CO2, 37°C, and saturated humidity.

6. According to the method of claim 1, the D-Hanks solution is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4·12H2O, 0.35g NaHCO3, and water to 1000ml.

7. According to claim 1, the DMEM-F12 culture medium is formulated as follows: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, and L-phenylalanine 35.48 mg. g, 1,4-Butanediamine dihydrochloride 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, vitamin H 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous sodium dihydrogen phosphate 5 4.35 mg, L-aspartic acid 6.65 mg, folic acid 2.65 mg, disodium hydrogen phosphate 71.02 mg, L-cysteine ​​hydrochloride 17.56 mg, i-inositol 12.6 mg, zinc sulfate heptahydrate 0.432 mg, L-glutamate 7.35 mg, nicotinamide 2.02 mg, L-arginine hydrochloride 147.5 mg, L-proline 17.25 mg, pyridoxal hydrochloride 2 mg, L-cysteine ​​hydrochloride 31.29 mg, L-tryptophan 9.02 mg g, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water added to 1000mL.

8. According to the method of claim 1, in step (1), the culture flask contains (0.5~5)×10 4 cells / cm 2 Increase cell density.

9. According to the method of claim 1, step (1) uses T75 culture flasks, with each flask inoculated with (2~10) × 10 5 Add 10-20 ml of culture medium to each cell.

10. According to the method of claim 1, in step (1), the cells are cultured for 20 to 30 hours to allow them to adhere to the culture vessel.

11. According to the method of claim 1, in step (1), after adding IL-1β, continue culturing for 20 to 30 hours.

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

13. According to the method of claim 1, in step (4), the liquid is first centrifuged at 250g and 4℃ for 12 minutes, then centrifuged at 2500g and 4℃ for 18 minutes, then centrifuged at 8000g and 4℃ for 35 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 120000g and 4℃ for 75 minutes.

14. According to the method of claim 1, in step (4), the mixture is first centrifuged at 350g and 4℃ for 8 minutes, then centrifuged at 1500g and 4℃ for 25 minutes, then centrifuged at 12000g and 4℃ for 25 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 80000g and 4℃ for 120 minutes.

15. According to the method of claim 1, in step (4), the mixture is first centrifuged at 300g and 4℃ for 10 minutes, then centrifuged at 2000g and 4℃ for 20 minutes, then centrifuged at 10000g and 4℃ for 30 minutes, and the supernatant is filtered through a 0.22μm filter membrane and then centrifuged at 100000g and 4℃ for 90 minutes.

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

17. The method according to claim 1, wherein, In step (1), sodium tartrate and lysine hydrochloride were added to the culture medium along with IL-1β, with concentrations of 0.15 mg / mL and 2.2 mg / mL, respectively.

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