Preparation method of extracellular small vesicles of functional homogeneity mesenchymal stem cells

Abstract

The invention discloses a preparation method of extracellular small vesicles of functional homogeneity mesenchymal stem cells. The preparation method comprises the following steps: separately and statically culturing tissues from different individual sources for 8-12 days through a 3D culture microcarrier; and carrying out mixed dynamic culture on the culture product obtained in the step S100, collecting a supernatant, and separating the extracellular vesicles from the supernatant. The sEVs obtained by the preparation method have high-homogeneity mesenchymal stem cell sEVs proteome, and in addition, compared with sEVs purified by independently culturing mesenchymal stem cells, the sEVs have stronger immune regulation and control capabilities (including inflammatory reaction inhibition, proinflammatory factor reduction and inflammatory inhibition factor increase) and regeneration and repair promotion capabilities.

Description

Technical Field

[0001] The present application relates to the technical field of cell biology, and in particular to a method for preparing functionally homogeneous extracellular vesicles of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of multipotent stem cell that originates from a wide range of sources, including the placenta, umbilical cord, bone marrow, adipose tissue, and foreskin. They possess the ability to self-renew and multidirectionally differentiate, as well as possess immunomodulatory and anti-inflammatory properties. These characteristics make MSCs an ideal cell source for treating a variety of immune and traumatic diseases, as well as for health care. Recent studies have shown that the mechanism of action of the therapeutic effects of MSCs is more related to their paracrine effects than to their direct differentiation. The paracrine effects of MSCs are closely related to the extracellular vesicles (EVs) released by MSCs, particularly small extracellular vesicles (sEVs). They exhibit similar biological functions to MSCs and are therefore considered to be a potential alternative to MSCs for the treatment of various diseases.

[0003] sEVs are a major type of extracellular vesicle (sEV) released by cells. They carry a rich reservoir of bioactive components, such as mRNA, miRNA, lipids, and proteins. Their structure consists of a lipid bilayer and ranges from approximately 30 to 200 nm in diameter. sEVs play a crucial role in intercellular communication, signal transduction, and disease regulation, regulating diverse biological processes such as cell migration, proliferation, and apoptosis. MSC-derived sEVs have shown great potential in medical applications. First, due to their cell-free nature and minimal immunogenicity, their therapeutic use avoids the ethical and safety concerns associated with direct MSC use. Second, MSC-derived sEVs can deliver bioactive molecules through targeted delivery, potentially enabling tissue repair and regeneration, immune regulation, cardiovascular disease treatment, and tissue and organ damage treatment. A key advantage is that sEVs lack the potential for mutation and carcinogenesis that cells may harbor, and are easily stored and transported, offering enormous clinical application prospects.

[0004] At present, various mesenchymal stem cell sEVs are cultured in 2D or 3D, and the culture supernatant is collected and separated by centrifugation, kits, microfluidics and other methods for research and clinical trials. However, there is an unsolvable problem with the mesenchymal stem cell sEVs currently obtained. Because of the differences in genetic DNA background and epigenetics between different individuals, their MSCs are a type of stem cell with great heterogeneity. Correspondingly, there are huge differences in composition and function in sEVs. This leads to different efficacy of the total amount (per batch) of sEVs obtained from each individual tissue culture, which affects the clinical efficacy and its judgment. Therefore, it is necessary to provide a method for preparing extracellular vesicles of mesenchymal stem cells, which has better efficacy and functional uniformity of the extracellular vesicles obtained by this method. Summary of the Invention

[0005] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a method for preparing functionally homogeneous extracellular vesicles of mesenchymal stem cells.

[0006] In a first aspect of the present application, a method for preparing extracellular vesicles is provided, comprising the following steps:

[0007] S100: Tissues from different sources are mixed with microcarriers and statically cultured in separate areas for 10 to 14 days.

[0008] S200: The culture products from different areas of S100 were mixed for 3D dynamic culture, the supernatant was collected, and extracellular vesicles were isolated from the supernatant.

[0009] The preparation method according to the embodiment of the present application has at least the following beneficial effects:

[0010] The sEVs obtained by this preparation method have a highly homogeneous mesenchymal stem cell sEVs protein group. In addition, compared with sEVs purified by culturing mesenchymal stem cells alone, they have stronger immune regulation ability (including inhibiting inflammatory response, reducing proinflammatory factors and increasing anti-inflammatory factors) and promoting regeneration and repair ability.

[0011] Although mesenchymal stem cells from different sources do not express HLA class II antigens, they still express some HLA class I antigens, potentially leading to insufficient production of sEVs or insufficient sEV function. Therefore, this protocol separates tissue blocks at the beginning of the preparation process to minimize tissue rejection between tissue blocks with different genetic backgrounds and different HLA expression. After a period of static culture, tissues from different individual sources are mixed and cultured, allowing the mesenchymal stem cells from different sources to grow in a mixed and competitive manner, thereby driving the expression of a more complete and complex proteome by the sEVs within them.

[0012] In addition, sEVs isolated and purified from mixed cultured mesenchymal stem cells were identified by nanoflow cytometry to have a purity of >98% and were positive for CD9 and CD81; proteomic analysis results demonstrated the presence of a more complete proteome.

[0013] In some embodiments of the present application, S200 includes:

[0014] S210: The culture products from different regions of S100 were mixed and dynamically cultured in 3D for 2 to 6 days to obtain mixed cultured P0 mesenchymal stem cells;

[0015] S220: The P0 mesenchymal stem cells are subcultured without digestion, the supernatant is collected after 3 to 5 days, and the extracellular vesicles are separated from the supernatant.

[0016] In some embodiments of the present application, the supernatant is collected every 3 to 5 days.

[0017] In some embodiments of the present application, the subculture is performed once every 5 to 15 days without digestion.

[0018] In some embodiments of the present application, the digestion-free subculture comprises inoculating the microcarriers with cells separately into new culture containers, and adding new microcarriers into the new culture containers.

[0019] In some embodiments of the present application, the digestion-free subculture comprises dividing the microcarriers that are full of cells into bottles and adding new microcarriers.

[0020] In some embodiments of the present application, separate static cultures are performed by separating different areas from the same culture container for separate static cultures.

[0021] In some embodiments of the present application, different areas are separated from the same culture container for static culture, including placing tissues from different individual sources at the bottom of different areas separated by stirring blades in the culture container, adding microcarriers and culture medium while avoiding confusion of tissues in different areas, and then statically culturing.

[0022] In some embodiments of the present application, mixing the culture products from different regions of S100 for 3D dynamic culture includes mixing the culture products from different regions under the rotation of a stirring blade to perform 3D dynamic culture.

[0023] In some embodiments of the present application, the dynamic culture method includes at least one of stirring, perfusion, and rotation.

[0024] In some embodiments of the present application, the 3D dynamic culture method includes applying vibration and rotation to the culture container.

[0025] In some embodiments of the present application, the rotation speed is 10 to 100 rpm, for example, it can be 10 rpm, 20 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, or 100 rpm.

[0026] In some embodiments of the present application, the tissue includes at least one of bone marrow, periosteum, synovium, skeletal muscle, dental pulp, umbilical cord, umbilical cord blood, placenta, peripheral blood, fat, and foreskin.

[0027] In some embodiments of the present application, static culture and dynamic culture are cultured using serum-free medium.

[0028] In some embodiments of the present application, the serum-free culture medium includes a basal culture medium and additives.

[0029] In some embodiments of the present application, the basal culture medium includes any one of DMEM medium, RPMI 1640 medium, IMDM medium, and MEM medium.

[0030] In some embodiments of the present application, the additive includes at least one of essential amino acids, non-essential amino acids, vitamins, hormones, growth factors, and minerals.

[0031] In some embodiments of the present application, immunogenic components are pre-cultured prior to static culture. Multiple tissues can be mixed and cultured to obtain sEVs, but these tissues may be susceptible to immune rejection. Therefore, pre-culture of immunogenic components prior to static culture avoids immune rejection that could affect cell growth, sEV production, and uniform, high-quality protein expression.

[0032] In some embodiments of the present application, the immunogenic portion comprises at least one of the epidermis and the inner plate of the foreskin.

[0033] In some embodiments of the present application, the immunogenic portion includes at least one of a blood vessel of the umbilical cord and an amniotic membrane.

[0034] In some embodiments of the present application, separating the extracellular vesicles from the supernatant comprises transferring the supernatant into a hollow fiber column for concentration, and then separating the extracellular vesicles by column chromatography.

[0035] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1These are FSMSCs from different individuals in this application, labeled with different fluorescent proteins. A through C show ZSgreen-FSMSCs, BFP-FSMSCs, and Mcheery-FSMSCs, respectively, 72 hours after transfection. Scale bar: 200 μm.

[0037] Figure 2 The results of mixed culture and passage of FSMSCs from different individual sources in this application are shown in Figure 1. Figure A shows a fluorescence image of FSMSCs at passage 3, and Figure B shows the changes in the proportions of FSMSCs from different individual sources at different cell passages. The scale bar in Figure A is 200 μm.

[0038] Figure 3 This is a Venn diagram showing the number of sEVs proteins and the number of co-expressed proteins in foreskin mesenchymal stem cells from four different individuals in this application.

[0039] Figure 4 This is a Venn diagram showing the number of sEVs proteins and the number of co-expressed proteins in umbilical cord mesenchymal stem cells from four different individual sources in this application.

[0040] Figure 5 This is a schematic diagram of a 3D culture tank used in one embodiment of the present application.

[0041] Figure 6 1 is the growth curve of mesenchymal stem cells during the culture process of the experimental group and the control group in one embodiment of the present application.

[0042] Figure 7 This is a bar graph of the OD values of mesenchymal stem cells during the culture process of the experimental group and the control group in one embodiment of the present application. The vertical axis is the OD value at 450nm, and the horizontal axis is the column from left to right every day. Figure 6 The difference between the two groups was not significant, followed by control group 1c, control group 1a, control group 1b, and experimental group. ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0043] Figure 8 ] is the proliferation index of the experimental group and the control group in one embodiment of the present application, wherein ns indicates no significant difference, ** indicates p<0.01, and *** indicates p<0.001.

[0044] Figure 9 The T cell inhibition rates of the experimental group and the control group in one embodiment of the present application are shown in Table 1. ns indicates no significant difference, ** indicates p<0.01, and *** indicates p<0.001.

[0045] Figure 10The results of the test for the ability to inhibit T cell differentiation in the experimental and control groups in one embodiment of the present application are shown. A represents the result of inhibiting T cell differentiation and pro-inflammatory Th1 cell differentiation, and B represents the result of inhibiting T cell differentiation and pro-inflammatory Th17 cell differentiation. ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0046] Figure 11 This is the proteomic detection result of sEVs prepared from foreskin mesenchymal stem cells from different individual sources according to Example 1 or Comparative Example 1 in an embodiment of the present application.

[0047] Figure 12 This is the proteomic detection result of sEVs prepared from umbilical cord mesenchymal stem cells from different individuals according to Example 1 or Comparative Example 1 in an embodiment of the present application.

[0048] Figure 13 This is the detection result of the ability of sEVs to regulate macrophage polarization in one embodiment of the present application.

[0049] Figure 14 This is the test result of the repair ability of sEVs on alveolar epithelial cells damaged by lipopolysaccharide in one embodiment of the present application.

[0050] Figure 15 This is a photograph of the test results of the angiogenesis-promoting ability of sEVs in one embodiment of the present application.

[0051] Figure 16 This is the quantitative result of the detection of the angiogenesis-promoting ability of sEVs in one embodiment of the present application. DETAILED DESCRIPTION

[0052] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0053] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0054] In the description of this application, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0055] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0056] In the description of this application, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.

[0057] During the experiment, it was found that MSCs from the same tissue but different individuals are also a type of stem cell with great heterogeneity, and the sEVs they generate also have great differences in composition and function. Mixed culture of foreskin mesenchymal stem cells from three different individual sources found that in the mixed culture system, the growth and proliferation capabilities of foreskin mesenchymal stem cells from each different source were different. The specific process is as follows:

[0058] First, three foreskin mesenchymal stem cells from different individuals were transfected with lentivirus containing three fluorescent proteins and labeled. The cells were screened to make the fluorescence positive rate greater than 90%. Figure 1 Then, the three foreskin mesenchymal stem cells (FSMSCs) from different individuals were mixed and cultured in equal proportions. The results were as shown in Figure 2 As shown in the figure, the three types of FSMSCs do not grow uniformly, but rather exhibit individual dominance, with one individual exhibiting a clear numerical advantage, which becomes more pronounced with increasing generations. This result suggests that foreskin mesenchymal stem cells from different sources have significant differences in their biological properties, with more actively growing mesenchymal stem cells secreting more and more functional extracellular vesicles.

[0059] Based on the above results, foreskin mesenchymal stem cells from four different individuals were cultured in 3D dynamic culture under the same conditions (the specific process can be referred to the subsequent examples), and the obtained sEVs were subjected to proteomics detection. The results are as follows Figure 3 As shown, the four sEVs contained 1809, 1879, 1936, and 2130 proteins, respectively. The combined protein count was 2610, but only 1194 proteins were expressed in common, accounting for 45.7% of the total. Each foreskin mesenchymal stem cell-derived sEV contained 615 to 936 unique proteins, which may have different functions.

[0060] Under the same conditions, umbilical cord mesenchymal stem cells from four different individuals were cultured in 3D dynamic culture (the specific process can be referred to the subsequent examples). The obtained sEVs were subjected to proteomics detection. The results are as follows Figure 4 As shown, the four sEVs expressed 1757, 1875, 2155, and 2160 proteins, respectively. The combined protein pool of the four individually cultured umbilical cord sEVs yielded 2542 proteins, of which 1429 shared common proteins, accounting for 56.2% of the total pool. Each umbilical cord mesenchymal stem cell-derived sEV expressed 328 to 731 unique proteins, suggesting distinct functions.

[0061] Based on the above results, current methods for obtaining sEVs have distinct and highly variable proteomes from different individuals, failing to achieve the uniform composition and stable function required for industrialized pharmaceutical production. Therefore, this approach was proposed. For detailed implementation details, please refer to the following examples.

[0062] Example 1

[0063] This embodiment provides a method for preparing extracellular vesicles, the steps are as follows:

[0064] (1) Foreskins from different individuals were obtained, and the epidermis and inner plate with strong immune rejection reactions were removed as completely and thoroughly as possible. Then, the internal fibrous tissue of the foreskin was cut into tissue blocks with a diameter of 0.1 to 0.2 cm.

[0065] (2)Reference Figure 5 Adjust the position of the stirring blade of the 3D culture tank and place it at the bottom of the culture tank. Use the stirring blade to physically separate the bottom of the 3D culture tank, and place different foreskin fiber tissue blocks in different areas at the bottom of the 3D culture tank to avoid contact between foreskin tissue blocks from different individuals.

[0066] (3) Gently add 3D culture microcarriers (Cytiva, catalog number 17044802) into the 3D culture flask to avoid the impact of microcarriers that may cause confusion of tissue blocks from different regions and different individuals;

[0067] (4) Slowly add an appropriate amount of serum-free culture medium (serum-free culture medium was purchased from Shanghai Yuanpei Company and Shenzhen Side Cell Science Co., Ltd.) to avoid confusion of tissue blocks from different individuals during the operation.

[0068] (5) Culture for 10 days under conditions that avoid shaking and rotation as much as possible to give the P0 cells time and space to grow.

[0069] (6) On the 10th day, microcarrier microscopy was performed to confirm the growth of P0 cells. Then, the cells were gently shaken and rotated at 30 rpm according to conventional 3D dynamic culture. During this process, P0 cells in different areas were mixed under the rotation of the blades.

[0070] (7) On the 14th day, all microcarriers were mixed and collected to obtain P0 generation mesenchymal stem cells with mixed growth, stronger proliferation activity and sEVs secretion ability.

[0071] (8) The P0 mesenchymal stem cells on the microcarriers are not digested, but directly divided into microcarrier bottles. One bottle of microcarriers full of P0 mesenchymal stem cells is divided into 4 to 5 3D culture bottles for further culture.

[0072] (9) Starting from the P1 generation cells, continue to culture the cells using the same serum-free medium as in step (4), harvest the supernatant every 3 to 5 days, and divide the mesenchymal stem cells into flasks every 10 to 15 days. One flask is divided into 4 to 5 3D culture flasks for continued subculture, and the supernatant is collected repeatedly.

[0073] (10) The collected supernatant was separated and purified by the following methods:

[0074] The peristaltic pump was started at 25 rpm to circulate the supernatant to a 500 kD polyethersulfone hollow fiber column (Repligen, catalog number K05-E500-05N). After circulating for 2 h, the concentration of extracellular vesicles in the concentrated supernatant was 10 to 20 times that before concentration.

[0075] The concentrated supernatant was loaded onto a protein separation and purification system and separated and purified using a HiScreen Capto Core 700 gel column with a column head pressure of 0.8 MPa and a flow rate of 1.5 mL / min. 1× phosphate buffer was used for column equilibration and flushing of the pipeline. The fractions with an absorption peak at 280-320 nm were collected, which were the purified extracellular vesicles.

[0076] The purified extracellular vesicles were transferred into a 50 mL 100 kD ultrafiltration tube and centrifuged at 4°C and 3500×g for 20 min to obtain pure extracellular vesicles.

[0077] Example 2

[0078] Three foreskin samples from different individuals (a, b, and c) were used to prepare sEVs according to the method of Example 1, serving as the experimental group. Separately, three foreskin samples from these three different individuals were used to prepare sEVs according to the method of Comparative Example 1 (control groups 1a, 1b, and 1c, respectively). Three replicates were performed in each group.

[0079] The preparation method of Comparative Example 1 differs from that of Example 1 in that steps (2) to (7) are to place the corresponding foreskin tissue blocks in a 3D culture tank, then add 3D culture microcarriers and serum-free culture medium, and perform mild shaking and rotation according to conventional 3D dynamic culture. On the 14th day, all microcarriers are mixed and collected together.

[0080] 1. Cell proliferation test

[0081] During the above culture process, 100 μL of sample was taken every day to measure cell proliferation using the CCK-8 method, and the proliferation index was calculated according to the following formula:

[0082] Proliferation index = (OD value on the 7th day - OD value of the blank group) / (OD value on the 1st day - OD value of the blank group).

[0083] The results are as follows Figures 6 to 8 As shown in the figure, it can be seen that the 3D mixed cultured foreskin mesenchymal stem cells obtained according to the embodiment of the present application have a better growth curve. Figure 6 and Figure 7 Day 1 is the first day of conventional 3D dynamic culture after 10 days of static culture. The OD values of the experimental group on days 4 to 6 were higher than those of the three control groups. This indicates that the FSMSCs prepared using the method of Example 1 have a faster proliferation capacity than those in Comparative Example 1.

[0084] 2. T cell inhibitory ability detection

[0085] T cell proliferation inhibition assay:

[0086] ① Isolation of peripheral blood mononuclear cells (PBMC)

[0087] PBMCs were isolated from donor peripheral blood. First, peripheral blood samples were collected in EDTA anticoagulant tubes, the blood was placed in centrifuge tubes, an equal volume of PBS (without calcium and magnesium) was added, and PBMCs were isolated by Ficoll-Paque density gradient centrifugation. The centrifugation conditions were 400×g, 20 minutes, room temperature, slow rise and fall. The buffy coat layer was collected and transferred to a new tube. The cells were washed with PBS and the centrifugation conditions were: 300×g, 10 minutes, room temperature. After removing the supernatant, the PBMC cells were suspended in RPMI-1640 medium, the cells were counted, and the cell concentration was adjusted.

[0088] ② Magnetic bead sorting of T cells

[0089] Wash solution (DPBS buffer containing 2 v / v% FBS and EDTA with a final concentration of 1 mM) and culture medium (RPMI-1640 + 10 v / v% FBS) were prepared.

[0090] The PBMC in step ① were resuspended in the washing solution to a cell concentration of 1×10 8 / mL, add 100μL / mL Selection Cocktail to the flow tube and mix well, incubate in the dark for 3 minutes. TM (Magnetic beads) were vortexed for 30 seconds to mix them into evenly dispersed particles. 60 μL / mL of RapidSpheres was added to each tube of flow cytometry sample. TM , at room temperature and away from light for 3 minutes. The wash solution was diluted to 2.5 mL, mixed, and placed in EasySep TM Place the cells in the magnet. After incubation at room temperature for 3 minutes, invert the magnet with the flow cytometer tube inserted and pour out the supernatant. Do not shake the flow cytometer tube or aspirate any droplets from the tube opening to prevent reducing the sorting efficiency. Remove the flow cytometer tube and adjust the volume to 2.5 mL again with wash solution. Place the cells in the magnet for sorting and discard the supernatant. Repeat this process twice, for a total of three sorting cycles. Finally, resuspend the CD3+ T cells adhered to the tube wall in culture medium and mix thoroughly for cell counting.

[0091] ③CFSE-labeled T cells

[0092] CFDA SE stored at -20°C was restored to room temperature and centrifuged briefly to allow the powder to fully settle to the bottom of the tube. 100 μL of CFDA SE solvent was added and the powder was fully dissolved to prepare a CFDA SE stock solution (1000×). T cells were collected by centrifugation and resuspended in 1 mL of 1× CFDA SE Buffer in a 15 mL centrifuge tube to adjust the cell concentration to 1×10 6CFDASE working solution (1000×) was diluted to 2× with 1× CFDASE Buffer to obtain CFDASE working solution. 1 mL of CFDASE working solution was added to 1 mL of the T cell suspension to be labeled, and the mixture was mixed by inversion. Incubate in a cell culture incubator at 37°C and 5% CO2 for 10 minutes in the dark. Immediately add 5 times the incubation volume of prewarmed complete culture medium (RPMI-1640 + 10 v / v% FBS) to the centrifuge tube and mix by inversion to terminate the labeling reaction. Centrifuge at 300g for 5 minutes at room temperature, aspirate and discard the supernatant, and wash once with an appropriate amount of complete culture medium. Add 10 mL of complete culture medium and incubate in a cell culture incubator at 37°C and 5% CO2 for 5 minutes in the dark to promote the retention of CFSE in the cells and the entry of unreacted CFSE into the complete culture medium. Centrifuge at 300g for 5 minutes at room temperature, aspirate and discard the supernatant to obtain CFSE-labeled T cells.

[0093] ④ PHA-L and IL-2 stimulate T cells

[0094] CFSE-labeled T cells were divided into multiple experimental groups, and all cells were cultured in culture medium containing PHA-L (final concentration 5 μg / mL) and IL-2 (final concentration 1000 IU / mL) as well as sEVs of the experimental or control groups at 37°C and 5% CO2 for 4 days to ensure T cell proliferation in this environment.

[0095] ⑤Flow cytometry detection

[0096] After 4 days of culture, flow cytometry was used to analyze the proliferation of T cells. First, the culture medium was centrifuged, the cells were collected, and washed with PBS. The CFSE fluorescence intensity was analyzed using a flow cytometer, and the proliferation state of CFSE-labeled cells was reflected by the weakening of their fluorescence intensity. The proliferation of T cells is judged by the degree of CFSE attenuation. Generally speaking, the CFSE fluorescence intensity of proliferating T cells will decrease, and a secondary distribution of fluorescence intensity will appear with each division. The proliferation index and proliferation ratio of cells can be analyzed by the flow cytometer software, and the flow cytometer test follows the conventional test process.

[0097] The results are as follows Figure 9 As shown, sEVs from control groups 1a-1c, prepared from single tissue samples according to Comparative Example 1, and sEVs from the experimental group, prepared from mixed tissue samples from different individuals, both inhibited T cell proliferation. Furthermore, while the T cell inhibition rates of control groups 1a-1c varied among individuals, the T cell inhibition rates of the experimental group sEVs were significantly higher than those of control groups 1a-1c.

[0098] 3. Detection of the ability to inhibit T cell differentiation

[0099] The results are as follows Figure 10 As shown in the results, the sEVs prepared in the control group and the experimental group can inhibit the differentiation of T cells into pro-inflammatory Th1 and Th17 cells; however, the sEVs obtained in the experimental group have a higher ability to inhibit the differentiation of Th1 and Th7 than the control group.

[0100] 4. Detection of the regulation of inflammatory factors released by immune cells

[0101] The results are shown in Table 1. The sEVs obtained using the method in Example 1 significantly increased the level of the anti-inflammatory cytokine IL-10, while significantly decreasing the level of the pro-inflammatory cytokine TNF-α. This demonstrates that the sEVs obtained using this method have a stronger ability to inhibit inflammatory responses.

[0102] Table 1. Results of different groups inhibiting T cell differentiation into Th1 and Th7

[0103] TNF-α(A8) IL-10(B4) Experimental group <1.81 <5.48 Control group 1b <1.81 <5.48 Control group 1c <1.81 <5.48 Control group 1a <1.81 <5.48 Experimental group + TC 4.63 229.96 Control group 1b+TC 7.62 207.36 Control group 1c+TC 2.58 177.06 Control group 1a+TC 9.35 132.61 T cell stimulation group 61.19 83.75 T cell unstimulated group 38.22 40.05

[0104] TC in the table refers to T cells.

[0105] Example 3: Detection of protein homogeneity and integrity of foreskin mesenchymal stem cell sEVs

[0106] ① Four foreskin tissue samples were collected from different individuals, and sEVs were prepared from these four foreskin tissue samples according to the method of Example 1, repeated three times. ② Four foreskin tissue samples were collected from different individuals, and sEVs were prepared from each foreskin tissue sample according to the method of Comparative Example 1 in Example 2. The sEVs prepared by the two methods were identified by proteomics testing (sent to Hangzhou Jingjie Company for testing). The protein detection results of the three replicates in ① were recorded as FM3Dexo1, FM3Dexo2, and FM3Dexo3, respectively. The protein detection results of the four sEVs in ② were combined and recorded as Single Fexo.

[0107] Protein test results such as Figure 11As shown in the figure, the total number of proteins in Single Fexo is 2610. FM3Dexo1, FM3Dexo2, and FM3Dexo3 identified 3293, 3311, and 3238 proteins, respectively, showing high protein homogeneity. Among them, the intersection of Single Fexo and FM3Dexo1 accounts for 1909 proteins, accounting for 73.14% of the total number of Single Fexo proteins; the intersection of Single Fexo and FM3Dexo2 accounts for 1910 proteins, accounting for 73.18% of the total number of Single Fexo proteins; and the intersection of Single Fexo and FM3Dexo3 accounts for 1899 proteins, accounting for 72.76% of the total number of Single Fexo proteins.

[0108] The above results show that the protein content of sEVs obtained by mixed culture of foreskin mesenchymal stem cells in Example 1 is higher, which can highly cover the protein group obtained by culturing foreskin mesenchymal stem cells alone to obtain sEVs.

[0109] In addition, samples of foreskin tissues from four different individuals were directly taken and, referring to the method of Example 1, directly mixed with microcarriers for 3D dynamic culture after removing the inner plate of the epidermis. The results showed that P0 cells had difficulty growing, and only a few P0 cells grew after two weeks.

[0110] Example 4: Detection of protein homogeneity and integrity of umbilical cord mesenchymal stem cell sEVs

[0111] ① Four umbilical cord tissues from different individuals were sampled separately, and then sEVs were prepared from the four umbilical cord tissue samples according to the method of Example 1, repeated three times; ② Four umbilical cord tissues from different individuals were sampled separately, and sEVs were prepared from each umbilical cord tissue sample according to the method of Comparative Example 1 in Example 2. The sEVs prepared by the two methods were identified by proteomics detection (sent to Hangzhou Jingjie Company for testing). The protein detection results of the three replicates in ① were recorded as HM3Dexo1, HM3Dexo2 and HM3Dexo3, respectively, and the protein detection results of the four sEVs in ② were taken and recorded as Single Hexo.

[0112] Protein test results such as Figure 12As shown in the figure, the total number of proteins in Single Hexo is 2542. The number of proteins identified in HM3Dexo1, HM3Dexo2, and HM3Dexo3 is 4635, 4659, and 4625, respectively. Of these, 2247 proteins intersect with HM3Dexo1, accounting for 88.39% of the total number of proteins in Single Hexo; 2249 proteins intersect with HM3Dexo2, accounting for 88.47% of the total number of proteins in Single Hexo; and 2258 proteins intersect with HM3Dexo3, accounting for 88.82% of the total number of proteins in Single Hexo.

[0113] The above results show that the protein content of sEVs obtained by mixed culture of umbilical cord mesenchymal stem cells in Example 1 is higher, which can highly cover the protein group obtained by culturing umbilical cord mesenchymal stem cells alone to obtain sEVs.

[0114] Example 5: Detection of the ability of sEVs to regulate macrophage polarization

[0115] LPS (500 ng / mL) and IFN-γ (20 ng / mL) were added to M0 macrophages (CD86 positive rate 27.2%) and stimulated for 24 hours, which successfully induced them into pro-inflammatory M1 macrophages (CD86 positive rate 68.7%). Different concentrations of sEVs were added to the M1 macrophages that had been induced into pro-inflammatory type in each experimental group (mixed foreskin group concentration: 10 8 / mL; single foreskin group concentration: 10 8 / mL; Concentration of mixed umbilical cord group: 10 8 / mL; single umbilical cord group concentration: 10 8 / mL) for 24 hours, and a control group (M0 macrophages without any treatment) and an M1 activation group (M1 macrophages treated only with LPS and IFN-γ) were established. Among them, according to the different experimental groups added with sEVs, they can be divided into a mixed foreskin group (i.e., sEVs obtained from a replicated sEV in ① of Example 3), a single foreskin group (i.e., sEVs obtained from a foreskin in ② of Example 3), a mixed umbilical cord group (i.e., sEVs obtained from a replicated sEV in ① of Example 4), and a single umbilical cord group (i.e., sEVs obtained from a umbilical cord in ② of Example 4). The positive rate of M1 (PE-CD86) was detected by flow cytometry.

[0116] The results are as follows Figure 13As shown, mesenchymal stem cell sEVs from different experimental groups can regulate macrophage polarization. However, regardless of foreskin or umbilical cord, the CD86 positivity rate of sEVs obtained by mixed culture using the method of Example 1 (38.7% in the mixed foreskin group and 48.3% in the mixed umbilical cord group) is lower than the CD86 positivity rate of sEVs obtained by culture according to the method of Comparative Example 1 (45.6% in the single foreskin group and 52.4% in the single umbilical cord group). In other words, the M1 cell inhibition rate of sEVs obtained by mixed culture using the method of Example 1 is higher than that of sEVs obtained by culture according to the method of Comparative Example 1. The above results indicate that mesenchymal stem cell sEVs obtained by the method of Example 1 are more effective in regulating macrophage polarization.

[0117] Example 6: Detection of the repair ability of sEVs on lipopolysaccharide-damaged alveolar epithelial cells

[0118] A549 cells were seeded in 6-well plates and replaced with serum-free medium when the cells grew to 80-90% confluence. Lipopolysaccharide (LPS, 500 μg / mL) was added to the culture medium and the cells were cultured for 24 hours to induce cell inflammatory damage. After the induction, sEVs from four different experimental groups as in Example 9 were added to the LPS-treated A549 cells and cultured for 24 hours. A control group (untreated cells) and an LPS damage group (only LPS-treated cells) were set up at the same time. Cell viability and apoptosis of the cells were detected by cell viability assay (CCK-8 method) and cell apoptosis analysis (flow cytometry).

[0119] The results are as follows Figure 14 As shown in the figure, after 24 hours of treatment with 500 μg / mL LPS, cell viability decreased significantly. Compared with the LPS-injured group, the viability of the mesenchymal stem cell sEVs in the four LPS+ groups increased and the apoptosis rate decreased. Whether for foreskin or umbilical cord tissue, the sEVs obtained using the preparation method of Example 1 had a stronger ability to repair LPS-damaged cells than the sEVs obtained using the method provided in Comparative Example 1. These results indicate that the sEVs obtained using Example 1 are more effective in reversing inflammatory damage to alveolar epithelial cells.

[0120] Example 7: Detection of sEVs' ability to promote angiogenesis

[0121] HUVEC cells (3×10 per well) 4 HUVECs were seeded onto Matrigel plates (Matrigel 356234, BD Biosciences) and cultured at 37°C under 5% CO2. Angiogenesis was observed over 24 hours using an inverted microscope. The networks formed by HUVECs were quantified using Image J software.

[0122] The results are as follows Figure 15 As shown in Figure 2, the mesenchymal stem cell sEVs from different experimental groups can promote angiogenesis. The angiogenesis-promoting ability of the mesenchymal stem cell sEVs prepared according to the preparation method provided in Example 1 is significantly higher than that of the mesenchymal stem cell sEVs obtained according to the method of Comparative Example 1. The quantitative results are shown in Figure 2. Figure 16 As shown in the results, the number of tubes in cells treated with single sEVs and mixed sEVs was significantly increased compared with the control samples. In particular, mixed sEVs induced the formation of more tube-like structures compared with single sEVs (P < 0.001).

[0123] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.

Claims

1. A method for preparing extracellular vesicles, characterized in that: The following steps are involved: S100: Tissues from different sources are mixed evenly with microcarriers and statically cultured in separate areas for 10 to 14 days; S200: The culture products from different regions of S100 are mixed for 3D dynamic culture, the supernatant is collected, and the extracellular vesicles are separated from the supernatant.

2. The preparation method according to claim 1, characterized in that S200 includes: S210: The culture products from different regions of S100 were mixed and dynamically cultured in 3D for 2 to 6 days to obtain mixed cultured P0 mesenchymal stem cells; S220: subculturing the P0 mesenchymal stem cells without digestion, collecting the supernatant every 3 to 5 days, and separating the extracellular vesicles from the supernatant.

3. The preparation method according to claim 2, characterized in that The subculture is performed once every 5-15 days without digestion.

4. The preparation method according to claim 1, characterized in that The separate static culture is to separate different areas in the same culture container for separate static culture.

5. The preparation method according to claim 1, characterized in that The dynamic culture method includes at least one of stirring, perfusion, and rotation.

6. The preparation method according to claim 1, characterized in that The tissue includes at least one of bone marrow, periosteum, synovium, skeletal muscle, dental pulp, umbilical cord, umbilical cord blood, placenta, peripheral blood, fat, and foreskin.

7. The preparation method according to claim 1, characterized in that The static culture and the dynamic culture are cultured in serum-free medium.

8. The preparation method according to claim 7, characterized in that The serum-free culture medium includes a basal culture medium and additives, wherein the basal culture medium includes any one of DMEM culture medium, RPMI 1640 culture medium, IMDM culture medium, and MEM culture medium, and the additives include at least one of essential amino acids, non-essential amino acids, vitamins, hormones, growth factors, and minerals.

9. The preparation method according to claim 1, characterized in that The tissues were pre-cleaned of immunogenic components before static culture.

10. The preparation method according to claim 1, characterized in that The method of separating the extracellular vesicles from the supernatant comprises transferring the supernatant into a hollow fiber column for concentration, and then separating the extracellular vesicles by column chromatography.

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