Active mitochondria-containing extracellular vesicles and methods of making same

Abstract

The application belongs to the technical field of cell culture, and particularly relates to a kind of extracellular vesicles containing active mitochondria and a preparation method thereof.The preparation method specifically comprises the following steps: culturing mitochondrial donor cells until the fusion degree reaches 70-80%, discarding the original culture medium, adding DMEM / F12 culture medium containing exosome-free fetal bovine serum, verbascoside and lupeol for continuous culture; collecting cell culture supernatant, and after multiple centrifugation and microporous filter filtration, centrifuging to collect the precipitate, thereby obtaining extracellular vesicles rich in active mitochondria.The extracellular vesicles obtained by the preparation method of the application can significantly promote the proliferation of UVB-induced photoaging human skin fibroblasts, inhibit the expression of senescence-associated proteins p16 and p21, have the effects of repairing photoaging damage and reversing cell senescence, and have a broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to an extracellular vesicle containing active mitochondria and its preparation method. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale membrane structures actively secreted by cells. They possess natural biocompatibility, low immunogenicity, and cross-tissue delivery capabilities, serving as natural delivery carriers for endogenous bioactive substances. Recent studies have confirmed that cells can achieve intercellular mitochondrial transfer and mitochondrial quality regulation by secreting EVs carrying intact mitochondria or functional mitochondrial components. Furthermore, these vesicles can protect mitochondrial structural stability through membrane encapsulation, effectively maintaining their respiratory activity and membrane potential, significantly improving mitochondrial survival in the extracellular environment and target cell internalization efficiency, providing a safer and more efficient new pathway for mitochondrial function repair and related disease treatment.

[0003] However, current technologies for preparing mitochondrial-containing extracellular vesicles still face numerous technical challenges, primarily the following bottlenecks: Under physiological conditions, the release efficiency of spontaneously secreted mitochondrial vesicles is low, and the mitochondrial activity carried in these vesicles is low, making it difficult to meet the needs of basic research and clinical translation. Conventional induction methods easily induce stress responses or even apoptosis in donor cells, resulting in a significant decrease in the purity of vesicles due to the presence of large amounts of cell debris, apoptotic bodies, and protein polymer products, affecting the safety of subsequent applications. Furthermore, the preparation process lacks standardization; there are no unified specifications for the entire process from induction to separation and purification, making it difficult to maintain the integrity of mitochondrial structure and function while achieving efficient induction, thus restricting the large-scale preparation and precise application of mitochondrial-containing vesicles.

[0004] Therefore, developing a mild and efficient method to induce the generation of functional mitochondrial vesicles, with standardized operation, high purity, and good reproducibility, has become a breakthrough in overcoming the dual bottlenecks of mitochondrial delivery and vesicle production. This method holds significant theoretical value and application prospects for promoting mitochondrial targeted therapy and cell-free regenerative medicine. Based on the above objectives, this invention provides an extracellular vesicle containing active mitochondria and its preparation method. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the primary objective of this invention is to provide a method for preparing extracellular vesicles containing active mitochondria.

[0006] A second objective of this invention is to provide an extracellular vesicle containing active mitochondria.

[0007] The objective of this invention is achieved through the following technical solution: A method for preparing extracellular vesicles containing active mitochondria, the method specifically comprising the following steps: (1) The mitochondrial donor cells were cultured in DMEM / F12 complete medium; (2) When the mitochondrial donor cells from step (1) reach a cell confluence of 70-80%, discard the original culture medium, wash twice with PBS, and add conditioned medium to continue culturing; the conditioned medium is DMEM / F12 medium containing exosome-free fetal bovine serum, verbascoside, and lupeol. (3) After completing step (2), collect the cell culture supernatant and centrifuge at low speed to collect the supernatant; (4) After completing step (3), collect the supernatant by medium-speed centrifugation; (5) After completing step (4), the supernatant is collected by high-speed centrifugation and the filtrate is obtained by filtration using a microporous membrane. (6) After completing step (5), the precipitate is collected by ultracentrifugation to obtain extracellular vesicles rich in active mitochondria.

[0008] Further, in step (1), the mitochondrial donor cells are umbilical cord mesenchymal stem cells; the DMEM / F12 complete culture medium is a DMEM / F12 complete culture medium containing 10% FBS, 100U / mL penicillin, and 100μg / mL streptomycin.

[0009] Further, the culture time in step (2) is 45-52h; the culture conditions are 37℃ and 5% CO2; the conditioned medium contains 1-5% (v / v) exosome-free fetal bovine serum; 10-20μM verbascoside; and 5-10μM lupeol.

[0010] Furthermore, the conditions for low-speed centrifugation in step (3) are 4℃, 200-400g for 4-6 minutes.

[0011] Furthermore, the conditions for medium-speed centrifugation in step (4) are 4℃, 1000-3000g for 8-12 minutes.

[0012] Furthermore, the conditions for high-speed centrifugation in step (5) are 4°C, 9000-11000g for 20-40 minutes; the microporous filter membrane is a 100-500nm microporous filter membrane.

[0013] Furthermore, the conditions for ultracentrifugation in step (6) are 4°C, 90,000-110,000g for 50-70 minutes.

[0014] An extracellular vesicle containing active mitochondria was prepared according to the above-described method for preparing extracellular vesicles containing active mitochondria.

[0015] Compared with the prior art, the main advantages of the present invention are as follows: This invention provides a method for preparing extracellular vesicles containing active mitochondria. Mitochondrial donor cells are cultured in a conditioned medium containing verbascoside and lupeol, where the two work synergistically. Verascoside activates the ERK1 / 2 signaling pathway to enhance mitochondrial division, while lupeol increases cytoplasmic free calcium. 2+ Concentration triggers vesicle fusion with the plasma membrane, thereby efficiently promoting the generation of extracellular vesicles rich in mitochondria. This method also achieves efficient induction under low serum concentration conditions, effectively reducing production costs. Experimental results show that the extracellular vesicles containing active mitochondria prepared in this invention can significantly promote the proliferation of UVB-induced photoaged human skin fibroblasts and effectively inhibit the expression of aging-related proteins p16 and p21. They possess the efficacy of repairing photoaging damage and reversing cellular aging, and can be used to prepare subcutaneous injection formulations for the face. They have broad market application prospects in the anti-aging and cosmetic fields such as wrinkle removal, skin tightening, improving skin elasticity, and inhibiting skin aging. Attached Figure Description

[0016] Figure 1 These are P3 generation umbilical cord mesenchymal stem cells; Figure 2 Morphology of extracellular vesicles under a transmission electron microscope; Figure 3 The expression results of extracellular vesicle marker proteins CD63 and ALIX; Figure 4 The relative ATP content of extracellular vesicles; Figure 5 Percentage of functional mitochondria in extracellular vesicles; Figure 6 The results are for CCK-8 cell viability assay. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0018] Example 1 Preparation of an extracellular vesicle containing active mitochondria.

[0019] S1: Preparation of umbilical cord mesenchymal stem cells: Umbilical cord tissue was washed with PBS, Wharton jelly was peeled off, and the jelly was cut into small pieces. The pieces were washed three times with physiological saline. The tissue pieces were then placed in DMEM medium supplemented with 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) and cultured in a 37°C, 5% CO2 cell culture incubator, with half the medium changed every 2 days. When umbilical cord mesenchymal stem cells were observed around the tissue pieces, the medium was changed again, and this was designated as generation P0. When cell confluence reached 70%, the umbilical cord tissue pieces and culture medium were discarded. The tissue was digested with 0.25% trypsin solution, centrifuged to remove the digestive fluid, and resuspended in DMEM medium for passage culture to obtain generation P3 umbilical cord mesenchymal stem cells. The morphology of the obtained generation P3 umbilical cord mesenchymal stem cells was observed under a microscope, and the results are as follows: Figure 1 As shown, from Figure 1 As can be seen, the cells are elongated spindle-shaped and uniform in size, consistent with the typical characteristics of umbilical cord mesenchymal stem cells. Therefore, this invention has successfully obtained umbilical cord mesenchymal stem cells.

[0020] S2: A method for preparing extracellular vesicles containing active mitochondria, the preparation method specifically including the following steps: (1) The P3 generation umbilical cord mesenchymal stem cells prepared by S1 were used at 5×10 3 The cells were seeded at a density of 10 cells / well in 6-well plates and cultured in DMEM / F12 complete medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0021] (2) When the umbilical cord mesenchymal stem cells from step (1) reach 80% cell fusion, discard the original culture medium, wash twice with PBS, and add conditioned culture medium and continue to culture for 48h at 37℃ and 5% CO2. The conditioned culture medium is DMEM / F12 medium containing 3 (v / v)% exosome-free fetal bovine serum, 15 μM verbascoside, and 7 μM lupeol.

[0022] (3) After completing step (2), collect the cell culture supernatant and centrifuge at 4℃ and 300g for 5 minutes to collect the supernatant; (4) After completing step (3), centrifuge at 4℃ and 2000g for 10 min to collect the supernatant; (5) After completing step (4), the supernatant was collected by centrifugation at 4℃ and 10000g for 30 min, and the filtrate was obtained by filtration using a 220nm microporous membrane. (6) After completing step (5), the precipitate was collected by centrifugation at 4℃ and 100000g for 60 min to obtain extracellular vesicles rich in active mitochondria.

[0023] An extracellular vesicle containing active mitochondria was prepared according to the above-described method for preparing extracellular vesicles containing active mitochondria.

[0024] S3: Identification of an extracellular vesicle containing active mitochondria, specifically including the following steps: The extracellular vesicle precipitate obtained in step S2 was resuspended in an appropriate amount of PBS. 10 μL of the extracellular vesicle suspension was added dropwise onto a copper grid covered with a Formvar membrane. The grid was allowed to stand at room temperature for 10 min to absorb the adsorption. Excess liquid was blotted from the edge with filter paper, and 2.5% glutaraldehyde was added for fixation at room temperature for 5 min. The copper grid was then gently washed three times with PBS, 1 min each time. A 1% uranium acetate solution was added for negative staining at room temperature for 5 min. The staining solution was blotted off with filter paper, and the grid was air-dried at room temperature. The morphology of the extracellular vesicles was observed under a transmission electron microscope. The results are as follows: Figure 2 As shown. From Figure 2 It can be seen that the prepared samples contain typical cup-shaped or spherical vesicle structures with complete double membrane outlines, which are consistent with the typical characteristics of extracellular vesicles.

[0025] Example 2 The method for preparing umbilical cord mesenchymal stem cells in this invention is the same as in Example 1.

[0026] A method for preparing extracellular vesicles containing active mitochondria, the method specifically comprising the following steps: (1) The P3 generation umbilical cord mesenchymal stem cells prepared by S1 were used at 5×10 3 The cells were seeded at a density of 10 cells / well in 6-well plates and cultured in DMEM / F12 complete medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0027] (2) When the umbilical cord mesenchymal stem cells from step (1) reach 80% cell fusion, discard the original culture medium, wash twice with PBS, and add conditioned culture medium to continue culturing at 37°C and 5% CO2 for 45h; the conditioned culture medium is DMEM / F12 medium containing 1% (v / v) exosome-free fetal bovine serum, 10 μM verbascoside, and 5 μM lupeol.

[0028] (3) After completing step (2), collect the cell culture supernatant and centrifuge at 4℃ and 200g for 4 min to collect the supernatant; (4) After completing step (3), centrifuge at 4℃ and 1000g for 8 minutes to collect the supernatant; (5) After completing step (4), centrifuge at 4℃ and 9000g for 20min to collect the supernatant, and filter it with a 220nm microporous membrane to obtain the filtrate; (6) After completing step (5), the precipitate was collected by centrifugation at 4℃ and 90000g for 50 minutes to obtain extracellular vesicles rich in active mitochondria.

[0029] An extracellular vesicle containing active mitochondria was prepared according to the above-described method for preparing extracellular vesicles containing active mitochondria.

[0030] Example 3 The method for preparing umbilical cord mesenchymal stem cells in this invention is the same as in Example 1.

[0031] A method for preparing extracellular vesicles containing active mitochondria, the method specifically comprising the following steps: (1) The P3 generation umbilical cord mesenchymal stem cells prepared by S1 were used at 5×10 3 The cells were seeded at a density of 10 cells / well in 6-well plates and cultured in DMEM / F12 complete medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0032] (2) When the umbilical cord mesenchymal stem cells from step (1) reach 80% cell fusion, discard the original culture medium, wash twice with PBS, and add conditioned culture medium to continue culturing at 37°C and 5% CO2 for 52 hours; the conditioned culture medium is DMEM / F12 medium containing 5% (v / v) exosome-free fetal bovine serum, 20 μM verbascoside, and 10 μM lupeol.

[0033] (3) After completing step (2), collect the cell culture supernatant and centrifuge at 400g for 6 minutes at 4℃ to collect the supernatant; (4) After completing step (3), collect the supernatant by centrifugation at 4℃ and 3000g for 12 min; (5) After completing step (4), the supernatant was collected by centrifugation at 4℃ and 11000g for 40 min, and the filtrate was obtained by filtration using a 220nm microporous membrane. (6) After completing step (5), the precipitate was collected by centrifugation at 4℃ and 110000g for 70min to obtain extracellular vesicles rich in active mitochondria.

[0034] An extracellular vesicle containing active mitochondria was prepared according to the above-described method for preparing extracellular vesicles containing active mitochondria.

[0035] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that verbascoside in the conditioned medium of step (2) is omitted, while the rest are the same as in Example 1.

[0036] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that lupeol in the conditioned medium component of step (2) is omitted, while all other components are the same as in Example 1.

[0037] Experimental Example 1 This study used Western blotting to detect the expression of extracellular vesicle marker proteins CD63 and ALIX, and identified extracellular vesicles. The specific experimental steps are as follows: Extracellular vesicle pellets prepared in Examples 1-3, Comparative Examples 1 and 2 were added with RIPA lysis buffer containing protease inhibitors. Total protein was extracted, and the protein concentration was determined by BCA method to ensure consistency. Simultaneously, an equal volume of untreated umbilical cord mesenchymal stem cells was collected to extract total protein as a control group. Protein samples were separated by 12% SDS-PAGE gel electrophoresis, wet-transferred to PVDF membranes, blocked with 5% skim milk powder, and incubated with primary antibodies: CD63 and ALIX, respectively, overnight at 4°C. After washing with TBST, incubation with HRP-labeled secondary antibody was performed, and finally, imaging was achieved with ECL chemiluminescence buffer. Results are shown below. Figure 3 As shown.

[0038] The results are as follows Figure 3 The image shows the expression results of extracellular vesicle marker proteins CD63 and ALIX. Figure 3 It can be seen that, compared with the control group, the protein extracts of Examples 1-3, Comparative Example 1, and Comparative Example 2 of this invention all expressed CD63 and ALIX extracellular vesicle marker proteins. This indicates that the preparation methods of Examples 1-3, Comparative Example 1, and Comparative Example 2 successfully obtained extracellular vesicles from the culture supernatant of umbilical cord mesenchymal stem cells, laying the foundation for subsequent functional comparisons.

[0039] Experimental Example 2 Functional identification of extracellular vesicles rich in active mitochondria To evaluate the function of the extracellular vesicles rich in active mitochondria prepared in Examples 1-3, Comparative Example 1, and Comparative Example 2, this study comprehensively assessed the function by detecting two indicators: ATP content and the positive rate of active mitochondria in the extracellular vesicles. The specific detection methods are as follows: The ATP concentration of extracellular vesicles prepared in Examples 1-3, Comparative Examples 1 and 2 was detected using an ATP assay kit. Total vesicle protein was normalized using the BCA method and expressed as relative ATP content. Results are shown below. Figure 4 As shown in the figure. Secondly, the proportion of functional mitochondrial positive vesicles with intact membrane potential was detected by flow cytometry using the mitochondrial membrane potential-dependent dye Mitotracker Deep-Red labeling method. The results are expressed as a percentage of functional mitochondria, as shown in the figure. Figure 5 As shown.

[0040] The results are as follows Figure 4The figure shows the relative ATP content of extracellular vesicles. Compared with Comparative Example 1, the ATP content in the extracellular vesicles prepared in Examples 1-3 was significantly increased, indicating that the extracellular vesicles prepared in Example 1 of this invention carry mitochondria with stronger energy synthesis activity.

[0041] The results are as follows Figure 5 The figure shows the percentage of functional mitochondria in extracellular vesicles. Compared with Comparative Example 1, the percentage of functional mitochondria in the extracellular vesicles prepared in Examples 1-3 of the present invention is significantly increased, indicating that the extracellular vesicles prepared in Example 1 of the present invention have a high proportion of active mitochondria with intact membrane potential.

[0042] The above results indicate that the extracellular vesicles prepared in Example 1 of this invention contain a higher proportion of functional mitochondria and have stronger ATP synthesis capabilities, confirming that the preparation method of this invention can effectively enrich extracellular vesicles containing highly active mitochondria. It is speculated that this may be due to the synergistic effect of verbascoside and lupeol in the conditioned medium, where verbascoside can activate the ERK1 / 2 signaling pathway to enhance mitochondrial division, and lupeol can increase cytoplasmic free calcium. 2+ Concentration triggers vesicle fusion with the plasma membrane, thereby efficiently promoting the generation of extracellular vesicles rich in mitochondria.

[0043] Experimental Example 3 Evaluation of the anti-aging effects of cells rich in active mitochondrial extracellular vesicles To evaluate the anti-aging efficacy of the active mitochondrial extracellular vesicles prepared in Examples 1-3, Comparative Examples 1 and 2, a UVB-induced human skin fibroblast (HSF) photoaging model was used. Cell proliferation activity was detected by the CCK-8 assay, and the expression levels of aging-related proteins p16 and p21 were detected by Western blotting. The specific experimental steps are as follows: (1) Divide the experiment into seven groups: Control group: HSF cells at 2×10 4 / cm 2 Density seeding in six-well plates, normal culture, without UVB irradiation or vesicle treatment.

[0044] Model group: HSF cells at 2×10 4 / cm 2 Density seeding was performed on six-well plates, and when cultured to 80% confluence, 100 mJ / cm² was administered. 2 A photoaging model was established using UVB irradiation.

[0045] Example 1 group: HSF cells at 2×10 4 / cm 2 Density seeding was performed on six-well plates, and when cultured to 80% confluence, 100 mJ / cm² was administered. 2Photoaging was established by UVB irradiation, and 20 μg / mL of extracellular vesicles prepared in Example 1 were added after UVB irradiation.

[0046] Example 2 group: HSF cells at 2×10 4 / cm 2 Density seeding was performed on six-well plates, and when cultured to 80% confluence, 100 mJ / cm² was administered. 2 Photoaging was established by UVB irradiation, and 20 μg / mL of extracellular vesicles prepared in Example 2 were added after UVB irradiation.

[0047] Example 3 group: HSF cells at 2×10 4 / cm 2 Density seeding was performed on six-well plates, and when cultured to 80% confluence, 100 mJ / cm² was administered. 2 Photoaging was established by UVB irradiation, and 20 μg / mL of extracellular vesicles prepared in Example 3 were added after UVB irradiation.

[0048] Comparative Example 1: HSF cells were used at a rate of 2 × 10⁻⁶ 4 / cm 2 Density seeding was performed on six-well plates, and when cultured to 80% confluence, 100 mJ / cm² was administered. 2 Photoaging was established by UVB irradiation, and extracellular vesicles prepared in Comparative Example 1 were added after UVB irradiation.

[0049] Comparative Example 2: HSF cells were used at a rate of 2 × 10⁻⁶ 4 / cm 2 Density seeding was performed on six-well plates, and when cultured to 80% confluence, 100 mJ / cm² was administered. 2 Photoaging was established by UVB irradiation, and extracellular vesicles prepared in Comparative Example 2 were added after UVB irradiation.

[0050] (2) After 48 hours of treatment, each well was incubated with CCK-8 solution for 4 hours. The absorbance was measured at 450 nm, and the relative cell viability was calculated. The results are as follows: Figure 6 As shown.

[0051] (3) After 72 hours of treatment, total protein was extracted from each group of cells. The protein expression levels of p16 and p21 were detected by Western Blot. The results are shown in Table 1.

[0052] The results are as follows Figure 6The results of CCK-8 cell viability testing are shown. Compared with the control group, the cell viability of the model group was significantly reduced, indicating that the UVB-induced photoaging model was successfully constructed. Compared with the model group, Comparative Example 1, and Comparative Example 2, the viability of human skin fibroblasts treated with extracellular vesicles in Examples 1-3 of this invention was restored, with the highest cell viability in Example 1, indicating that the extracellular vesicles prepared in Example 1 can effectively promote the proliferation of photoaged cells.

[0053] Table 1. Relative expression levels of aging-related proteins p21 and p16 in cells of each group. The results are shown in Table 1, which presents the relative expression levels of senescence-related proteins p21 and p16 in each group of cells. The expression levels of p21 and p16 proteins are closely related to cellular senescence and are often used to characterize the degree of cellular senescence. As shown in Table 1, compared with the control group, the expression levels of p16 and p21 proteins in the model group were significantly increased, confirming that photoaging induces cellular senescence. Compared with the model group, Comparative Example 1, and Comparative Example 2, the extracellular vesicles in Examples 1-3 of this invention can significantly reduce the relative expression levels of p21 and p16 proteins in human skin fibroblasts, with Example 1 showing the best effect. This indicates that the extracellular vesicles prepared in Example 1 can inhibit the expression of senescence-related proteins, thereby inhibiting the senescence of human skin fibroblasts.

[0054] The above results indicate that the extracellular vesicles containing active mitochondria prepared in Example 1 of this invention can more effectively promote the proliferation of photo-aged damaged cells and inhibit the expression of aging-related proteins, thereby inhibiting the aging of human skin fibroblasts.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A method for preparing extracellular vesicles containing active mitochondria, characterized in that, The preparation method specifically includes the following steps: (1) The mitochondrial donor cells were cultured in DMEM / F12 complete medium; (2) When the mitochondrial donor cells from step (1) reach a cell confluence of 70-80%, discard the original culture medium, wash twice with PBS, and add conditioned medium to continue culturing; the conditioned medium is DMEM / F12 medium containing exosome-free fetal bovine serum, verbascoside, and lupeol. (3) After completing step (2), collect the cell culture supernatant and centrifuge at low speed to collect the supernatant; (4) After completing step (3), collect the supernatant by medium-speed centrifugation; (5) After completing step (4), the supernatant is collected by high-speed centrifugation and the filtrate is obtained by filtration using a microporous membrane. (6) After completing step (5), the precipitate is collected by ultracentrifugation to obtain extracellular vesicles containing active mitochondria.

2. The method for preparing extracellular vesicles containing active mitochondria according to claim 1, characterized in that, In step (1), the mitochondrial donor cells are umbilical cord mesenchymal stem cells; the DMEM / F12 complete culture medium is a DMEM / F12 complete culture medium containing 10% FBS, 100U / mL penicillin, and 100μg / mL streptomycin.

3. The method for preparing extracellular vesicles containing active mitochondria according to claim 1, characterized in that, The culture time in step (2) is 45-52h; the culture conditions are 37℃ and 5% CO2; the conditioned medium contains 1-5% (v / v) exosome-free fetal bovine serum; 10-20μM verbascoside; and 5-10μM lupeol.

4. The method for preparing extracellular vesicles containing active mitochondria according to claim 1, characterized in that, The conditions for low-speed centrifugation in step (3) are 4℃, 200-400g for 4-6 minutes.

5. The method for preparing extracellular vesicles containing active mitochondria according to claim 1, characterized in that, The conditions for medium-speed centrifugation in step (4) are 4℃, 1000-3000g for 8-12 minutes.

6. The method for preparing extracellular vesicles containing active mitochondria according to claim 1, characterized in that, The conditions for high-speed centrifugation in step (5) are 4℃, 9000-11000g for 20-40min; the microporous filter membrane is a 100-500nm microporous filter membrane.

7. The method for preparing extracellular vesicles containing active mitochondria according to claim 1, characterized in that, The conditions for ultracentrifugation in step (6) are 4℃, 90000-110000g for 50-70 minutes.

8. An extracellular vesicle containing active mitochondria, characterized in that, The extracellular vesicles containing active mitochondria were prepared according to any one of claims 1-7.

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