Culture method of high-concentration composite exosome

Abstract

The invention discloses a culture method of a high-concentration composite exosome. In order to solve the problem of continuous high-yield culture of exosome culture, an exosome culture scheme is improved, and through cross passage and starvation sequential control, proliferation of umbilical cord mesenchymal stem cells and collection of exosomes can be synchronously realized in a short time, so that the survival rate of the umbilical cord mesenchymal stem cells is increased, and the survival rate of the umbilical cord mesenchymal stem cells is increased. In addition, the differential centrifugal purification process set in the subsequent steps can also remove fragments with different sizes in the supernate through multi-stage different-gradient centrifugal rates, so that the yield of the exosome is increased, and the purity of the exosome is ensured at the same time.

Description

Technical Field

[0001] This invention relates to the field of exosome technology, specifically a method for culturing high-concentration composite exosomes. Background Technology

[0002] Exosomes, tiny membrane vesicles widely found in cellular secretions, range in diameter from 30 to 150 nm. Based on a lipid bilayer, they contain various functional molecules, including cytokines, growth factors, lipids, and mRNA-encoding molecules. They effectively mediate intercellular communication and reshape the local tissue microenvironment, playing an indispensable regulatory role in many physiological and pathological processes such as immune regulation and tissue repair. However, current exosome culture protocols often suffer from low yields, high heterogeneity, and significant purification losses. Existing technologies often employ single-stage starvation culture without cross-passing, leading to asynchrony between cell proliferation and exosome secretion. Purification often relies on a single centrifugation rate, which fails to effectively remove cell debris, hindering large-scale exosome culture and preventing continuous production. Summary of the Invention

[0003] The purpose of this invention is to provide a method for culturing high-concentration complex exosomes to solve the problems raised in the prior art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for culturing high-concentration complex exosomes, comprising the following steps: S1. Cell culture; S11. Cell seeding and culture; Thawing, resuscitation, and initial culture of P3 generation umbilical cord mesenchymal stem cells; S12. Cell passage culture; After the initial culture is completed, cell growth is monitored. When the cells have reached 80-90% confluence, they are passaged to the third generation to obtain third-generation T-225 cell culture flasks. S2. Exosome isolation and purification; S21. Select 60-70% of the total number of third-generation T-225 cell culture flasks, replace them with serum-free culture medium, and starve them for 48-72 hours to obtain third-generation starved T-225 cell culture flasks. The remaining third-generation T-225 cell culture flasks were processed according to step S12 and further passaged until the number of cells was the same as the number of third-generation T-225 cell culture flasks in step S12. After culturing for 48-72 hours, fourth-generation T-225 cell culture flasks were obtained. S22. Collect the supernatant from the third-generation starved T-225 cell culture flask in step S21, centrifuge it, mix it evenly, and obtain the first batch of exosome supernatant; Select 60-70% of the fourth-generation T-225 cell culture flasks again, replace them with serum-free medium, add the first batch of exosome supernatant, and starve them for 48-72 hours to obtain the fourth-generation starved T-225 cell culture flasks. The remaining fourth-generation T-225 cell culture flasks were processed according to step S12 and further passaged until the number was the same as the number of fourth-generation starved T-225 cell culture flasks. After culturing for 48-72 hours, fifth-generation T-225 cell culture flasks were obtained. S23. Collect the supernatant from the fourth generation starved culture T-225 cell culture flask in step S22, centrifuge and mix them evenly to obtain the second batch of exosome supernatant; Replace the fifth-generation T-225 cell culture flask with serum-free medium, add the second batch of exosome supernatant, starve the cells for 48-72 hours, collect the supernatant from the cell culture flask, centrifuge to obtain high-concentration composite exosome supernatant. S3. The high-concentration complex exosome supernatant obtained in step S2 is purified by differential centrifugation to obtain high-concentration complex exosomes.

[0005] Furthermore, in step S11, the thawing, revival, and initial culture specifically include the following steps: P3 generation umbilical cord mesenchymal stem cells were taken, thawed in a constant temperature water bath at 37°C, and then poured into a 50mL centrifuge tube containing 30mL DPBS buffer. After sealing the tube, the cells were repeatedly inverted and shaken to mix, and then centrifuged. After centrifugation, the supernatant was taken for sterility testing. After the test showed no contamination, the excess supernatant in the centrifuge tube was poured out. The centrifuged cells were resuspended in the culture medium and mixed well. The mixture was then divided and seeded into two T-225 cell culture flasks. The culture medium volume was added to 40 mL. The T-225 cell culture flasks were then placed in a cell culture incubator at 37°C and 5% CO2 concentration for 48-72 h to complete the initial culture.

[0006] Furthermore, in steps S1 and S2, the specific steps for subculturing are as follows: After discarding the original culture medium in the T-225 cell culture flask, add DPBS buffer at a ratio of 8-12 mL / T-225 cell culture flask to wash the cells. Discard the DPBS buffer and add recombinant trypsin at a ratio of 4-6 mL / T-225 cell culture flask to digest the cells for 2-3 minutes. Then, add DPBS buffer at a ratio of 8-12 mL / T-225 cell culture flask to stop the digestion. Add the cell suspension to a centrifuge tube and centrifuge at 1200-1800 rpm for 4-6 minutes. Resuspend the cells in culture medium and add them to a new T-225 cell culture flask, adding culture medium to bring the volume to 40 mL / T-225 cell culture flask.

[0007] Furthermore, in steps S1 and S2, the subculture ratio is 1:2~4.

[0008] Furthermore, in step S1, during the initial culture and subculture, the culture medium formulation is serum-free α-MEN medium containing 4-6% UltraGRO-Advanced.

[0009] Furthermore, in step S2, during subculture, the culture medium formulation is serum-free α-MEN medium containing 5% UltraGRO-Advanced.

[0010] Furthermore, in step S2, during starvation culture, the serum-free culture medium is formulated as α-MEN serum-free culture medium containing 0~0.1μmol / L lacuncurin A and 0~5μmol / L Rho kinase inhibitor.

[0011] Furthermore, in step S2, when preparing the first and second batches of high-concentration complex exosome supernatant, the centrifugation separation operation is as follows: Add the supernatant to a centrifuge tube, cool to 4°C, centrifuge at 300-400g for 8-12 minutes, then centrifuge at 1800-2500g for 20 minutes. Take the supernatant from the centrifuge tube, filter it through a 100-200μm cell filter, collect the filtrate, aliquot it, and complete the centrifugation.

[0012] Furthermore, in step S3, the specific steps for differential centrifugation purification are as follows: Add the high-concentration complex exosome supernatant to a centrifuge tube, cool to 4°C, centrifuge at 300-400g for 8-12 min, centrifuge at 9000-10000g for 25-40 min, and centrifuge again at 90000-100000g for 70-90 min. Remove the centrifuge tube, aspirate the supernatant after centrifugation, and retain a 0.5 cm liquid layer to avoid exosome loss. This completes the differential centrifugation purification.

[0013] Furthermore, a high-concentration complex exosome was obtained by culturing using the above-mentioned method.

[0014] Compared with the prior art, the beneficial effects of the present invention are: To address the challenge of continuous, high-yield exosome culture, this invention improves the exosome culture protocol. Through crossover passage and controlled starvation, umbilical cord mesenchymal stem cells can simultaneously proliferate and collect exosomes within a short timeframe, ultimately producing composite exosomes containing multiple generations of cytokines and growth factors. Furthermore, to further enhance exosome yield, this invention adds leucovorin A and Rho kinase inhibitors, which act as cytoskeleton regulators, during starvation culture. These substances reduce the rigidity and stability of the cytoskeleton. Leucovorin A acts on actin microfilaments, thereby disrupting the structural stability of the cytoskeleton, reducing the difficulty of exosome secretion and release, and weakening the physical barriers during exosome release and migration. Rho... Kinase inhibitors can inhibit the contractile function of the cytoskeleton, thereby further reducing the resistance to exosome release. However, while the addition of lacuncurin A and Rho kinase inhibitors can increase exosome secretion, it also leads to a decrease in cell strength, resulting in an increase in the content of broken cells in the culture flask supernatant. The differential centrifugation purification process set in the subsequent steps of this invention can perfectly solve this defect. By using multiple centrifugation rates of different gradients, fragments of different sizes in the supernatant are removed, thereby increasing the exosome yield while ensuring the purity of the exosomes. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a particle size-average concentration graph of exosome concentration and particle size NTA detection in Example 1 of the present invention; Figure 2 This is a particle size-intensity scatter plot of exosome concentration particle size NTA detection in Example 1 of the present invention; Figure 3 This is a comparison chart of Western marker detection in Embodiment 1 of the present invention; Figure 4 This is an electron micrograph of exosomes under negative staining as described in Example 1 of the present invention; Figure 5 These are the results of quantitative detection of exosome BCA protein in Examples 1-2 and Comparative Examples 1-2 of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1. A method for culturing high-concentration complex exosomes, comprising the following steps: S1. Cell culture; S11. Cell seeding and culture; P3 generation umbilical cord mesenchymal stem cells were taken, thawed in a constant temperature water bath at 37°C, and then poured into a 50mL centrifuge tube containing 30mL DPBS buffer. After sealing the tube, the cells were repeatedly inverted and shaken to mix, and then centrifuged. After centrifugation, the supernatant was taken for sterility testing. After the test showed no contamination, the excess supernatant in the centrifuge tube was poured out. The centrifuged cells were resuspended in the culture medium and mixed well. The cells were then divided and seeded into two T-225 cell culture flasks. The culture medium volume was added to 40 mL. The T-225 cell culture flasks were then placed in a cell culture incubator at 37°C and 5% CO2 concentration for 72 h to complete the initial culture. S12. Cell passage culture; After the initial culture is completed, cell growth is monitored. When the cells have reached 80-90% confluence, they are passaged to the third generation to obtain third-generation T-225 cell culture flasks. During subculturing, the original culture medium in the two T-225 cell culture flasks was discarded. DPBS buffer was added at a ratio of 10 mL / T-225 cell culture flask to wash the cells. The DPBS buffer was then discarded. Recombinant trypsin was added at a ratio of 5 mL / T-225 cell culture flask to digest the cells for 3 min. DPBS buffer was then added at a ratio of 10 mL / T-225 cell culture flask to stop the digestion. The cell suspension was then added to a centrifuge tube and centrifuged at 1500 rpm for 5 min. The cells were then resuspended in culture medium and added to four new T-225 cell culture flasks. Culture medium was added to bring the volume to 40 mL / T-225 cell culture flask to complete the subculturing. After subculturing, the cells were cultured for 72 h and then subcultured again to obtain the first generation of T-225 cell culture flasks. After culturing for 72 hours, the above steps were repeated to passage 4 first-generation T-225 cell culture flasks into 12 second-generation T-225 cell culture flasks; and the above steps were repeated to passage 12 second-generation T-225 cell culture flasks into 36 third-generation T-225 cell culture flasks. The culture medium is α-MEN serum-free culture medium containing 5% UltraGRO-Advanced; S2. Exosome isolation and purification; S21. Select 25 third-generation T-225 cell culture flasks, replace them with serum-free culture medium, and starve them for 72 hours to obtain third-generation starved T-225 cell culture flasks; The remaining 11 third-generation T-225 cell culture flasks were processed according to step S12 and further passaged into 36 T-225 cell culture flasks. After culturing for another 72 hours, 36 fourth-generation T-225 cell culture flasks were obtained. S22. Collect the supernatant from 25 third-generation starved T-225 cell culture flasks in step S21, centrifuge them, mix them evenly, and obtain the first batch of exosome supernatant. Another 25 fourth-generation T-225 cell culture flasks were selected, replaced with serum-free culture medium, and the first batch of exosome supernatant was added to them. They were starved cultured for 72 hours to obtain fourth-generation starved culture T-225 cell culture flasks. The remaining 11 fourth-generation T-225 cell culture flasks were processed according to step S12 and further passaged to 25 flasks. After culturing for another 72 hours, fifth-generation T-225 cell culture flasks were obtained. S23. Collect the supernatant from the fourth generation starved culture T-225 cell culture flask in step S22, centrifuge and mix them evenly to obtain the second batch of exosome supernatant; The fifth-generation T-225 cell culture flask was replaced with serum-free medium, and the second batch of exosome supernatant was added. After starvation culture for 72 hours, the supernatant in the cell culture flask was collected, centrifuged, and a high-concentration composite exosome supernatant was obtained. The specific procedures for centrifuging the supernatant are as follows: Add the supernatant to a centrifuge tube, cool to 4°C, centrifuge at 300g for 10 min, then centrifuge at 2000g for 20 min. Take the supernatant from the centrifuge tube, pass it through a 100μm cell filter, collect the filtrate, collect it, dispense it, and complete the centrifugation. The serum-free culture medium is α-MEN serum-free culture medium; S3. The high-concentration complex exosome supernatant obtained in step S2 is purified by differential centrifugation. The high-concentration complex exosome supernatant is added to a centrifuge tube, cooled to 4°C, and centrifuged at 300g for 10 min. The precipitate is discarded, and the remaining supernatant is centrifuged at 10000g for 30 min, and then centrifuged again at 100000g for 70 min. The centrifuge tube is removed, the supernatant after centrifugation is aspirated, and a 0.5 cm liquid layer is retained to avoid exosome loss. Differential centrifugation purification is completed to obtain high-concentration complex exosomes.

[0018] Example 2. A method for culturing high-concentration complex exosomes, comprising the following steps: Compared with Example 1, this example increases the content of lacucolin A and Rho kinase inhibitor in serum-free culture medium, while the other steps remain unchanged; S2. Exosome isolation and purification; S21. Select 25 third-generation T-225 cell culture flasks, replace them with serum-free culture medium, and starve them for 72 hours to obtain third-generation starved T-225 cell culture flasks; The remaining 11 third-generation T-225 cell culture flasks were processed according to step S12 and further passaged into 36 T-225 cell culture flasks. After culturing for another 72 hours, 36 fourth-generation T-225 cell culture flasks were obtained. S22. Collect the supernatant from 25 third-generation starved T-225 cell culture flasks in step S21, centrifuge them, mix them evenly, and obtain the first batch of exosome supernatant. Another 25 fourth-generation T-225 cell culture flasks were selected, replaced with serum-free culture medium, and the first batch of exosome supernatant was added to them. They were starved cultured for 72 hours to obtain fourth-generation starved culture T-225 cell culture flasks. The remaining 11 fourth-generation T-225 cell culture flasks were processed according to step S12 and further passaged to 25 flasks. After culturing for another 72 hours, fifth-generation T-225 cell culture flasks were obtained. S23. Collect the supernatant from the fourth generation starved culture T-225 cell culture flask in step S22, centrifuge and mix them evenly to obtain the second batch of exosome supernatant; The fifth-generation T-225 cell culture flask was replaced with serum-free medium, and the second batch of exosome supernatant was added. After starvation culture for 72 hours, the supernatant in the cell culture flask was collected, centrifuged, and a high-concentration composite exosome supernatant was obtained. The specific procedures for centrifuging the supernatant are as follows: Add the supernatant to a centrifuge tube, cool to 4°C, centrifuge at 300g for 10 min, then centrifuge at 2000g for 20 min. Take the supernatant from the centrifuge tube, pass it through a 100μm cell filter, collect the filtrate, collect it, dispense it, and complete the centrifugation. The serum-free culture medium is α-MEN serum-free culture medium containing 0.1 μmol / L lacuncurin A and 5 μmol / L Rho kinase inhibitor.

[0019] Comparative Example 1. A method for culturing high-concentration complex exosomes, comprising the following steps: Compared with Example 1, this comparative example did not use differential centrifugation for purification, but only performed centrifugation at 100,000g for 60 minutes, with the remaining steps unchanged; S3. The high-concentration complex exosome supernatant obtained in step S2 is purified by differential centrifugation. The high-concentration complex exosome supernatant is added to a centrifuge tube, cooled to 4°C, and centrifuged at 300g for 10 min. The precipitate is discarded, and the remaining supernatant is centrifuged at 10000g for 30 min, and then centrifuged again at 100000g for 70 min. The centrifuge tube is removed, the supernatant after centrifugation is aspirated, and a 0.5 cm liquid layer is retained to avoid exosome loss. Differential centrifugation purification is completed to obtain high-concentration complex exosomes.

[0020] Comparative Example 2. A method for culturing high-concentration complex exosomes, comprising the following steps: Compared with Example 1, this comparative example did not undergo multiple passages of starvation culture in step S2; S2. Exosome isolation and purification; S21. Select 36 third-generation T-225 cell culture flasks, replace them with serum-free culture medium, starve them for 72 hours to obtain third-generation starved T-225 cell culture flasks, collect the supernatant in the cell culture flasks, centrifuge to obtain high-concentration complex exosome supernatant. The specific procedures for centrifuging the supernatant are as follows: Add the supernatant to a centrifuge tube, cool to 4°C, centrifuge at 300g for 10 min, then centrifuge at 2000g for 20 min. Take the supernatant from the centrifuge tube, pass it through a 100μm cell filter, collect the filtrate, collect it, dispense it, and complete the centrifugation. The serum-free culture medium is α-MEN serum-free culture medium; S3. The high-concentration complex exosome supernatant obtained in step S2 is purified by differential centrifugation. The high-concentration complex exosome supernatant is added to a centrifuge tube, cooled to 4°C, and centrifuged at 300g for 10 min. The precipitate is discarded, and the remaining supernatant is centrifuged at 10000g for 30 min, and then centrifuged again at 100000g for 70 min. The centrifuge tube is removed, the supernatant after centrifugation is aspirated, and a 0.5 cm liquid layer is retained to avoid exosome loss. Differential centrifugation purification is completed to obtain high-concentration complex exosomes.

[0021] Detection: The NTA concentration of exosomes in Example 1 and the comparative example was measured. The results are shown in [Figure Number]. Figure 1 , Figure 2 ; Western blot analysis was performed on the high-concentration composite exosomes prepared in Example 1 to detect biomarkers. The results are shown in [Figure 1]. Figure 3 ; The high-concentration composite exosomes prepared in Example 1 were examined using negative staining electron microscopy. The results were... Figure 4 ; The high-concentration composite exosomes prepared in Examples 1-2 and Comparative Examples 1-2 were subjected to quantitative detection of exosomal BCA protein, and their protein content was determined. The results are shown in [Figure 1]. Figure 5 ; Exosome recovery rates were detected for Examples 1-2 and Comparative Examples 1-2, respectively. The exosome recovery rates were obtained by nanoparticle tracking analysis combined with BCA protein quantification. The results are shown in the table below. It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0022] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for culturing high-concentration complex exosomes, characterized in that, Includes the following steps: S1. Cell culture; S11. Cell seeding and culture; Thawing, resuscitation, and initial culture of P3 generation umbilical cord mesenchymal stem cells; S12. Cell passage culture; After the initial culture is completed, cell growth is monitored. When the cells have reached 80-90% confluence, they are passaged to the third generation to obtain third-generation T-225 cell culture flasks. S2. Exosome isolation and purification; S21. Select 60-70% of the total number of third-generation T-225 cell culture flasks, replace them with serum-free culture medium, and starve them for 48-72 hours to obtain third-generation starved T-225 cell culture flasks. The remaining third-generation T-225 cell culture flasks were processed according to step S12 and further passaged until the number of cells was the same as the number of third-generation T-225 cell culture flasks in step S12. After culturing for 48-72 hours, fourth-generation T-225 cell culture flasks were obtained. S22. Collect the supernatant from the third-generation starved T-225 cell culture flask in step S21, centrifuge it, mix it evenly, and obtain the first batch of exosome supernatant; Select 60-70% of the fourth-generation T-225 cell culture flasks again, replace them with serum-free medium, add the first batch of exosome supernatant, and starve them for 48-72 hours to obtain the fourth-generation starved T-225 cell culture flasks. The remaining fourth-generation T-225 cell culture flasks were processed according to step S12 and further passaged until the number was the same as the number of fourth-generation starved T-225 cell culture flasks. After culturing for 48-72 hours, fifth-generation T-225 cell culture flasks were obtained. S23. Collect the supernatant from the fourth generation starved culture T-225 cell culture flask in step S22, centrifuge and mix them evenly to obtain the second batch of exosome supernatant; Replace the fifth-generation T-225 cell culture flask with serum-free medium, add the second batch of exosome supernatant, starve the cells for 48-72 hours, collect the supernatant from the cell culture flask, centrifuge to obtain high-concentration composite exosome supernatant. S3. The high-concentration complex exosome supernatant obtained in step S2 is purified by differential centrifugation to obtain high-concentration complex exosomes.

2. The method for culturing high-concentration composite exosomes according to claim 1, characterized in that: In step S11, the thawing, recovery, and initial culture process specifically includes the following steps: P3 generation umbilical cord mesenchymal stem cells were taken, thawed in a constant temperature water bath at 37°C, and then poured into a 50mL centrifuge tube containing 30mL DPBS buffer. After sealing the tube, the cells were repeatedly inverted and shaken to mix, and then centrifuged. After centrifugation, the supernatant was taken for sterility testing. After the test showed no contamination, the excess supernatant in the centrifuge tube was poured out. The centrifuged cells were resuspended in the culture medium and mixed well. The mixture was then divided and seeded into two T-225 cell culture flasks. The culture medium volume was added to 40 mL. The T-225 cell culture flasks were then placed in a cell culture incubator at 37°C and 5% CO2 concentration for 48-72 h to complete the initial culture.

3. The method for culturing high-concentration complex exosomes according to claim 1, characterized in that: In steps S1 and S2, the specific steps for subculture are as follows: After discarding the original culture medium in the T-225 cell culture flask, add DPBS buffer at a ratio of 8-12 mL / T-225 cell culture flask to wash the cells. Discard the DPBS buffer and add recombinant trypsin at a ratio of 4-6 mL / T-225 cell culture flask to digest the cells for 2-3 minutes. Then, add DPBS buffer at a ratio of 8-12 mL / T-225 cell culture flask to stop the digestion. Add the cell suspension to a centrifuge tube and centrifuge at 1200-1800 rpm for 4-6 minutes. Resuspend the cells in culture medium and add them to a new T-225 cell culture flask, adding culture medium to bring the volume to 40 mL / T-225 cell culture flask.

4. The method for culturing high-concentration complex exosomes according to claim 1, characterized in that: In steps S1 and S2, the subculture ratio is 1:2 to 4.

5. A method for culturing high-concentration complex exosomes according to claim 1, characterized in that: In step S1, during the initial culture and subculture, the culture medium formula is serum-free α-MEN medium containing 4-6% UltraGRO-Advanced.

6. A method for culturing high-concentration complex exosomes according to claim 1, characterized in that: In step S2, during subculture, the culture medium is formulated as serum-free α-MEN medium containing 4-6% UltraGRO-Advanced.

7. A method for culturing high-concentration complex exosomes according to claim 1, characterized in that: In step S2, during starvation culture, the serum-free culture medium is formulated as α-MEN serum-free medium containing 0~0.1 μmol / L lacuncurin A and 0~5 μmol / L Rho kinase inhibitor.

8. The method for culturing high-concentration complex exosomes according to claim 1, characterized in that: In step S2, the centrifugation process for preparing the first and second batches of high-concentration complex exosome supernatant is as follows: Add the supernatant to a centrifuge tube, cool to 4°C, centrifuge at 300-400g for 8-12 minutes, then centrifuge at 1800-2500g for 20 minutes. Take the supernatant from the centrifuge tube, filter it through a 100-200μm cell filter, collect the filtrate, aliquot it, and complete the centrifugation.

9. The method for culturing high-concentration composite exosomes according to claim 1, characterized in that: In step S3, the specific steps for differential centrifugation purification are as follows: Add the high-concentration complex exosome supernatant to a centrifuge tube, cool to 4°C, centrifuge at 300-350g for 10-12 min, centrifuge at 9000-10000g for 25-40 min, and centrifuge again at 90000-100000g for 70-90 min. Remove the centrifuge tube, aspirate the supernatant after centrifugation, and retain a 0.5 cm liquid layer to avoid exosome loss. This completes the differential centrifugation purification.

10. A high-concentration complex exosome cultured by the culture method according to any one of claims 1 to 9.

Images