CN117866890A - Preparation method for extracting and purifying adipose-derived mesenchymal stem cell exosomes - Google Patents
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Abstract
The invention discloses a preparation method for extracting and purifying an exosome of a adipose-derived mesenchymal stem cell, which belongs to the technical field of exosomes and comprises the following steps: s1, fusion and subculture; step S2, liquid exchange culture; s3, concentrating; s4, enzymolysis; s5, column chromatography; in the invention, an enzymatic agent is added in the purification process of exosomes, the enzymatic agent contains sulfonic acid groups, active hydroxyl groups, quaternary ammonium salt structures and benzene rings, the sulfonic acid groups can be used as acid promoters for enzymolysis reaction, so that the enzymolysis reaction is more thorough, the active hydroxyl groups and the quaternary ammonium salt structures can generate good hydrogen bonding effect with amino acid structures in exosome suspension, in addition, the benzene rings can generate pi-pi interaction with delocalized pi bonds on small molecular peptides, so that the small molecular peptides are better dissolved in the enzymatic agent, and the extraction rate of exosome purified substances is improved.
Description
Technical Field
The invention relates to the technical field of exosomes, in particular to a preparation method for extracting and purifying an exosome of a adipose-derived mesenchymal stem cell.
Background
The exosomes are vesicles surrounded by a bilayer phospholipid molecular layer, exhibiting a typical tea cup-like structure. The exosomes contain within them a number of proteins, such as the four transmembrane protein family (CD 63, CD81 and CD 9), the tumor susceptibility gene 101 protein, and some nucleic acids and lipid molecules. Compared with mesenchymal stem cells, the exosomes have the advantages of being more stable, safer and less immunogenic, and are more convenient to transport and store, so that the exosomes become a more excellent new choice based on MSC.
The existing exosome extraction method comprises an ultracentrifugation method, a size exclusion chromatography method and an immunoadsorption method, wherein the ultracentrifugation method has a considerable extraction amount, is convenient to operate and low in cost, and is favored by people. However, the simple use of ultracentrifugation is difficult to remove impurities that are not close in size to exosomes, and at the same time, it is difficult to achieve extraction of certain effective components, such as oligopeptides, from exosomes, so it is particularly necessary to develop a method for extracting and purifying adipose-derived mesenchymal stem cell exosomes.
Disclosure of Invention
The invention aims to provide a preparation method for extracting and purifying adipose-derived stem cell exosomes, which aims to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a preparation method for extracting and purifying adipose-derived mesenchymal stem cell exosomes comprises the following steps:
step S1, fusion and subculture: when the primary adipose-derived stem cells of the mice are cultured to 80-90% of fusion degree, subculturing to the adipose-derived stem cells of the mice of the third to sixth generation;
step S2, liquid-changing culture: culturing the third to sixth generation mouse fat stem cells in a culture bottle of T75, wherein the changed solution is a complete culture medium, adding a mixed culture medium when culturing and proliferating to 85-90% confluence, continuously culturing for 48 hours, collecting cell supernatant after culturing, sub-packaging the cell supernatant in a 50mL centrifuge tube, and preserving at-80 ℃ for later use;
step S3, concentration: concentrating the thawed cell supernatant by using a 1000kDa ultrafiltration tube, centrifuging the concentrated cell supernatant for 0.5-1h at 2000-4000 Xg to remove cells and fragments, adding 50% of total exosome separating agent by volume of cell culture medium, mixing uniformly, incubating overnight in a refrigerator at 4 ℃, centrifuging at 4 ℃ at 10000-20000 Xg for 1.5-2.5h, re-suspending the precipitate on the tube wall with 200 mu L of PBS, and storing the obtained exosome suspension at-80 ℃ for later use;
step S4, enzymolysis: adding an enzymatic agent into the unfrozen exosome suspension, stirring uniformly to obtain a suspension, carrying out primary enzymolysis and nanofiltration membrane filtration to obtain a primary enzymolysis product, carrying out secondary enzymolysis and nanofiltration membrane filtration, and collecting a primary permeate and a secondary permeate, wherein the mass ratio of the enzymatic agent to the unfrozen exosome suspension is (1-2): 50-80 parts;
step S5, column chromatography: adding the primary permeate and the secondary permeate into a Sephadex G-50 column for chromatographic purification, using deionized water to perform gradient elution on a sample at a flow rate of 1mL/min, collecting the 51 st-150 th eluent, adding the eluent into a C8 reverse chromatographic column, performing gradient elution by using methanol and deionized water, washing the column with deionized water, gradually increasing the methanol content to 100%, collecting the 30 th-60 th eluent, merging the eluents, and performing low-temperature vacuum concentration and spray drying to obtain an exosome purified product.
Further, in step S1, the cell seeding concentration of the fusion culture was 1X 10 6 /mL。
Further, in step S2, the interval between the culture with the liquid change is 2 d.
Further, in step S3, the total exosome separation reagent is sameifeishier technologies.
Further, the complete culture medium is prepared from 10% of mesenchymal stem cells FBS, 1% of double antibody solution and DMEM/F12 culture medium according to the mass ratio of 14-15:4:1, and mixing.
Further, the mixed culture medium is 10% of mesenchymal stem cells FBS without exosomes, 1% of double-antibody solution and DMEM/F12 culture medium according to the mass ratio of 14-15:4:1, and mixing.
In the operation process, the collagen peptide with the molecular weight smaller than 1kD is reserved after primary enzymolysis, secondary enzymolysis is carried out on the collagen peptide with the molecular weight larger than 1kD, then the collagen peptide with the molecular weight smaller than 1kD is obtained through nanofiltration, the yield of the micromolecular collagen peptide can be improved through the secondary enzymolysis process, meanwhile, the utilization rate of protease is improved, and the production cost is reduced.
Further, in the step S4, primary enzymolysis is carried out by adding pepsin for enzymolysis to obtain primary enzymolysis liquid, wherein the mass of the pepsin is 0.3-0.5% of that of the suspension, the enzymolysis temperature is 40 ℃ and the enzymolysis time is 1-1.5h, then the primary enzymolysis liquid is heated to 85-90 ℃ and kept for 8-10min, and the enzyme is inactivated; and filtering the inactivated primary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain primary permeate liquid and primary cutoff solution.
Further, secondary enzymolysis is to collect the primary interception solution obtained in the step S4, deionized water is added into the primary interception solution, the mass of the deionized water is 1-2 times of that of the primary interception solution, alkali is added into the primary interception solution to adjust the pH value of the solution to 7-7.5, the solution is stirred uniformly, then papain is added into the solution for enzymolysis, the secondary enzymolysis solution is obtained, wherein the mass of the papain is 0.8-1% of that of the primary interception solution, the enzymolysis temperature is 50 ℃ and the enzymolysis time is 35-45min, then the primary enzymolysis solution is heated to 85-90 ℃ and kept for 8-10min, and enzymes are inactivated; and filtering the inactivated secondary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain secondary permeate and secondary cutoff solution.
Further, the preparation method of the enzymatic agent comprises the following steps:
adding citric acid and hydroxyethyl trimethylammonium p-toluenesulfonate into anhydrous DMF, heating to 65-75 ℃ under stirring, reacting for 3-5h, standing for 4-6h after the reaction is finished, carrying out suction filtration, respectively washing a filter cake with absolute ethanol and deionized water for 3-5 times, and freeze-drying to obtain an enzymatic agent, wherein the dosage ratio of the citric acid to the hydroxyethyl trimethylammonium p-toluenesulfonate to the DMF is 2-4g:2.2-8.4mL: and (3) carrying out esterification reaction on carboxyl on citric acid and hydroxyl on hydroxyethyl trimethylammonium p-toluenesulfonate in the reaction process to obtain the enzymatic agent in 35-45mL.
Compared with the prior art, the invention has the following beneficial effects: (1) According to the technical scheme, an enzymatic agent is added in the purification process of an exosome, the enzymatic agent is prepared by esterification reaction of citric acid and hydroxyethyl trimethyl ammonium p-toluenesulfonate, the prepared enzymatic agent contains sulfonic acid groups, active hydroxyl groups, quaternary ammonium salt structures and benzene rings, the sulfonic acid groups can be used as acid promoters for enzymolysis reaction, so that the enzymolysis reaction is more thorough, the existence of the active hydroxyl groups and the quaternary ammonium salt structures can generate good hydrogen bonding with amino acid structures in the exosome suspension, in addition, the benzene rings can generate pi-pi interaction with delocalized pi bonds on small molecule peptides, so that the small molecule peptides are better dissolved in the enzymatic agent, and the extraction rate of the exosome purified matters is improved.
(2) In the technical scheme of the invention, twice enzymolysis is adopted, collagen peptide with molecular weight smaller than 1kD is reserved after one enzymolysis, twice enzymolysis is carried out on collagen peptide with molecular weight larger than 1kD, then nanofiltration is carried out to obtain collagen peptide with molecular weight smaller than 1kD, the yield of micromolecular collagen peptide can be improved in the twice enzymolysis process, meanwhile, the utilization rate of protease is improved, and the production cost is reduced.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Embodiment 1 preferably, this embodiment provides a method for preparing an enzymatic agent, comprising the steps of:
3g of citric acid and 5.3mL of hydroxyethyl trimethylammonium p-toluenesulfonate are added into 40mL of anhydrous DMF, the temperature is raised to 70 ℃ while stirring, the stirring reaction is carried out for 4 hours, after the reaction is finished, the mixture is stood for 5 hours, suction filtration is carried out, filter cakes are respectively washed for 4 times by using anhydrous ethanol and deionized water, and the mixture is frozen and dried to obtain the enzymatic agent.
Example 2 this example provides a method for the preparation of extraction and purification of adipose-derived mesenchymal stem cell exosomes comprising the following steps:
step S1, fusion and subculture: culturing the primary adipose-derived stem cells of the mice to 80% fusion degree, and subculturing to obtain adipose-derived stem cells of the third mice;
step S2, liquid-changing culture: the concentration was 1X 10 6 culturing/mL third mouse fat stem cells in T75 culture flask with exchange solution of every 2dWhen the complete culture medium is cultivated and proliferated to 85% confluence, adding 10% of mesenchymal stem cells FBS without exosomes, 1% of double-antibody solution and DMEM/F12 culture medium according to the mass ratio of 14:4:1, continuously culturing the mixed culture medium for 48 hours, collecting cell supernatant after the culture is finished, sub-packaging the cell supernatant into 50mL centrifuge tubes, and preserving the cell supernatant at the temperature of minus 80 ℃ for later use;
wherein the complete culture medium is prepared from 10% of mesenchymal stem cells FBS, 1% of double-antibody solution and DMEM/F12 culture medium according to the mass ratio of 14:4:1, mixing;
step S3, concentration: concentrating the thawed cell supernatant with a 1000kDa ultrafiltration tube, centrifuging the concentrated cell supernatant at 2000 x g for 0.5h to remove cells and debris, adding 50% of total exosome separation reagent by volume of cell culture medium, mixing well, incubating overnight at 4deg.C in a refrigerator, centrifuging at 4deg.C for 1.5h at 10000 x g, re-suspending the precipitate on the tube wall with 200 μl of PBS, and storing the obtained exosome suspension at-80deg.C;
step S4, enzymolysis: the enzymatic agent prepared in example 1 was prepared in a mass ratio of 1:50 adding the solution into the thawed exosome suspension, stirring uniformly to obtain a suspension, adding pepsin accounting for 0.3% of the mass of the suspension, carrying out enzymolysis for 1.h at 40 ℃, heating to 88 ℃ and keeping for 9min, inactivating enzymes, filtering the inactivated primary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain primary permeate and primary cutoff solution, adding deionized water accounting for 1 time of the mass of the primary cutoff solution, adding alkali to adjust the pH value of the solution to 7, stirring uniformly, adding papain accounting for 0.8% of the mass of the primary cutoff solution, carrying out enzymolysis for 35min at 50 ℃, heating to 85 ℃ and keeping for 8min, inactivating enzymes, and filtering the inactivated secondary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain secondary permeate and secondary cutoff solution;
step S5, column chromatography: adding the primary permeate and the secondary permeate into a Sephadex G-50 column for chromatographic purification, using deionized water to perform gradient elution on a sample at a flow rate of 1mL/min, collecting the 51 st tube of eluent, adding the eluent into a C8 reverse chromatographic column, performing gradient elution by using methanol and deionized water, washing the column with deionized water, gradually increasing the methanol content to 100%, collecting the 30 th tube of eluent, merging the eluents, and performing low-temperature vacuum concentration and spray drying to obtain an exosome purified product.
Example 3 this example provides a method for the preparation of extraction and purification of adipose-derived mesenchymal stem cell exosomes comprising the steps of:
step S1, fusion and subculture: culturing the primary adipose-derived stem cells of the mice to 80% fusion degree, and subculturing to obtain adipose-derived stem cells of the third mice;
step S2, liquid-changing culture: the concentration was 1X 10 6 Carrying out liquid change culture on/mL third mouse adipose-derived stem cells in a culture bottle of T75, wherein the changed solution is a complete culture medium, and when the culture and proliferation are carried out to 85% confluence, adding 10% of mesenchymal stem cells FBS without exosomes, 1% of double antibody solution and DMEM/F12 culture medium according to the mass ratio of 14.5:4:1, continuously culturing the mixed culture medium for 48 hours, collecting cell supernatant after the culture is finished, sub-packaging the cell supernatant into 50mL centrifuge tubes, and preserving the cell supernatant at the temperature of minus 80 ℃ for later use;
wherein the complete culture medium is prepared from 10% of mesenchymal stem cells FBS, 1% of double-antibody solution and DMEM/F12 culture medium according to the mass ratio of 14.5:4:1, mixing;
step S3, concentration: concentrating the thawed cell supernatant with a 1000kDa ultrafiltration tube, centrifuging the concentrated cell supernatant at 3000 x g for 0.75h to remove cells and debris, adding 50% of total exosome separation reagent by volume of cell culture medium, mixing well, incubating overnight at 4deg.C in a refrigerator, centrifuging at 4deg.C 15000 x g for 2.0h, resuspending the precipitate on the tube wall with 200 μl of PBS, and storing the resulting exosome suspension at-80deg.C;
step S4, enzymolysis: the enzymatic agent prepared in example 1 was prepared in a mass ratio of 1:50 adding the solution into the thawed exosome suspension, stirring uniformly to obtain a suspension, adding pepsin accounting for 0.4% of the mass of the suspension, carrying out enzymolysis for 1.25h at 40 ℃, heating to 88 ℃ and keeping for 9min, inactivating enzymes, filtering the inactivated primary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain primary permeate and primary cutoff solution, adding deionized water accounting for 1.5 times of the mass of the primary cutoff solution, adding alkali to adjust the pH value of the solution to 7.2, stirring uniformly, adding papain accounting for 0.9% of the mass of the primary cutoff solution, carrying out enzymolysis for 40min at 50 ℃, heating to 88 ℃ and keeping for 9min, inactivating enzymes, and filtering the inactivated secondary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain secondary permeate and secondary cutoff solution;
step S5, column chromatography: adding the primary permeate and the secondary permeate into a Sephadex G-50 column for chromatographic purification, using deionized water to perform gradient elution on a sample at a flow rate of 1mL/min, collecting the 100 th eluent, adding the eluent into a C8 reverse chromatographic column, performing gradient elution by using methanol and deionized water, washing the column with deionized water, gradually increasing the methanol content to 100%, collecting the 45 th eluent, combining the eluents, and performing low-temperature vacuum concentration and spray drying to obtain an exosome purified product.
Example 4 this example provides a method for the preparation of extraction and purification of adipose-derived mesenchymal stem cell exosomes comprising the steps of:
step S1, fusion and subculture: culturing the primary adipose-derived stem cells of the mice to 80% fusion degree, and subculturing to obtain adipose-derived stem cells of the third mice;
step S2, liquid-changing culture: the concentration was 1X 10 6 Carrying out liquid change culture on/mL third mouse adipose-derived stem cells in a culture bottle of T75, wherein the changed solution is a complete culture medium, and when the culture and proliferation are carried out to 85% confluence, adding 10% of mesenchymal stem cells FBS without exosomes, 1% of double antibody solution and DMEM/F12 culture medium according to the mass ratio of 15:4:1, continuously culturing for 48 hours, collecting cell supernatant after culturing, sub-packaging the cell supernatant in a 50mL centrifuge tube, and preserving the cell supernatant at the temperature of minus 80 ℃ for standby, wherein the complete culture medium is prepared from 10% by volume of mesenchymal stem cell FBS, 1% by mass of double antibody solution and DMEM/F12 culture medium according to the mass ratio of 15:4:1, mixing;
step S3, concentration: concentrating the thawed cell supernatant by using a 1000kDa ultrafiltration tube, centrifuging the concentrated cell supernatant for 1h at 4000×g to remove cells and fragments, adding a total exosome separation reagent in an amount of 50% of the volume of the cell culture medium, uniformly mixing, incubating overnight at 4 ℃ in a refrigerator, centrifuging at 4 ℃ for 2.5h at 20000×g, re-suspending the precipitate on the tube wall with 200 μl of PBS, and storing the obtained exosome suspension at-80 ℃ for later use;
step S4, enzymolysis: the enzymatic agent prepared in example 1 was prepared in a mass ratio of 1:50 adding the solution into the thawed exosome suspension, stirring uniformly to obtain a suspension, adding pepsin accounting for 0.4% of the mass of the suspension, carrying out enzymolysis for 1.5 hours at 40 ℃, heating to 90 ℃ and keeping for 10 minutes, inactivating enzymes, filtering the inactivated primary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain primary permeate and primary cutoff solution, adding deionized water accounting for 2 times of the mass of the primary cutoff solution, adding alkali to adjust the pH value of the solution to 7.5, stirring uniformly, adding papain accounting for 1.0% of the mass of the primary cutoff solution, carrying out enzymolysis for 45 minutes at 50 ℃, heating to 90 ℃ and keeping for 10 minutes, inactivating enzymes, and filtering the inactivated secondary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain secondary permeate and secondary cutoff solution;
step S5, column chromatography: adding the primary permeate and the secondary permeate into a Sephadex G-50 column for chromatographic purification, using deionized water to perform gradient elution on a sample at a flow rate of 1mL/min, collecting the 150 th tube of eluent, adding the eluent into a C8 reverse chromatographic column, performing gradient elution by using methanol and deionized water, washing the column with deionized water, gradually increasing the methanol content to 100%, collecting the 60 th tube of eluent, merging the eluents, and performing low-temperature vacuum concentration and spray drying to obtain an exosome purified product.
Comparative example 1
The citric acid in example 1 was removed, the remaining materials and preparation were kept unchanged, and the prepared material was replaced with the enzymatic agent in example 3, the remaining materials and preparation were kept unchanged.
Comparative example 2
The enzymatic agent of example 3 was removed and the remaining starting materials and preparation were kept unchanged.
And (3) performance detection:
and detecting the purity and extraction rate of the adipose-derived mesenchymal stem cell exosomes.
The purity and extraction rate of the adipose-derived mesenchymal stem cell exosomes prepared in examples 2 to 4 and comparative example 12 were measured by using a high performance liquid chromatograph under the following specific measurement conditions:
by C 18 HD (4.6 x 150 mm, 3.5 μm) column, eluting with acetonitrile, column temperature 35 ℃, flow rate 0.3ml. Min -1 Sample injection quantity 10 mu L, and detection wavelength 220nm; and determining the content of the adipose-derived mesenchymal stem cell exosome by using a peak area normalization method.
Extraction rate (%) = (mass of adipose mesenchymal stem cell exosome purification ≡wheat adipose mesenchymal stem cell content) ×100%.
Wherein the quality of the adipose-derived mesenchymal stem cell exosome purification is approximately equal to the exosome content in the adipose-derived mesenchymal stem cell exosome. Therefore, in the extraction yield calculation formula, the quality of the exosome purified material is used for replacing the exosome content in the adipose mesenchymal stem cells, and the test results are shown in table 1.
Table 1 purity and extraction yield of exosome purified products in examples 2 to 4 and comparative examples 1 to 2 adipose-derived mesenchymal stem cells.
TABLE 1
As can be seen from Table 1, the exosomes obtained by the method of extracting and purifying adipose-derived mesenchymal stem cell exosomes in examples 2-3 have higher purity and extraction yield than those of comparative examples 1-2.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A preparation method for extracting and purifying adipose-derived mesenchymal stem cell exosomes comprises the following steps;
step S1, fusion and subculture: when the primary adipose-derived stem cells of the mice are cultured to 80-90% of fusion degree, subculturing to the adipose-derived stem cells of the mice of the third to sixth generation;
step S2, liquid-changing culture: culturing the third to sixth generation mouse fat stem cells in a culture bottle of T75, wherein the changed solution is a complete culture medium, adding a mixed culture medium when culturing and proliferating to 85-90% confluence, continuously culturing for 48 hours, collecting cell supernatant after culturing, sub-packaging the cell supernatant in a 50mL centrifuge tube, and preserving at-80 ℃ for later use;
step S3, concentration: concentrating the thawed cell supernatant with a 1000kDa ultrafiltration tube, centrifuging the concentrated cell supernatant at 2000 x g for 0.5h to remove cells and debris, adding 50% of total exosome separation reagent by volume of cell culture medium, mixing well, incubating overnight at 4deg.C in a refrigerator, centrifuging at 4deg.C for 1.5h at 10000 x g, re-suspending the precipitate on the tube wall with 200 μl of PBS, and storing the obtained exosome suspension at-80deg.C;
step S4, enzymolysis: adding the enzymatic agent prepared in the embodiment 1 into the unfrozen exosome suspension, uniformly stirring to obtain a suspension, carrying out primary enzymolysis and nanofiltration membrane filtration to obtain a primary enzymolysis product, carrying out secondary enzymolysis and nanofiltration membrane filtration, and collecting a primary permeate and a secondary permeate;
step S5, column chromatography: adding the primary permeate and the secondary permeate into a Sephadex G-50 column for chromatographic purification, using deionized water to perform gradient elution on a sample at a flow rate of 1mL/min, collecting the 51 st tube of eluent, adding the eluent into a C8 reverse chromatographic column, performing gradient elution by using methanol and deionized water, washing the column with deionized water, gradually increasing the methanol content to 100%, collecting the 30 th tube of eluent, merging the eluents, and performing low-temperature vacuum concentration and spray drying to obtain an exosome purified product.
2. The method for preparing the extraction and purification of adipose-derived mesenchymal stem cell exosomes according to claim 1, wherein the method comprises the following steps: the preparation method of the enzymatic agent comprises the following steps:
adding citric acid and hydroxyethyl trimethyl ammonium p-toluenesulfonate into anhydrous DMF, heating to 65-75 ℃ while stirring, stirring for 3-5h, standing for 4-6h after the reaction is finished, carrying out suction filtration, respectively washing a filter cake with absolute ethanol and deionized water for 3-5 times, and freeze-drying to obtain the enzymatic agent.
3. The method for preparing the extraction and purification of adipose-derived mesenchymal stem cell exosomes according to claim 1, wherein the method comprises the following steps: in the step S1, the cell inoculation concentration of the fusion culture is 1×10 6 /mL。
4. The method for preparing the extraction and purification of adipose-derived mesenchymal stem cell exosomes according to claim 1, wherein the method comprises the following steps: in the step S2, the interval between the liquid-changing culture is 2 d.
5. The method for preparing the extraction and purification of adipose-derived mesenchymal stem cell exosomes according to claim 1, wherein the method comprises the following steps: the complete culture medium is prepared from 10% of mesenchymal stem cells FBS, 1% of double-antibody solution and a DMEM/F12 culture medium according to the mass ratio of 14-15:4:1, wherein the mixed culture medium is prepared by mixing 10% of mesenchymal stem cells FBS without exosomes, 1% of double-antibody solution and a DMEM/F12 culture medium according to the mass ratio of 14-15:4:1, and mixing.
6. The method for preparing the extraction and purification of adipose-derived mesenchymal stem cell exosomes according to claim 1, wherein the method comprises the following steps: in the step S4, the primary enzymolysis is carried out by adding pepsin for enzymolysis, thus obtaining primary enzymolysis liquid.
7. The method for preparing the extraction and purification of adipose-derived mesenchymal stem cell exosomes according to claim 1, wherein the method comprises the following steps: and (3) secondary enzymolysis is to collect the primary trapped solution obtained in the step (S4), adding deionized water into the solution, wherein the mass of the deionized water is 1-2 times that of the primary trapped solution, adding alkali into the solution to adjust the pH value of the solution to 7-7.5, uniformly stirring, and then adding papain for enzymolysis to obtain secondary enzymolysis solution.
8. The method for preparing the fat mesenchymal stem cell exosome extraction and purification according to claim 6, wherein the method comprises the following steps: the mass of the pepsin is 0.3-0.5% of that of the suspension, the enzymolysis temperature is 40 ℃, the enzymolysis time is 1-1.5 hours, then the primary enzymolysis liquid is heated to 85-90 ℃ and kept for 8-10 minutes, and the enzyme is inactivated; and filtering the inactivated primary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain primary permeate liquid and primary cutoff solution.
9. The method for preparing the fat mesenchymal stem cell exosome extraction and purification according to claim 7, wherein the method comprises the following steps: the mass of the papain is 0.8-1% of that of the primary intercepting solution, the enzymolysis temperature is 50 ℃, the enzymolysis time is 35-45min, then the primary enzymolysis solution is heated to 85-90 ℃ and kept for 8-10min, and the enzyme is inactivated; and filtering the inactivated secondary enzymolysis liquid through a nanofiltration membrane with the molecular weight cutoff of 1kD to obtain secondary permeate and secondary cutoff solution.
10. The method for preparing the extraction and purification of adipose-derived mesenchymal stem cell exosomes according to claim 2, wherein the method comprises the following steps: the dosage ratio of the citric acid, the hydroxyethyl trimethyl ammonium p-toluenesulfonate and the DMF is 2-4g:2.2-8.4mL:35-45mL.
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