CN113502262A - Culture method of exosome for improving Treg cell activity - Google Patents
Culture method of exosome for improving Treg cell activity Download PDFInfo
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Abstract
The invention provides a culture method of exosomes for improving Treg cell activity, wherein in the culture process of mesenchymal stem cells, stem cell growth factors, fibroblast growth factors, heparin sodium, prostaglandin E2, TGF-b and vitamin C are added into a basic culture solution, and the obtained culture solution supernatant is subjected to ultracentrifugation to obtain exosomes capable of improving Treg cell activity. The stem cell growth factor and the fibroblast growth factor improve the dryness of cells, the addition of the vitamin C mainly plays a role in antioxidation, the replicative senescence in the cell passage process is reduced, the addition of the anticoagulant mainly aims at reducing the adhesion of the cells, the addition of other fibroblast growth factors, TGF-b, prostaglandin E2 and unsaturated fatty acid mainly can enhance the capability of an exosome secreted by the mesenchymal stem cells for activating the activity of the Treg cells, and the application range and the effect of the exosome are effectively improved.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a culture method of exosomes for improving Treg cell activity.
Background
Exosomes refer to small membrane vesicles (30-150nm) containing complex RNAs and proteins, which today refer specifically to discoidal vesicles with diameters between 40-100 nm. In 1983, exosomes were first found in sheep reticulocytes, which were named "exosomes" by Johnstone in 1987. Various cells can secrete exosomes under normal and pathological states, mainly come from a multivesicular body formed by invagination of intracellular lysosome particles, and are released into extracellular matrix after fusion of outer membranes and cell membranes of the multivesicular body.
Patent No. CN201810168252.3 separation and purification method of human umbilical cord mesenchymal stem cell exosome and application of human umbilical cord mesenchymal stem cell exosome provides a sucrose density gradient centrifugation and purification method of human umbilical cord mesenchymal stem cell exosome and treatment of applying human umbilical cord mesenchymal stem cell exosome to a type II diabetes model, the exosome is obtained from simple culture of culture solution, and improvement of other functions of exosome is not mentioned. Therefore, the prior art mostly focuses on the improvement and optimization of exosome separation methods, and further exploration is needed for the functional improvement of exosomes per se.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a culture method of exosome for improving the activity of Treg cells.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
a method for culturing exosome for improving the activity of Treg cells comprises the steps of adding growth factors, TGF-b, vitamin C, anticoagulant and unsaturated fatty acid into a basic culture solution in the process of culturing mesenchymal stem cells, and ultracentrifuging cell supernatant obtained by culturing to obtain the exosome for improving the activity of the Treg cells.
Preferably, the basal medium is an aMEM medium containing 10% fetal bovine serum.
Preferably, the growth factor is selected from at least one of stem cell growth factor or fibroblast growth factor.
Preferably, the anticoagulant is selected from heparin sodium; the unsaturated fatty acid is selected from prostaglandin E2.
Preferably, the manner of adding the growth factor, TGF-b, vitamin C, anticoagulant and unsaturated fatty acid to the basic culture solution is as follows:
(1) during the culture process of the mesenchymal stem cells of the generation P1-P3, stem cell growth factors and fibroblast growth factors are added into a culture solution;
(2) during the culture process of the mesenchymal stem cells of the generations P4 and P5, fibroblast growth factor, TGF-b, vitamin C, prostaglandin E2, anticoagulant and unsaturated fatty acid are added into a culture solution.
The stem cell factor and the fibroblast growth factor are added in the step (1) to mainly improve the dryness of P1-P3 generation cells and enhance the proliferation capacity of the cells, the fibroblast growth factor, TGF-b, vitamin C, an anticoagulant and unsaturated fatty acid are added in the culture solution in the step (2), wherein the vitamin C is added to mainly play a role in resisting oxidation and reducing replicative senescence in the process of cell passage, the anticoagulant is added to mainly reduce cell adhesion, and the other fibroblast growth factors, TGF-b, prostaglandin E2 and unsaturated fatty acid are added to mainly enhance the capacity of an exosome secreted by the mesenchymal stem cells for activating the activity of the Treg cells.
Preferably, the concentration of both the stem cell growth factor and the fibroblast growth factor in step (1) is 1-30 ng/mL. For example, 1ng/mL, 3ng/mL, 7ng/mL, 10ng/mL, 15ng/mL, 22ng/mL, 27ng/mL, or 30 ng/mL.
Preferably, the concentrations of fibroblast growth factor, TGF-b and heparin sodium in step (2) are each 1-30ng/mL, for example 1ng/mL, 3ng/mL, 7ng/mL, 10ng/mL, 15ng/mL, 22ng/mL, 27ng/mL, or 30 ng/mL. Prostate E2 has a concentration of 1-30mg/mL, such as 1ng/mL, 3ng/mL, 7ng/mL, 10ng/mL, 15ng/mL, 22ng/mL, 27ng/mL, or 30 ng/mL. The concentration of vitamin C is 10-50ug/mL, such as 1ng/mL, 3ng/mL, 7ng/mL, 10ng/mL, 15ng/mL, 22ng/mL, 27ng/mL, 30ng/mL, 36ng/mL, 38ng/mL, 42ng/mL, 47ng/mL, 50 ng/mL.
The invention provides an exosome for improving the activity of Treg cells, which is cultured by the culture method.
An application of the exosome for improving the activity of the Treg cells in a medicine for promoting the Treg.
Compared with the prior art, the invention has the beneficial effects that:
according to the method for culturing the mesenchymal stem cells for improving the activity of the Treg cells, the ability of the mesenchymal stem cells for activating the activity of the Treg cells can be effectively enhanced by adding the cell factors in stages, and the dryness of the cells is improved by adding the stem cell growth factors in the culture process of the mesenchymal stem cells from the P1 generation to the P3 generation of the cells. Fibroblast growth factors, prostaglandin E2, TGF-b, vitamin C and heparin sodium are added in the culture process of the mesenchymal stem cells of P4 and P5 generations of cell expansion, wherein the addition of the vitamin C mainly plays a role in oxidation resistance and reduces replicative senescence in the process of cell passage, the addition of an anticoagulant mainly aims at reducing cell adhesion, the addition of other fibroblast growth factors, TGF-b, prostaglandin E2 and unsaturated fatty acid mainly can enhance the capability of an exosome secreted by the mesenchymal stem cells for activating the activity of Treg cells, and the application range and the effect of the exosome are effectively improved.
Drawings
Fig. 1 is an electron micrograph of exosomes enhancing Treg cell activity in example 1 of the present invention;
FIG. 2 shows the flow-type detection results of different exosomes co-cultured with PBMC, respectively;
FIG. 3 is a graph showing the effect of different exosomes on fibroblast proliferation S phase.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
Recovering the frozen and stored mesenchymal stem cells of P0 generation, centrifuging to obtain mesenchymal stem cells of P1 generation, adding serum-free cell culture solution, adding 10ng/mL stem cell growth factors and 10ng/mL fibroblast growth factors into the culture solution, shaking uniformly by a cross method, and then placing the mixture into a cell culture box for culture;
and (3) culturing the subcultured P1 mesenchymal stem cells for 3 days until the cells grow to reach 80% of fusion degree, digesting the cells, digesting and centrifuging to obtain the P2 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 10ng/mL stem cell growth factors and 10ng/mL fibroblast growth factors into the culture solution, shaking uniformly by a cross method, and then placing the cells in a cell culture box for culturing.
And (3) culturing the subcultured P2 mesenchymal stem cells for 3 days until the cells grow to reach 80% of fusion degree, digesting the cells, digesting and centrifuging to obtain the P3 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 10ng/mL stem cell growth factors and 10ng/mL fibroblast growth factors into the culture solution, shaking uniformly by a cross method, and then placing the cells in a cell culture box for culturing.
Culturing the subcultured P3 mesenchymal stem cells for 3 days until the cell growth reaches 80%, digesting the cells, digesting and centrifuging to obtain the P4 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 15ng/mL fibroblast growth factor, 10mg/mL prostaglandin E2, 10ng/mL TGF-b, 30ug/mL vitamin C and 10ng/mL heparin sodium into the culture solution, shaking uniformly by a cross method, and placing the mixture into a cell culture box for culturing.
Culturing the subcultured P4 mesenchymal stem cells for 3 days until the cell growth reaches 80%, digesting the cells, digesting and centrifuging to obtain the P5 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 15ng/mL fibroblast growth factor, 10mg/mL prostaglandin E2, 10ng/mL TGF-b, 30ug/mL vitamin C and 10ng/mL heparin sodium into the culture solution, shaking uniformly by a cross method, and placing the mixture into a cell culture box for culturing.
After culturing for 3 days, when the cell grows until the fusion degree reaches 85%, collecting cell supernatant, centrifuging by 300 Xg for 10 minutes, collecting supernatant after centrifuging, centrifuging by 2000 Xg for 10 minutes, collecting supernatant after centrifuging, centrifuging by 10000 Xg for 30 minutes for the third time, collecting supernatant after centrifuging, centrifuging for the fourth time, centrifuging by 100000 Xg for 70 minutes, discarding supernatant, adding phosphate buffer solution, blowing and mixing uniformly, centrifuging for the fifth time, and centrifuging by 100000 Xg for 70 minutes to obtain precipitate as exosome.
Comparative example
This comparative example differs from example 1 in that: 10ng/mL stem cell growth factor and 10ng/mL fibroblast growth factor are not added in a culture solution in the culture process of the mesenchymal stem cells of the P1-P3 generation; 15ng/mL fibroblast growth factor, 10mg/mL prostaglandin E2, 10ng/mL TGF-b, 30ug/mL vitamin C and 10ng/mL heparin sodium in a culture solution in the culture process of the P4-P5 generation mesenchymal stem cells.
Fig. 1 is an electron microscope image of the exosome for improving Treg cell activity obtained in example 1, which illustrates that the exosome for improving Treg cell activity obtained by the method has a complete structure, uniform size, a diameter of 90-120nm, high purity and no other impurities.
Fig. 2 shows the Treg cell activation of different exosomes after co-culture with PBMCs, the fraction of Treg cells of the exosomes of the factor-treated group in fig. 2 is 21%, the fraction of Treg cells of the exosomes of the untreated group is 12.3%, and the fraction of Treg cells of the exosomes of the non-treated group is 3.2%, so that it can be seen that the exosomes after factor induction have the ability to activate the Treg cell activity.
FIG. 3 shows the effect of different exosomes on the S-phase of fibroblast proliferation, in FIG. 3 the S-phase of fibroblasts was 13.6%, and fibroblasts were co-cultured with exosomes secreted by untreated cells
The S phase was 19.8%, and the S phase of fibroblasts was 30% after co-culturing exosomes secreted from factor-treated cells with fibroblasts, indicating that the factor-treated cell exosomes promote proliferation of fibroblasts.
Example 2
Recovering the frozen and stored mesenchymal stem cells of P0 generation, centrifuging to obtain mesenchymal stem cells of P1 generation, adding a serum-free cell culture solution, adding 1ng/mL stem cell growth factor and 1ng/mL fibroblast growth factor into the culture solution, shaking uniformly by a cross method, and then placing the mixture into a cell culture box for culture;
and (3) culturing the subcultured P1 mesenchymal stem cells for 3 days until the cells grow to reach 80% of fusion degree, digesting the cells, digesting and centrifuging to obtain the P2 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 1ng/mL stem cell growth factor and 1ng/mL fibroblast growth factor into the culture solution, shaking uniformly by a cross method, and then placing in a cell culture box for culturing.
And (3) culturing the subcultured P2 mesenchymal stem cells for 3 days until the cells grow to reach 80% of fusion degree, digesting the cells, digesting and centrifuging to obtain the P3 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 1ng/mL stem cell growth factor and 1ng/mL fibroblast growth factor into the culture solution, shaking uniformly by a cross method, and then placing in a cell culture box for culturing.
Culturing the subcultured P3 mesenchymal stem cells for 3 days until the cell growth reaches 80%, digesting the cells, digesting and centrifuging to obtain the P4 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 1ng/mL fibroblast growth factor, 1mg/mL prostaglandin E2, 1ng/mL TGF-b, 10ug/mL vitamin C and 1ng/mL heparin sodium into the culture solution, shaking uniformly by a cross method, and placing the mixture into a cell culture box for culturing.
Culturing the subcultured P4 mesenchymal stem cells for 3 days until the cell growth reaches 80%, digesting the cells, digesting and centrifuging to obtain the P5 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 1ng/mL fibroblast growth factor, 1mg/mL prostaglandin E2, 1ng/mL TGF-b, 10ug/mL vitamin C and 1ng/mL heparin sodium into the culture solution, shaking uniformly by a cross method, and placing the mixture into a cell culture box for culturing.
After culturing for 3 days, when the cell grows until the fusion degree reaches 85%, collecting cell supernatant, centrifuging by 300 Xg for 10 minutes, collecting supernatant after centrifuging, centrifuging by 2000 Xg for 10 minutes, collecting supernatant after centrifuging, centrifuging by 10000 Xg for 30 minutes for the third time, collecting supernatant after centrifuging, centrifuging for the fourth time, centrifuging by 100000 Xg for 70 minutes, discarding supernatant, adding phosphate buffer solution, blowing and mixing uniformly, centrifuging for the fifth time, and centrifuging by 100000 Xg for 70 minutes to obtain precipitate which is an exosome.
Example 3
Recovering the frozen and stored mesenchymal stem cells of P0 generation, centrifuging to obtain mesenchymal stem cells of P1 generation, adding a serum-free cell culture solution, adding 1ng/mL stem cell growth factor and 1ng/mL fibroblast growth factor into the culture solution, shaking uniformly by a cross method, and then placing the mixture into a cell culture box for culture;
and (3) culturing the subcultured P1 mesenchymal stem cells for 3 days until the cells grow to reach 80% of fusion degree, digesting the cells, digesting and centrifuging to obtain the P2 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 30ng/mL stem cell growth factors and 30ng/mL fibroblast growth factors into the culture solution, shaking uniformly by a cross method, and then placing the cells in a cell culture box for culturing.
And (3) culturing the subcultured P2 mesenchymal stem cells for 3 days until the cells grow to reach 80% of fusion degree, digesting the cells, digesting and centrifuging to obtain the P3 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 30ng/mL stem cell growth factors and 30ng/mL fibroblast growth factors into the culture solution, shaking uniformly by a cross method, and then placing the cells in a cell culture box for culturing.
Culturing the subcultured P3 mesenchymal stem cells for 3 days until the cell growth reaches 80%, digesting the cells, digesting and centrifuging to obtain the P4 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 30ng/mL fibroblast growth factor, 30mg/mL prostaglandin E2, 30ng/mL TGF-b, 50ug/mL vitamin C and 30ng/mL heparin sodium into the culture solution, shaking uniformly by a cross method, and placing the mixture into a cell culture box for culturing.
Culturing the subcultured P4 mesenchymal stem cells for 3 days until the cell growth reaches 80%, digesting the cells, digesting and centrifuging to obtain the P5 generation mesenchymal stem cells, adding a serum-free cell culture solution, adding 30ng/mL fibroblast growth factor, 30mg/mL prostaglandin E2, 30ng/mL TGF-b, 50ug/mL vitamin C and 30ng/mL heparin sodium into the culture solution, shaking uniformly by a cross method, and placing the mixture into a cell culture box for culturing.
After culturing for 3 days, when the cell grows until the fusion degree reaches 85%, collecting cell supernatant, centrifuging by 300 Xg for 10 minutes, collecting supernatant after centrifuging, centrifuging by 2000 Xg for 10 minutes, collecting supernatant after centrifuging, centrifuging by 10000 Xg for 30 minutes for the third time, collecting supernatant after centrifuging, centrifuging for the fourth time, centrifuging by 100000 Xg for 70 minutes, discarding supernatant, adding phosphate buffer solution, blowing and mixing uniformly, centrifuging for the fifth time, and centrifuging by 100000 Xg for 70 minutes to obtain precipitate which is an exosome.
Example 4
This example provides an exosome for increasing Treg cell activity, which is cultured by the culture method of example 1 described above.
The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (9)
1. A method for culturing exosomes for improving the activity of Treg cells is characterized in that growth factors, TGF-b, vitamin C, anticoagulant and unsaturated fatty acid are added into a basic culture solution in the process of culturing mesenchymal stem cells, and the exosomes for improving the activity of the Treg cells are obtained after ultracentrifugation of mesenchymal cell supernatant obtained by culture.
2. The culture method according to claim 1, wherein the basal medium is a serum-free medium.
3. The culture method of claim 1, wherein the growth factor is selected from at least one of stem cell growth factor or fibroblast growth factor.
4. The culture method according to claim 1, wherein the anticoagulant is selected from the group consisting of heparin sodium; the unsaturated fatty acid is selected from prostaglandin E2.
5. The culture method according to claim 1, wherein the growth factor, TGF-b, vitamin C, anticoagulant and unsaturated fatty acid are added to the basic culture solution in the following manner:
(1) during the culture process of the mesenchymal stem cells of the generation P1-P3, stem cell growth factors and fibroblast growth factors are added into a culture solution;
(2) during the culture process of the mesenchymal stem cells of the generations P4 and P5, fibroblast growth factor, TGF-b, vitamin C, anticoagulant and unsaturated fatty acid are added into a culture solution.
6. The culture method according to claim 5, wherein the concentration of the stem cell growth factor and the fibroblast growth factor in step (1) is 1 to 30 ng/mL.
7. The culture method according to claim 5, wherein the fibroblast growth factor, TGF-b and heparin sodium in step (2) are each present at a concentration of 1-30ng/mL, the prostate E2 is present at a concentration of 1-30mg/mL, and the vitamin C is present at a concentration of 10-50 ug/mL.
8. An exosome for increasing Treg cell activity, which is cultured by the culture method according to any one of claims 1 to 7.
9. Use of an exosome according to claim 8 for increasing Treg cell activity in a medicament for promoting tregs.
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