Method for efficiently promoting maturation of dendritic cells and application
By adding specific inducing factors in the dendritic cell culture process, the problem of insufficient maturity of dendritic cells in the prior art was solved, and efficient acquisition of mature dendritic cells was achieved, which improved its application effectiveness in anti-tumor immune response.
Patent Information
- Application Number
- CN202411916006.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art is difficult to efficiently induce and obtain a large number of mature dendritic cells in vitro, resulting in limited application of their anti-tumor immune response.
The maturation of dendritic cells is promoted by adding inducing factors such as IL-β, TNFα, IGF, triptylanide and cyperine during dendritic cell culture. The specific method is to add immature dendritic cells to the culture medium, and add induction factors on days 5 to 7, and continue to culture for 2 to 3 days to obtain mature dendritic cells.
This method can significantly improve the maturity and survival rate of dendritic cells, enhance the expression of their surface antigens, especially CD86, and thus improve their efficacy in anti-tumor immune response.
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell culture, and in particular to a method for efficiently promoting maturation of dendritic cells and its application. Background Art
[0002] Dendritic cells (DC) are the most powerful antigen-presenting cells in the human body. They can efficiently take up, process and present antigens. Immature DCs have strong migration ability. Mature DCs can effectively activate naive T cells. They are at the center of initiating, regulating and maintaining immune responses. They are the most powerful and the only professional antigen-presenting cells that can activate naive T cells. Recent studies have shown that DC-based immunotherapy has become a research hotspot, especially the in vitro large-scale expansion and cultivation of DCs for viral infection and tumor treatment has entered the clinical stage.
[0003] Dendritic cells originate from CD34+ cells in the bone marrow and are widely distributed in various organs of the body except the brain, but their number is extremely small, accounting for only 1%-2% of peripheral mononuclear cells. Currently, more research is on stimulating, inducing and transfecting dendritic cells with antigens or specific antigen genes extracted from viruses and tumors, so that they carry corresponding antigen information and further induce protective immunity for anti-virus, tumor, prevention and treatment of autoimmune diseases, transplant rejection and allergic reactions.
[0004] In the anti-tumor immune response, the Th1 immune response is believed to play an important role in anti-tumor immunity. In this process, the level of IL-12 secreted by dendritic cells plays a key role. High levels of IL-12 have been shown to effectively polarize the production of Th1 cells. Therefore, the ability to secrete high levels of IL-12 is one of the signs of high biological activity of dendritic cells. However, although many preparations have been found to be able to induce dendritic cells to secrete IL-12, the duration of this process is short-lived. After the stimulus is removed, the ability of dendritic cells to secrete IL-12 quickly disappears. This is due to the insufficient maturity of dendritic cells.
[0005] Therefore, how to provide more methods for obtaining dendritic cells and obtain a large number of mature dendritic cells by inducing differentiation in vitro through the action of cytokines is an urgent problem to be solved by those skilled in the art. Summary of the invention
[0006] The purpose of the present invention is to provide a highly efficient method for promoting dendritic cell maturation and its application. The present invention can effectively induce dendritic cells by adding induction factors during the dendritic cell culture process to obtain a large number of mature dendritic cells.
[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0008] The present invention provides a highly efficient method for promoting maturation of dendritic cells, wherein immature dendritic cells are added to a culture medium for culture, and when the culture reaches the 5th to 7th day, an induction factor is added, and the culture is continued for 2 to 3 days to obtain mature dendritic cells;
[0009] The culture medium includes: penicillin 90-100U / mL, streptomycin 90-100U / mL, macrophage colony stimulating factor 600-800U / mL, IL-4 300-400U / mL, IL-27 100-200U / mL, and the rest is the basic culture medium;
[0010] The inducing factors are: IL-β 30-50 ng / mL, TNFα 40-60 ng / mL, IGF 140-60 ng / mL, triptolide A 12-16 mg / mL, sinomenine 7-11 mg / mL (all expressed as the final concentration of the components after being added into the culture medium system).
[0011] Preferably, the culture temperature is 36-38° C. and the carbon dioxide concentration is 4-6%.
[0012] Preferably, during the culture process, half of the medium is replaced every 40 to 50 hours.
[0013] Preferably, the basal culture medium is X-VIVO-15 culture medium or AIM-V culture medium.
[0014] Preferably, the immature dendritic cells are peripheral blood mononuclear cells.
[0015] Preferably, the inoculation amount of the immature dendritic cells is 3 to 5×10 6 Pieces / mL.
[0016] The present invention also provides the use of mature dendritic cells prepared by the highly efficient method for promoting dendritic cell maturation in the preparation of anti-tumor drugs.
[0017] Preferably, the anti-tumor drug is one of drugs for liver cancer, lung cancer, kidney cancer, skin cancer or breast cancer.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention selects high-purity monocytes from peripheral blood mononuclear cells, and then uses macrophage colony stimulating factor, IL-4 and IL-27 to induce differentiation to obtain immature dendritic cells around the 5th day. Adding macrophage colony stimulating factor, IL-4 and IL-27 to adherent cells can stimulate the adherent cells to differentiate into dendritic cells.
[0020] Then, the present invention adds induction factors to the culture medium and continues to culture for a period of time to obtain mature dendritic cells. The induction factors used in the maturation-promoting culture medium of the present invention have a strong ability to promote the maturation of dendritic cells, and the survival rate of the dendritic cells after maturation is high.
[0021] Among the induction factors added by the present invention, mature dendritic cells are obtained mainly by stimulation with triptolide A and sinomenine, which also have the effect of upregulating the expression of mature dendritic cell surface antigens. Triptolide A can promote the expression of co-stimulatory molecules and MHC molecules on the surface of dendritic cells, and thus has the ability to promote the differentiation and maturation of dendritic cells. Sinomenine can stimulate monocytes to differentiate into immature DCs but can inhibit their further maturation. However, the applicant has found through previous experiments that the use of triptolide A and sinomenine together will significantly enhance the effect of the induction factor, and has a more excellent effect of promoting the maturation of dendritic cells than using triptolide alone. The results obtained in this experiment also verified this conclusion.
[0022] Through the experiments of the present invention, it was found that after immature dendritic cells were stimulated with induction factors, the expression of cell surface antigen CD86 increased significantly, and the expression of CD86 in exosomes secreted by dendritic cells also increased significantly, indicating that the method of the present invention can upregulate the expression of mature dendritic cell surface antigens and promote the maturation of dendritic cells. DETAILED DESCRIPTION
[0023] The present invention provides a highly efficient method for promoting maturation of dendritic cells, comprising adding immature dendritic cells to a culture medium, adding induction factors on the 5th to 7th day of culture, and continuing to culture for 2 to 3 days to obtain mature dendritic cells; preferably, adding induction factors on the 6th day of culture, and continuing to culture for 2 to 3 days to obtain mature dendritic cells; further preferably, adding induction factors on the 6th day of culture, and continuing to culture for 3 days to obtain mature dendritic cells.
[0024] In the present invention, the culture medium comprises: penicillin 90-100U / mL, streptomycin 90-100U / mL, macrophage colony stimulating factor 600-800U / mL, IL-43 00-400U / mL, IL-27 100-200U / mL, and the rest is the basic culture medium; preferably penicillin 92-98U / mL, streptomycin 92-98U / mL, macrophage colony stimulating factor 640-760U / mL, IL-43 20-380U / mL, IL-27 120-180 U / mL, and the rest are basal culture medium; further preferably, penicillin 94-96U / mL, streptomycin 94-96U / mL, macrophage colony stimulating factor 680-720U / mL, IL-43 40-360U / mL, IL-27 140-160U / mL, and the rest are basal culture medium; more preferably, penicillin 95U / mL, streptomycin 95U / mL, macrophage colony stimulating factor 700U / mL, IL-43 50U / mL, IL-27 150U / mL, and the rest are basal culture medium.
[0025] In the present invention, the inducing factors are: IL-β30-50ng / mL, TNFα40-60ng / mL, IGF 140-60ng / mL, triptolide A 12-16mg / mL, and sinomenine 7-11mg / mL; preferably IL-β34-46ng / mL, TNFα44-56ng / mL, IGF 144-56ng / mL, triptolide A 13-15mg / mL, and sinomenine 8-10mg / mL; further preferably IL-β38-42ng / mL, TNFα48-52ng / mL, IGF 148-52ng / mL, triptolide A 14mg / mL, and sinomenine 9mg / mL; more preferably IL-β40ng / mL, TNFα50ng / mL, IGF 150ng / mL, triptolide A 14mg / mL, and sinomenine 9mg / mL.
[0026] In the present invention, the culture temperature is 36-38°C, and the carbon dioxide concentration is 4-6%; preferably, the culture temperature is 37°C, and the carbon dioxide concentration is 4-6%; further preferably, the culture temperature is 37°C, and the carbon dioxide concentration is 5%.
[0027] In the present invention, during the culture process, half of the medium is replaced every 40 to 50 hours; preferably every 42 to 48 hours; more preferably every 44 to 46 hours; and more preferably every 45 hours.
[0028] In the present invention, the basal culture medium is X-VIVO-15 culture medium or AIM-V culture medium; preferably X-VIVO-15 culture medium.
[0029] In the present invention, the immature dendritic cells are peripheral blood mononuclear cells.
[0030] In the present invention, the inoculation amount of the immature dendritic cells is 3 to 5×10 6 / mL; preferably 4×10 6 Pieces / mL.
[0031] The present invention also provides the use of mature dendritic cells prepared by the highly efficient method for promoting dendritic cell maturation in the preparation of anti-tumor drugs.
[0032] In the present invention, the anti-tumor drug is one of drugs for liver cancer, lung cancer, kidney cancer, skin cancer or breast cancer; preferably, it is an anti-liver cancer drug.
[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0034] Example 1
[0035] A highly efficient method for promoting dendritic cell maturation, comprising the following steps:
[0036] (1) Healthy human peripheral blood mononuclear cells were added to the culture medium at a temperature of 36°C and a carbon dioxide concentration of 4%. The cell inoculation amount was 3×10 6 / mL; during the culture process, half of the medium was replaced every 40h;
[0037] (2) On the fifth day of culture, induction factors were added and culture was continued for 2 days to obtain mature dendritic cells;
[0038] The culture medium includes: penicillin 90U / mL, streptomycin 90U / mL, macrophage colony stimulating factor 600U / mL, IL-4 300U / mL, IL-27 100U / mL, and the rest is X-VIVO-15 culture medium;
[0039] The inducing factors are: IL-β 30 ng / mL, TNFα 40 ng / mL, IGF 140 ng / mL, triptolide A 12 mg / mL, and sinomenine 7 mg / mL.
[0040] Example 2
[0041] A highly efficient method for promoting dendritic cell maturation, comprising the following steps:
[0042] (1) Healthy human peripheral blood mononuclear cells were added to the culture medium at a temperature of 38°C and a carbon dioxide concentration of 6%. The cell inoculation amount was 5×10 6 / mL; during the culture process, half of the medium was replaced every 50h;
[0043] (2) On the seventh day of culture, induction factors were added and culture was continued for 3 days to obtain mature dendritic cells;
[0044] The culture medium includes: penicillin 100U / mL, streptomycin 100U / mL, macrophage colony stimulating factor 800U / mL, IL-4 400U / mL, IL-27 200U / mL, and the rest is AIM-V culture medium;
[0045] The inducing factors are: IL-β 50 ng / mL, TNFα 60 ng / mL, IGF 160 ng / mL, triptolide A 16 mg / mL, and sinomenine 11 mg / mL.
[0046] Example 3
[0047] A highly efficient method for promoting dendritic cell maturation, comprising the following steps:
[0048] (1) Healthy human peripheral blood mononuclear cells were added to the culture medium at a temperature of 37°C and a carbon dioxide concentration of 5%. The cell inoculation amount was 4×10 6 / mL; during the culture process, half of the medium was replaced every 45h;
[0049] (2) On the sixth day of culture, induction factors were added and culture was continued for 2 days to obtain mature dendritic cells;
[0050] The culture medium includes: penicillin 95U / mL, streptomycin 95U / mL, macrophage colony stimulating factor 700U / mL, IL-4350U / mL, IL-27150U / mL, and the rest is X-VIVO-15 culture medium;
[0051] The inducing factors are: IL-β40ng / mL, TNFα50ng / mL, IGF 150ng / mL, triptolide A 14mg / mL, and sinomenine 9mg / mL.
[0052] Control group 1
[0053] The other methods are the same as those in Example 3, except that no induction factor is added.
[0054] Control group 2
[0055] The other methods are the same as those in Example 3, except that triptolide A and sinomenine are not added to the induction factors.
[0056] Control group 3
[0057] The other methods are the same as those in Example 3, except that triptolide A is not added to the induction factor.
[0058] Control group 4
[0059] The other methods are the same as those in Example 3, except that the added amount of triptolide A is 25 mg / mL and the added amount of sinomenine is 20 mg / mL.
[0060] Control group 5
[0061] The other methods are the same as those in Example 3, except that the added amount of triptolide A is 2 mg / mL and the added amount of sinomenine is 3 mg / mL.
[0062] Experimental Example 1
[0063] Take the cells obtained in Example 3 and control groups 1 to 5, centrifuge the cells at 900 r / min, resuspend the cells in PBS after 5 min, add 5 μL of MHC II antibody, incubate on ice and away from light for 30 min, add PBS to wash away the excess antibody, and adjust the cell concentration to 5×10 6 / mL, take 500 μL to EP tube, mark, and use quantitative imaging analysis flow cytometer to measure the expression level of MHC II molecules on the cell surface. Each group is repeated three times, and the average value of the measurement results is shown in Table 1.
[0064] Table 1 Statistics of MHC II molecule expression levels
[0065] Group Expression level Example 3 23.41％ Control group 1 2.52％ Control group 2 8.36％ Control group 3 12.33％ Control group 4 18.25％ Control group 5 17.67％
[0066] From the contents recorded in Table 1, it can be seen that the expression level of MHC II molecules on the cell surface of mature dendritic cells in Example 3 group is 23.41%, which is significantly better than other control groups. From the data of the control group, it can be seen that whether triptolide A and sinomenine are added, and the concentration of addition will have a significant impact on the cell culture effect.
[0067] Experimental Example 2
[0068] Take the cells obtained in Example 3 and control groups 1 to 5, centrifuge the cells at 900 r / min, resuspend the cells with PBS after 5 min, add 5 μL of CD86 antibody, incubate on ice and away from light for 30 min, add PBS to wash away the excess antibody, and adjust the cell concentration to 5×10 6 / mL, take 500 μL into EP tube, mark it, and load it on quantitative imaging analysis flow cytometer.
[0069] The supernatant (40 mL) after cell culture in each test group was collected and centrifuged at 3000r / min for 10min, large cell debris was removed, and the supernatant was taken. The supernatant was centrifuged again at 11000r / min for 25min, and residual cell debris and organelles were removed, and the supernatant was taken. The supernatant was centrifuged again at 120000r / min for 65min, and the supernatant was discarded, and the tube wall reflux liquid was resuspended to obtain exosomes, and 10μL of CD63 antibody and 5μL of CD86 antibody were added, placed on ice, and incubated in the dark for 45min. PBS was added to wash away the excess antibody, and the ultracentrifuge was used again, centrifuged at 120000r / min for 65min, the supernatant was discarded, and the tube wall reflux liquid was resuspended, pipetted to the EP tube, marked, and used for quantitative imaging analysis flow cytometer. The expression level of CD86 was determined, and three groups were set up for each group. The average of the measurement results was taken, as shown in Table 2.
[0070] Table 2 Statistics of CD86 expression levels
[0071] Group Cellular expression level Exosome expression level Example 3 51.32％ 3.58％ Control group 1 9.63％ 0.11％ Control group 2 39.54％ 3.11％ Control group 3 43.78％ 3.50％ Control group 4 48.65％ 3.25％ Control group 5 46.87％ 3.47％
[0072] From the contents recorded in Table 2, it can be seen that the expression level of cell CD86 of mature dendritic cells in Example 3 group is 51.32%, which is significantly better than other control groups. The expression level of exosome CD86 can reach 3.58% (CD63 is an exosome marker, so the expression level of exosome CD86 can be seen from the double positive ratio of CD63 and CD86). It can be seen that after immature dendritic cells are stimulated with induction factors, the expression of cell surface antigen CD86 is significantly increased, and the expression of CD86 in exosomes secreted by dendritic cells is also significantly increased, indicating that the method of the present invention can upregulate the expression of mature dendritic cell surface antigens and promote the maturation of mature dendritic cells.
[0073] From the data of the control group, it can be seen that the added concentrations of triptolide A and sinomenine should be appropriate. Adding too much or too little will affect the stimulating effect of the induction factor.
[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for efficiently promoting dendritic cell maturation, characterized in that: Adding immature dendritic cells to the culture medium for culturing, adding induction factors on the 5th to 7th day of culturing, and continuing culturing for 2 to 3 days to obtain mature dendritic cells; The culture medium includes: penicillin 90-100U / mL, streptomycin 90-100U / mL, macrophage colony stimulating factor 600-800U / mL, IL-4 300-400U / mL, IL-27 100-200U / mL, and the rest is the basic culture medium; The inducing factors are: IL-β 30-50 ng / mL, TNFα 40-60 ng / mL, IGF 140-60 ng / mL, triptolide A 12-16 mg / mL, and sinomenine 7-11 mg / mL.
2. A method for efficiently promoting dendritic cell maturation according to claim 1, characterized in that: The culture temperature is 36-38° C., and the carbon dioxide concentration is 4-6%.
3. A highly efficient method for promoting dendritic cell maturation according to claim 1, characterized in that: During the culture process, half of the medium was replaced every 40 to 50 hours.
4. The method for efficiently promoting dendritic cell maturation according to claim 1, characterized in that: The basic culture medium is X-VIVO-15 culture medium or AIM-V culture medium.
5. The method for efficiently promoting dendritic cell maturation according to claim 1, characterized in that: The immature dendritic cells are peripheral blood mononuclear cells.
6. The method for efficiently promoting dendritic cell maturation according to claim 1, characterized in that: The inoculation amount of the immature dendritic cells is (3-5)×10 6 Pieces / mL.
7. Use of mature dendritic cells prepared by the method for promoting dendritic cell maturation according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.
8. The use according to claim 7, characterized in that: The anti-tumor drug is one of drugs for liver cancer, lung cancer, kidney cancer, skin cancer or breast cancer.