Factor composition for activating DC cells and application method thereof

Abstract

The invention provides a factor composition for activating DC cells and an application method thereof, and belongs to the technical field of cell preparations. The factor composition for activating the DC cells, provided by the invention, is prepared from the following components: rhGM-CSF, rhIL-4, rhSCF, rhTNF-alpha, IFN-alpha, insulin and lycium barbarum polysaccharide. Through the synergistic effect of all the components, PBMC (peripheral blood mononuclear cells) can be promoted to be differentiated into DC cells, the proportion of the DC cells is increased, the initial quantity of the DC cells is increased, proliferation of the DC cells is promoted, the viability of the DC cells is improved, expression of antigen costimulatory molecules can be improved, and the culture cycle of the DC cells is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of cell preparations, and in particular to a factor composition for activating DC cells and an application method thereof. Background Art

[0002] Dendritic cells (also known as DC cells) were discovered by Canadian scholar Steinman in 1973. They are the most powerful professional antigen presenting cells (Antigen presenting cells, APC) in the body. They can efficiently ingest, process and present antigens. Most DCs in the human body are in an immature state, expressing low levels of co-stimulatory factors and adhesion factors, and have a low ability to stimulate the proliferation of mixed lymphocytes in vitro. Mature DCs express high levels of co-stimulatory factors and adhesion factors. During the maturation process, DCs migrate from peripheral tissues that are in contact with antigens into secondary lymphoid organs, contact T cells and stimulate immune responses. The existing technology for preparing DC cells has low expression of antigen co-stimulatory molecules, poor activation of T cells, and a relatively long culture time. Therefore, it is hoped to provide a factor composition that can activate DC cells, promote DC cell proliferation, increase the expression of antigen co-stimulatory molecules, reduce the DC cell culture cycle, and increase DC cell viability. Summary of the invention

[0003] The object of the present invention is to provide a culture method that can promote DC cell proliferation, increase the expression of antigen co-stimulatory molecules, reduce the DC cell culture cycle, and improve the DC cell viability.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a factor composition for activating DC cells, comprising the following components: rhGM-CSF, rhIL-4, rhSCF, rhTNF-α, IFN-α, insulin and wolfberry polysaccharide;

[0006] Wherein, the concentration of rhGM-CSF is 650-850U / mL;

[0007] The concentration of the rhIL-4 is 150-250 U / mL:

[0008] The concentration of rhSCF is 60-80 ng / mL;

[0009] The concentration of rhTNF-α is 80-100 U / mL;

[0010] The concentration of IFN-α is 100-120 U / mL;

[0011] The concentration of the insulin is 10-20 nmol / L;

[0012] The concentration of the wolfberry polysaccharide is 68-80 μg / mL.

[0013] Preferably, the basic solvent in the factor composition is RPMI-1640 complete medium.

[0014] The present invention provides use of the above factor composition in activating DC cells.

[0015] The present invention also provides a method for activating DC cells, comprising the following steps:

[0016] (1) inoculating PBMC in the factor composition, adding 8-12% (v / t) serum, transferring to a culture bottle for culture, and after the culture is completed, transferring the suspended cells, and the remaining adherent cells are undifferentiated DC cells;

[0017] (2) adding the factor composition to undifferentiated DC cells and culturing them;

[0018] (3) adding a mixed solution of the factor composition and the OVA antigen solution, continuing the culture, and collecting mature DC cells.

[0019] Preferably, the seeding density of PBMC in step (1) is (2.0-2.5)×10 6 Pieces / mL;

[0020] The DC cell culture conditions in step (1) are: 4-6% CO2, 36-37°C, 1-2h.

[0021] Preferably, the amount of the factor composition added in step (2) is 50-70% of the volume of the culture bottle, and the culture conditions are 4-6% CO2, 36-37°C, and 10-20h.

[0022] Preferably, the mixed solution in step (3) is prepared by mixing the factor composition and the OVA antigen solution in a volume ratio of 1:1 to 2;

[0023] The amount of the mixed solution added is 4-6% of the volume of the factor composition in step (2).

[0024] Preferably, the conditions for continued culture in step (3) are 4-6% CO2, 36-37°C, and 10-15h.

[0025] It was found through experiments that the factor composition for activating DC cells provided by the present invention can not only promote PBMC differentiation into DC cells, increase the proportion of DC cells, increase the initial amount of DC cells, promote the proliferation of DC cells, improve DC cell viability, but also improve the expression of antigen co-stimulatory molecules, and reduce the DC cell culture cycle. The present invention stimulates cells to differentiate and grow into DC cells by adding rhTNF-α and IFN-α, promotes the development of DC cells by adding rhSCF and Lycium barbarum polysaccharides, induces cell maturation, and the addition of insulin can promote the metabolism of glucose by cells, which helps cells to further utilize the above substances. The components of the factor composition provided by the present invention cooperate with each other, can effectively promote DC cell proliferation, improve the expression of antigen co-stimulatory molecules, reduce the DC cell culture cycle, and enhance DC cell activity. DETAILED DESCRIPTION

[0026] In the present invention, rhGM-CSF, rhIL-4, rhSCF, rhTNF-α, and IFN-α were purchased from Peprotech; insulin and OVA antigens were purchased from Sigma; Lycium barbarum polysaccharides were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; RPMI-1640 complete culture medium and PBMC were purchased from Invitrogen; and interleukin 12 (IL-12) ELISA detection kit was purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0027] 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.

[0028] Example 1

[0029] A factor composition for activating DC cells is prepared by using RPMI-1640 complete medium as a basic medium, and adding the following components to obtain (the following are the final concentrations of each component): rhGM-CSF 650 U / mL, rhIL-4 150 U / mL, rhSCF 70 ng / mL, rhTNF-α 80 U / mL, IFN-α 100 U / mL, insulin 15 nmol / L and wolfberry polysaccharide 75 μg / mL).

[0030] Example 2

[0031] A factor composition for activating DC cells is prepared by using RPMI-1640 complete medium as a basic medium and adding the following components in concentrations: rhGM-CSF 700 U / mL, rhIL-4 250 U / mL, rhSCF 80 ng / mL, rhTNF-α 100 U / mL, IFN-α 120 U / mL, insulin 12 nmol / L and wolfberry polysaccharide 68 μg / mL.

[0032] Example 3

[0033] A factor composition for activating DC cells is prepared by using RPMI-1640 complete medium as a basic medium and adding the following components in concentrations: rhGM-CSF 800 U / mL, rhIL-4 200 U / mL, rhSCF 75 ng / mL, rhTNF-α 97 U / m, IFN-α 115 U / mL, insulin 18 nmol / L and wolfberry polysaccharide 72 μg / mL.

[0034] Example 4

[0035] Methods for activating DC cells:

[0036] (1) PBMCs were inoculated into 30 mL of the factor composition obtained in Example 1 at a density of 2×10 6 10 / mL, add 10% (v / t) FBS (Hyclone) of the factor composition, transfer all to a T75 cell culture flask, incubate at 37°C, 5% CO2 for 2 h, and after the incubation, transfer the suspended cells, leaving the adherent cells as undifferentiated DC cells;

[0037] (2) Add 45 mL of the factor composition described in Example 1 to the undifferentiated DC cells and resuspend them to adjust the cell concentration to 1×10 6 / mL, 37℃, 5% CO2, 24h;

[0038] (3) 1.8 mL of the mixed solution (the mixed solution was a mixture of the factor composition described in Example 1 and the OVA antigen solution at a volume ratio of 1:1, and the concentration of the OVA antigen was 25 μg / ml) was added, and the mixture was continued at 37°C and 5% CO. 2， After culturing for 24 h, mature DC cells were collected.

[0039] Example 5

[0040] Methods for activating DC cells:

[0041] (1) PBMCs were inoculated into 30 mL of the factor composition obtained in Example 2 at a density of 2×10 6 pcs / mL, add 8% (v / t) FBS of the factor composition, transfer all to a T75 cell culture flask, incubate at 37°C, 5% CO2 for 2h, after the incubation is complete, transfer the suspended cells, and the remaining adherent cells are the undifferentiated DC cells;

[0042] (2) Add 50 mL of the factor composition described in Example 2 to the undifferentiated DC cells and resuspend them to adjust the cell concentration to 1×10 6 / mL, 37℃, 5% CO2, culture for 20h;

[0043] (3) Add 2 mL of the mixed solution (the mixed solution is a mixture of the factor composition described in Example 2 and the OVA antigen solution at a volume ratio of 1:1) and continue to incubate at 37°C and 5% CO 2， After culturing for 20 h, mature DC cells were collected.

[0044] Example 6

[0045] Methods for activating DC cells:

[0046] (1) PBMCs were inoculated into 30 mL of the factor composition obtained in Example 1 at a density of 2.5×10 6 cells / mL, add 10% (v / t) FBS of the factor composition, transfer all to a T75 cell culture flask, incubate at 37°C, 5% CO2 for 2 h, and after the incubation is complete, transfer the suspended cells, and the remaining adherent cells are the undifferentiated DC cells;

[0047] (2) Add 46 mL of the factor composition described in Example 1 to the undifferentiated DC cells and resuspend them to adjust the cell concentration to 1×10 6 / mL, 37℃, 5% CO2, culture for 20h;

[0048] (3) Add 2 mL of the mixed solution (the mixed solution is a mixture of the factor composition described in Example 1 and the OVA antigen solution at a volume ratio of 1:1) and continue to incubate at 37°C and 5% CO 2， After culturing for 20 h, mature DC cells were collected.

[0049] Comparative Example 1

[0050] The difference from Example 4 is that in step (1), the factor composition is replaced by RPMI 1640 culture medium containing 5vt% FBS.

[0051] Comparative Example 2

[0052] The difference from Example 4 is that: Step (2) resuspended the adherent cells (DC cells) in RPMI-1640 medium containing 10% FBS and adjusted the cell concentration to 1×10 6 / mL, and added cytokines GM-CSF and IL-4 with a final concentration of 100 ng / mL (GM-CSF and IL-4 mass concentration ratio was 4:1), and cultured in a 37°C, 5% CO2 incubator.

[0053] Comparative Example 3

[0054] (1) PBMCs were resuspended in RPMI-1640 medium containing 1% fetal bovine serum (FBS) and the cell concentration was adjusted to 2×106 / mL, incubate for 2h at 37℃, 5% CO2, and discard the suspended cells;

[0055] (2) The adherent cells were resuspended in RPMI-1640 medium containing 10% FBS and the cell concentration was adjusted to 1×10 6 / mL, and added cytokines GM-CSF and IL-4 with a final concentration of 100 ng / mL (GM-CSF and IL-4 mass concentration 4:1), and cultured in a 37°C, 5% CO2 incubator for 7 days, during which half of the medium was replaced every 3 days;

[0056] (3) On the seventh day, IL-1β (10 ng / mL), IL-6 (10 ng / mL), TNF-α (10 ng / mL), and PGE2 (1 μg / mL) were added and incubated for 24 hours; mature DC cells were harvested on the eighth day.

[0057] Test Example 1

[0058] DC cells obtained after culture by the cell culture methods given in Examples 4 to 6 and Comparative Examples 1 to 3 were collected, and the collected mature DC cells were counted using a cell counter and 0.4% trypan blue staining method, and the cell activity was calculated. The results are shown in Table 1:

[0059] Table 1

[0060] <![CDATA[Cell count (10 7 / ml)]]> Cell viability (%) Example 4 2.89 98.43 Example 5 2.94 97.64 Example 6 2.89 97.99 Comparative Example 1 2.31 81.47 Comparative Example 2 1.95 75.74 Comparative Example 3 1.52 69.14

[0061] As can be seen from Table 1, the number and viability of DC cells cultured by the culture methods of Examples 4 to 6 are significantly higher than those of Comparative Examples 1 to 3. Compared with Comparative Example 1, it can be seen that the factor composition provided by the present invention can promote the differentiation of PBMC into DC cells, increase the proportion of DC cells, and increase the starting amount of DC cells; compared with Comparative Example 2, it can be seen that the factor composition provided by the present invention can effectively promote the proliferation of DC cells. In summary, it can be seen that the present invention helps the differentiation and proliferation of DC cells and reduces DC cell damage during the culture process.

[0062] Test Example 2

[0063] Beckman flow cytometer was used to detect the expression of surface markers of mature DC cells obtained in Examples 4 to 6 and Comparative Examples 1 to 3. The results are shown in Table 2:

[0064] Table 2 Surface marker expression

[0065] Example 4 Example 5 Example 6 Comparative Example 1 Comparative Example 2 Comparative Example 3 CD86 95.2％ 95.1％ 94.8％ 75.6％ 60.5％ 30.5％ CD83 98.3％ 97.9％ 98.2％ 85.4％ 65.9％ 37.7％ CD11c 96.4％ 95.8％ 96.8％ 78.1％ 63.6％ 20.5％

[0066] As shown in Table 2, the expression of DC mature cell surface markers obtained in Examples 4 to 6 is significantly higher than that in Comparative Examples 1 to 3. It can be seen that the factor composition provided by the present invention induces a higher positive expression rate of DC mature cells and can increase the expression level of antigen costimulatory molecules.

[0067] Test Example 3

[0068] The culture supernatants of the DC cells induced and cultured on the third day in Examples 4 to 6 and Comparative Examples 1 to 3 were collected respectively, and the IL-12 secreted by the mature DC cells in the culture supernatants was detected using an interleukin 12 (IL-12) ELISA detection kit. The specific operation was performed according to the ELISA detection manual. The results are shown in Table 3:

[0069] Table 3 IL-12 content (pg / ml)

[0070] Example 4 Example 5 Example 6 Comparative Example 1 Comparative Example 2 Comparative Example 3 IL-12 443 428 435 304 268 184

[0071] As can be seen from Table 3, the IL-12 content secreted by the mature DC cells induced and cultured in Examples 4 to 6 was significantly higher than the IL-12 content secreted by the mature DC cells induced and cultured in Comparative Examples 1 and 2, indicating that the factor composition provided by the present invention can effectively promote C cell maturation and the expression of related cytokine IL-12.

[0072] In summary, the factor composition for activating DC cells provided by the present invention can promote DC cell proliferation, improve DC cell viability, increase the expression of antigen co-stimulatory molecules, and shorten the DC cell culture period.

[0073] 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 factor composition for activating DC cells, characterized in that: It includes the following ingredients: rhGM-CSF, rhIL-4, rhSCF, rhTNF-α, IFN-α, insulin and wolfberry polysaccharides.

2. The factor composition according to claim 1, characterized in that In the factor composition, the concentration of rhGM-CSF is 650-850 U / mL; The concentration of the rhIL-4 is 150-250 U / mL: The concentration of rhSCF is 60-80 ng / mL; The concentration of rhTNF-α is 80-100 U / mL; The concentration of IFN-α is 100-120 U / mL; The concentration of the insulin is 10-20 nmol / L; The concentration of the wolfberry polysaccharide is 68-80 μg / mL.

3. The factor composition according to claim 2, characterized in that: The basic solvent in the factor composition is RPMI-1640 complete medium.

4. Use of the factor composition according to any one of claims 1 to 3 in activating DC cells.

5. The use according to claim 4, characterized in that: The method for activating DC cells is: (1) inoculating PBMC in the factor composition, adding 8-12% (v / t) serum, transferring to a culture bottle for culture, and after the culture is completed, transferring the suspended cells, and the remaining adherent cells are undifferentiated DC cells; (2) adding the factor composition to undifferentiated DC cells and culturing them; (3) adding a mixed solution of the factor composition and the OVA antigen solution, continuing the culture, and collecting mature DC cells.

6. The use according to claim 5, characterized in that The seeding density of PBMC in step (1) is (2.0-2.5)×10 6 Pieces / mL.

7. The use according to claim 6, characterized in that The DC cell culture conditions in step (1) are: 4-6% CO2, 36-37°C, 1-2h.

8. The use according to claim 7, characterized in that The amount of the factor composition added in step (2) is 50-70% of the volume of the culture bottle, and the culture conditions are 4-6% CO2, 36-37°C, and 10-20h.

9. The use according to claim 8, characterized in that The mixed solution in step (3) is prepared by mixing the factor composition and the OVA antigen solution in a volume ratio of 1:1 to 2; The amount of the mixed solution added is 4-6% of the volume of the factor composition in step (2).

10. The use according to claim 9, characterized in that The conditions for continuing the culture in step (3) are 4-6% CO2, 36-37°C, and 10-15h.