A composition of factors to activate DC cells and methods of use thereof

Abstract

The application provides a factor composition for activating DC cells and a method for application thereof, and belongs to the technical field of cell preparations.The factor composition for activating DC cells provided by the application comprises the following components: rhGM-CSF, rhIL-4, rhSCF, rhTNF-alpha, IFN-alpha, insulin and lycium barbarum polysaccharide.Through the synergistic effect between the components, the application can not only promote the differentiation of PBMC into DC cells, increase the proportion of DC cells, increase the initial amount of DC cells, promote the proliferation of DC cells, increase the activity of DC cells, but also can increase the expression of antigen costimulatory molecules and reduce the culture cycle of DC cells.

Description

Technical Field

[0001] This invention relates to the field of cell preparation technology, and in particular to a factor composition for activating DC cells and a method for applying it. Background Technology

[0002] Dendritic cells (DCs), discovered by Canadian scholar Steinman in 1973, are the body's most potent professional antigen-presenting cells (APCs), capable of efficiently uptake, processing, and presenting antigens. Most DCs in the human body are immature, expressing low levels of co-stimulatory factors and adhesion factors, resulting in a low ability to elicit allogeneic mixed lymphocyte proliferation responses in vitro. Mature DCs, however, express high levels of co-stimulatory factors and adhesion factors. During maturation, DCs migrate from antigen-contacting peripheral tissues to secondary lymphoid organs, where they contact T cells and elicit an immune response. Current techniques for preparing DCs result in low expression of antigen co-stimulatory molecules, poor T cell activation, and prolonged culture times. Therefore, there is a need to provide a factor composition that can activate DCs, promote DC proliferation, increase the expression of antigen co-stimulatory molecules, reduce the DC culture cycle, and improve DC cell viability. Summary of the Invention

[0003] The purpose of this invention is to provide a method for promoting DC cell proliferation, increasing the expression of antigen co-stimulatory molecules, reducing DC cell culture cycle, and improving DC cell viability.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a factor composition for activating dendritic cells, comprising the following components: rhGM-CSF, rhIL-4, rhSCF, rhTNF-α, IFN-α, insulin, and Lycium barbarum polysaccharide;

[0006] The concentration of rhGM-CSF is 650–850 U / mL;

[0007] The concentration of 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 insulin is 10–20 nmol / L;

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

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

[0014] This invention provides the application of the above-mentioned factor composition in activating DC cells.

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

[0016] (1) PBMCs are seeded into the factor composition, 8-12% (v / t) serum is added, and the cells are transferred to a culture flask for culture. After the culture is completed, the suspended cells are transferred and the remaining adherent cells are undifferentiated DC cells.

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

[0018] (3) Add the mixture of the factor composition and OVA antigen solution, continue culturing, and collect mature DC cells.

[0019] Preferably, the inoculation density of PBMCs in step (1) is (2.0~2.5)×10⁻¹⁰. 6 cells / mL;

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

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

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

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

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

[0025] Experiments have shown that the factor composition for activating dendritic cells (DCs) provided by this invention not only promotes the differentiation of PBMCs into DCs, increases the proportion of DCs, increases the initial number of DCs, promotes DC proliferation, and improves DC viability, but also enhances the expression of antigen co-stimulatory molecules and reduces the DC culture cycle. This invention stimulates cell differentiation and growth into DCs by adding rhTNF-α and IFN-α, promotes DC development and induces cell maturation by adding rhSCF and Lycium barbarum polysaccharides, and simultaneously promotes glucose metabolism, facilitating further utilization of these substances by the cells. The components of the factor composition provided by this invention work synergistically to effectively promote DC proliferation, increase the expression of antigen co-stimulatory molecules, reduce the DC culture cycle, and enhance DC activity. Detailed Implementation

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

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] A factor composition for activating DC cells, based on RPMI-1640 complete medium, with the following components added (the final concentrations of each component are as follows): rhGM-CSF 650U / mL, rhIL-4 150U / mL, rhSCF 70ng / mL, rhTNF-α 80U / mL, IFN-α 100U / mL, insulin 15nmol / L, and Lycium barbarum polysaccharide 75μg / mL.

[0030] Example 2

[0031] A factor composition for activating dendritic cells, based on RPMI-1640 complete medium, with the following components added at concentrations: rhGM-CSF 700U / mL, rhIL-4250U / mL, rhSCF 80ng / mL, rhTNF-α 100U / mL, IFN-α 120U / mL, insulin 12nmol / L, and Lycium barbarum polysaccharide 68μg / mL.

[0032] Example 3

[0033] A factor composition for activating dendritic cells, based on RPMI-1640 complete medium, with the following components added at concentrations: rhGM-CSF 800U / mL, rhIL-4200U / mL, rhSCF 75ng / mL, rhTNF-α 97U / mL, IFN-α 115U / mL, insulin 18nmol / L, and Lycium barbarum polysaccharide 72μg / mL.

[0034] Example 4

[0035] Methods to activate DC cells:

[0036] (1) PBMCs were inoculated into 30 mL of the factor composition obtained in Example 1 at an inoculation density of 2 × 10⁻⁶. 6 Add 10% (v / t) of FBS (Hyclone) of the factor composition per mL, and transfer all of it to a T75 cell culture flask. Incubate at 37°C and 5% CO2 for 2 hours. After incubation, transfer the suspended cells. The remaining adherent cells are the undifferentiated DC cells.

[0037] (2) Resuspend the undifferentiated DC cells in 45 mL of the factor composition described in Example 1 to adjust the cell concentration to 1 × 10⁻⁶. 6 Incubate at 37℃ with 5% CO2 for 24 hours at a concentration of 1 / mL;

[0038] (3) Add 1.8 mL of the mixed solution (the mixed solution is a 1:1 mixture of the factor composition described in Example 1 and the OVA antigen solution, with an OVA antigen concentration of 25 μg / mL), and continue incubation at 37°C with 5% CO2. 2， After culturing for 24 hours, mature DC cells were collected.

[0039] Example 5

[0040] Methods to activate DC cells:

[0041] (1) PBMCs were inoculated into 30 mL of the factor composition obtained in Example 2 at an inoculation density of 2 × 10⁻⁶. 6 Add 8% (v / t) of FBS to the factor composition, and transfer all of it to a T75 cell culture flask. Incubate at 37°C and 5% CO2 for 2 hours. After incubation, transfer the suspended cells. The remaining adherent cells are the undifferentiated DC cells.

[0042] (2) Resuspend the undifferentiated DC cells in 50 mL of the factor composition described in Example 2, adjusting the cell concentration to 1 × 10⁻⁶. 6Incubate at 37℃ with 5% CO2 for 20 h at a concentration of 1 / mL;

[0043] (3) Add 2 mL of the mixed solution (the mixed solution is a 1:1 volume ratio of the factor composition described in Example 2 and the OVA antigen solution) and continue incubation at 37°C with 5% CO2. 2， After culturing for 20 hours, mature DC cells were collected.

[0044] Example 6

[0045] Methods to activate DC cells:

[0046] (1) PBMCs were inoculated into 30 mL of the factor composition obtained in Example 1 at an inoculation density of 2.5 × 10⁻⁶. 6 Add 10% (v / t) of FBS to the factor composition per mL, and transfer all of it to a T75 cell culture flask. Incubate at 37°C and 5% CO2 for 2 hours. After incubation, transfer the suspended cells. The remaining adherent cells are the undifferentiated DC cells.

[0047] (2) Resuspend the undifferentiated DC cells in 46 mL of the factor composition described in Example 1 to adjust the cell concentration to 1 × 10⁻⁶. 6 Incubate at 37℃ with 5% CO2 for 20 h at a concentration of 1 / mL;

[0048] (3) Add 2 mL of the mixed solution (the mixed solution is a 1:1 volume ratio of the factor composition described in Example 1 and the OVA antigen solution) and continue incubation at 37°C with 5% CO2. 2， After culturing for 20 hours, mature DC cells were collected.

[0049] Comparative Example 1

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

[0051] Comparative Example 2

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

[0053] Comparative Example 3

[0054] (1) Resuspend PBMCs in RPMI-1640 medium containing 1% fetal bovine serum (FBS) and adjust the cell concentration to 2×10⁻⁶.6 Incubate at 37°C and 5% CO2 for 2 hours, then discard the suspended cells;

[0055] (2) Then the adherent cells were resuspended in RPMI-1640 medium containing 10% FBS, and the cell concentration was adjusted to 1×10⁻⁶. 6 / mL, and add cytokines GM-CSF and IL-4 to a final concentration of 100ng / mL (GM-CSF and IL-4 mass concentration 4:1), and incubate at 37℃ in a 5% CO2 incubator for 7 days, changing half the medium every 3 days during the period;

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

[0057] Experimental Example 1

[0058] DC cells obtained after culturing using the cell culture methods given in Examples 4-6 and Comparative Examples 1-3 were collected. Mature DC cells were counted using a cell counter and 0.4% trypan blue staining, and cell viability 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 shown in Table 1, the number and viability of DC cells cultured using the methods in Examples 4-6 were significantly higher than those in Comparative Examples 1-3. Compared to Comparative Example 1, it can be seen that the factor composition provided by this invention can promote the differentiation of PBMCs into DC cells, increase the proportion of DC cells, and increase the initial amount of DC cells; compared to Comparative Example 2, it can be seen that the factor composition provided by this invention can effectively promote the proliferation of DC cells. In summary, this invention helps in the differentiation and proliferation of DC cells and reduces DC cell damage during culture.

[0062] Experimental Example 2

[0063] The expression of surface markers in mature DC cells obtained in Examples 4-6 and Comparative Examples 1-3 was detected by Beckman flow cytometry. The results are shown in Table 2.

[0064] Table 2 Expression of surface markers

[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 surface markers on mature DC cells obtained in Examples 4-6 was significantly higher than that in Comparative Examples 1-3. This indicates that the factor composition provided in this invention induces a higher positive expression rate in mature DC cells and can enhance the expression level of antigen co-stimulatory molecules.

[0067] Experimental Example 3

[0068] Culture supernatants from DC cells induced and cultured in Examples 4-6 and Comparative Examples 1-3 were collected on day 3. The IL-12 secreted by mature DC cells in the culture supernatant was detected using an interleukin-12 (IL-12) ELISA kit. Specific procedures were performed according to the ELISA instructions. 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 shown in Table 3, the IL-12 content secreted by the induced mature DC cells in Examples 4-6 was significantly higher than that secreted by the induced mature DC cells 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 the related cytokine IL-12.

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

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a factor composition for activating dendritic cells (DCs) in activating DCs, characterized in that, The method for activating DC cells is as follows: (1) PBMCs are seeded into the factor composition, and serum of 8-12% of the volume of the factor composition is added. The cells are then transferred to a culture flask for culture. After the culture is completed, the suspended cells are transferred and the remaining adherent cells are the undifferentiated DC cells. (2) Add the factor composition to undifferentiated DC cells and culture them; (3) Add the mixture of factor composition and OVA antigen solution, continue culturing, and collect mature DC cells; The factor composition consists of the following components: rhGM-CSF, rhIL-4, rhSCF, rhTNF-α, IFN-α, insulin, wolfberry polysaccharide, and basic solvent; The concentration of rhGM-CSF is 650~850 U / mL; The concentration of 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 insulin is 10~20 nmol / L; The concentration of the wolfberry polysaccharide is 68~80 μg / mL; The base solvent is RPMI-1640 complete culture medium.

2. The application as described in claim 1, characterized in that, The inoculation density of PBMCs in step (1) is (2.0~2.5)×10⁻¹⁰. 6 per mL.

3. The application as described in claim 2, characterized in that, The cell culture conditions in step (1) are: 4~6% CO2, 36~37℃, 1~2 h.

4. The application as described in claim 3, characterized in that, The amount of factor composition added in step (2) is 50-70% of the volume of the culture flask, and the culture conditions are 4-6% CO2, 36-37℃, 10-20h.

5. The application as described in claim 4, characterized in that, The mixed solution in step (3) is prepared by mixing the factor composition and the OVA antigen solution at a volume ratio of 1:1 to 2.

6. The application as described in claim 5, characterized in that, The conditions for continued cultivation in step (3) are 4~6% CO2, 36~37℃, 10~15 h.