Culture method for enhancing secretion ability of DC-CIK cell factors
By mixing colon cancer cells with DC-CIK cells and blocking treatment with BrefeldinA and monenzyme, the problem of insufficient secretion of DC-CIK cytokine is solved, and its killing effect and immunity on tumor cells is significantly improved. Memory effect.
Patent Information
- Application Number
- CN202411653948.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2024-12-31
AI Technical Summary
The existing DC-CIK cell preparation methods have problems such as the CIK cell culture environment is not conducive to DC cell growth, slow DC cell maturation and short survival period, which limits the amplification and killing function of DC-CIK cells.
The cytokine secretion ability of DC-CIK cells was enhanced by mixing colon cancer cells with DC-CIK cells, and adding BrefeldinA solution and monenectin solution to the co-culture product for blocking treatment.
It significantly improves the killing effect of DC-CIK cells on tumor cells and enhances the immune memory effect.
Smart Images
Figure CN119220493A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell culture, and in particular to a culture method for enhancing the cytokine secretion ability of DC-CIK. Background Art
[0002] DC-CIK cells, i.e. cells co-cultured with dendritic cells and cytokine-induced killer cells, are immune cells that combine the antigen presentation function of dendritic cells (DC) with the killing function of CIK cells. DC cells can effectively present tumor antigens to T cells, while CIK cells have strong anti-tumor activity.
[0003] DC-CIK cells show great potential in tumor immunotherapy. They can effectively identify and attack tumor cells, while enhancing immune response by secreting cytokines. This cell therapy is considered one of the preferred options for adoptive immunotherapy of tumors. However, although DC-CIK cells show potential in tumor treatment, existing preparation methods have some limitations, such as the CIK cell culture environment is not conducive to DC cell growth, DC cell maturation is slow, and the survival period is short. These problems limit the expansion and killing function of DC-CIK cells. Summary of the invention
[0004] The purpose of the present invention is to provide a culture method for enhancing the cytokine secretion ability of DC-CIK cells, which can significantly improve the killing effect of DC-CIK cells on tumor cells and enhance the immune memory effect.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a method for cultivating DC-CIK cells to enhance their cytokine secretion ability, comprising the following steps:
[0007] (1) culturing colon cancer cells to obtain cultured colon cancer cells;
[0008] (2) mixing the cultured colon cancer cells and DC-CIK cells and co-culturing them to obtain a co-culture product;
[0009] (3) adding Brefeldin A solution and monensin solution to the co-culture product for blocking to obtain a blocking product;
[0010] (4) The blocking product is centrifuged, and the precipitate is resuspended and then blocked to obtain DC-CIK with enhanced cytokine secretion ability.
[0011] Preferably, the colon cancer cells in step (1) are SW480 colon cancer cells.
[0012] Preferably, the colon cancer cells in step (1) need to be pre-cultured before culturing;
[0013] The pre-culture method comprises the following steps:
[0014] A. Place colon cancer cells at 35-39°C and 4-6% CO2 for 44-52 hours, and replace the culture medium with 1640 culture medium containing 8-12% FBS;
[0015] B. When the cell confluence reaches 75-85%, cell passage is performed;
[0016] C. Remove the culture supernatant, wash the cells with PBS and then digest with trypsin;
[0017] D. Add 1640 culture medium containing 8-12% FBS, centrifuge and resuspend to obtain pre-cultured SW480 colon cancer cells;
[0018] The centrifugal conditions are: 300-500g centrifugation for 5-10 min.
[0019] Preferably, the culture conditions in step (1) are 35-39° C. and 4-6% CO 2 , and the culture time is 22-26 h.
[0020] Preferably, in step (2), the ratio of the number of colon cancer cells to the number of DC-CIK cells after culture is 1:0.8-1.2.
[0021] Preferably, the co-culture conditions in step (2) are 35-39° C. and 4-6% CO 2 , and the culture time is 18-22 h.
[0022] Preferably, in step (3), the final concentration of the Brefeldin A solution is 3-5 mg / mL, the final concentration of the monensin solution is 3-5 mg / mL, and the blocking time is 4-5 h.
[0023] Preferably, the centrifugation conditions in step (4) are: 280-320 g, 4-6 min, and FBS is used for resuspension.
[0024] Preferably, HumanBD Fc Block is used for blocking in step (4), and the blocking time is 8 to 12 minutes.
[0025] The present invention provides a culture method for enhancing the cytokine secretion ability of DC-CIK, comprising the following steps: (1) culturing colon cancer cells to obtain cultured colon cancer cells; (2) mixing the cultured colon cancer cells and DC-CIK cells for co-culture to obtain a co-culture product; (3) adding BrefeldinA solution and monensin solution to the co-culture product for blocking to obtain a blocking product; (4) centrifuging the blocking product, resuspending the precipitate and sealing to obtain DC-CIK with enhanced cytokine secretion ability. The present invention utilizes the stimulating effect of colon cancer cells on DC-CIK cells, and co-stimulates through specific culture conditions to obtain DC-CIK cells with enhanced cytokine secretion ability. The cytokine secretion ability is enhanced, the killing effect on tumor cells is significantly improved, and the immune memory effect is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the result of flow cytometry detection of cytokine secretion. DETAILED DESCRIPTION
[0027] The present invention provides a method for cultivating DC-CIK cells to enhance their cytokine secretion ability, comprising the following steps:
[0028] (1) culturing colon cancer cells to obtain cultured colon cancer cells;
[0029] (2) mixing the cultured colon cancer cells and DC-CIK cells and co-culturing them to obtain a co-culture product;
[0030] (3) adding Brefeldin A solution and monensin solution to the co-culture product for blocking to obtain a blocking product;
[0031] (4) The blocking product is centrifuged, and the precipitate is resuspended and then blocked to obtain DC-CIK with enhanced cytokine secretion ability.
[0032] In the present invention, the colon cancer cells in step (1) are preferably SW480 colon cancer cells.
[0033] In the present invention, the colon cancer cells in step (1) are preferably pre-cultured before culturing;
[0034] The pre-culture method preferably comprises the following steps:
[0035] A. Place colon cancer cells at 35-39°C and 4-6% CO2 for 44-52 hours, and replace the culture medium with 1640 culture medium containing 8-12% FBS;
[0036] B. When the cell confluence reaches 75-85%, cell passage is performed;
[0037] C. Remove the culture supernatant, wash the cells with PBS and then digest with trypsin;
[0038] D. Add 1640 culture medium containing 8-12% FBS, centrifuge and resuspend to obtain pre-cultured SW480 colon cancer cells.
[0039] In the present invention, the centrifugal conditions are preferably: 300-500g for 5-10 min, more preferably 350-450g for 6-9 min, and even more preferably 400g for 7-8 min.
[0040] In the present invention, the stationary conditions in step A are preferably 37° C. and 5% CO 2 .
[0041] In the present invention, the standing time in step A is preferably 48 hours.
[0042] In the present invention, the culture medium in step A is preferably 1640 culture medium containing 10% FBS.
[0043] In the present invention, the cell confluence degree in step B is preferably 80%.
[0044] In the present invention, the culture medium in step D is preferably 1640 culture medium containing 10% FBS.
[0045] In the present invention, the culture conditions in step (1) are preferably 35-39° C., 4-6% CO 2 , more preferably 37° C., 5% CO 2 , and the culture time is preferably 22-26 h, more preferably 24 h.
[0046] In the present invention, the ratio of the number of colon cancer cells to the number of DC-CIK cells after culture in step (2) is preferably 1:0.8-1.2, and more preferably 1:1.
[0047] In the present invention, the co-culture conditions in step (2) are preferably 35-39° C., 4-6% CO 2 , more preferably 37° C., 5% CO 2 , and the culture time is preferably 18-22 h, more preferably 20 h.
[0048] In the present invention, the final concentration of the Brefeldin A solution in step (3) is preferably 3-5 mg / mL, more preferably 4 mg / mL, the final concentration of the monensin solution is preferably 3-5 mg / mL, more preferably 4 mg / mL, and the blocking time is preferably 4-5 h, more preferably 4.5 h.
[0049] In the present invention, the centrifugation conditions in step (4) are preferably: 280-320 g, 4-6 min, more preferably 300 g, 5 min, and the resuspension is preferably performed using FBS.
[0050] In the present invention, HumanBD Fc Block is preferably used for the blocking in step (4), and the blocking time is preferably 8 to 12 minutes, more preferably 10 minutes.
[0051] 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.
[0052] The reagents and consumables used in the following examples are specifically shown in Table 1.
[0053] Table 1
[0054]
[0055]
[0056] Example 1 Preparation of SW480 colon cancer cells
[0057] SW480 colon cancer cells were purchased from CCTCC (China Center for Type Culture Collection). After receiving the cells, the culture flask was placed in a CO2 incubator for 2 days, and then the medium was replaced with 1640 culture medium containing 10% FBS; the cell fusion degree was observed under a microscope, and when it reached 80%, the cells were passaged; the supernatant was removed, washed once with PBS, and then trypsin was added, and the culture flask was placed in a CO2 incubator for 10 minutes; after digestion, 5 mL of 1640 culture medium containing 10% FBS was added to terminate digestion, the cells were collected, the supernatant was removed by centrifugation, the cells were resuspended, samples were taken and counted, and 3E6 cells to 6E6 cells were passaged according to the counting results.
[0058] Example 2 Preparation of DC-CIK cells
[0059] PBMCs of healthy people were purchased from Shanghai Aoneng and frozen; MNCs were separated by lymphocyte separation fluid (Ficoll) and the cell concentration was adjusted to 5×10 in AIM-V serum-free medium. 6 The cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. Non-adherent cells were collected and the cell concentration was adjusted to 2×10 cells / ml using IMDM containing 10% human AB serum. 6 / ml, and add 1000U / ml IFN-α suspension culture, add 1μg / ml CD3 monoclonal antibody and 1000U / ml rhIL-2 after 24 hours, change half of the medium every 3 days, supplement rhIL-2, and adjust the cell concentration to 2×106 / ml; add AIM-V culture medium containing 1000U / ml GM-CSF and 1000U / ml IL-4 to the adherent cells after PBMNC culture for 2 hours, and culture in a 37℃, 5% CO2 incubator; change the medium once every 3 days and supplement cytokines, add IL-1&beta, TNF-&alpha, PGE-2 and other cytokines together with the above cytokines on the 6th day to induce DC maturation, and culture for 9 days; the harvested DCs are counted and mixed with CIK at a ratio of 1:5, and cultured for 5 days to obtain DC-CIK cells.
[0060] Example 3 Cultivation method for enhancing the cytokine secretion ability of DC-CIK
[0061] ① SW480 cell plating: 24 h before adding DC-CIK cells, SW480 cells were plated at 2×10 6 The 6-well plate was plated with 5% CO2 at 37°C for 24 h in a carbon dioxide incubator.
[0062] ② 24 hours after plating, DC-CIK cells were obtained and cell counts were performed. According to the counting results, DC-CIK cells were divided into 2×10 6 The cells / well were added to a 6-well plate, and the 6-well plate was placed in a carbon dioxide incubator at 37°C and 5% CO2 for 20 h.
[0063] ③ After 20 hours of co-stimulation, take out the 6-well plate, add 3 μL of Brefeldin A solution (1000X) and monensin solution (1000X) respectively, mix well, and block for 5 hours.
[0064] ④ Fc blocking: transfer the cells to a 1.5mL centrifuge tube, centrifuge at 300g for 5min, remove the supernatant, add 100μL Stain Buffer (FBS) to resuspend the cells, then add 5μL Human BD Fc Block for blocking, leave at room temperature for 10min, and then proceed to the staining step without washing.
[0065] ⑤ Add 2.5 μL of 100-fold diluted live / dead dye, incubate at room temperature for 20 min, then add 1 mL of Stain Buffer for washing.
[0066] ⑥ Fixation and membrane permeabilization: Add 100μl of Fixation and Permealdehyde Solution, vortex to mix, and fix for 20 minutes at 4℃. After fixation, add 1mL of BD Perm / Wash Buffer (1x) to each tube, centrifuge at 300g for 5 minutes, and remove the supernatant.
[0067] ⑦ Intracellular factor staining: Add 2.5 μL of INF-γ, TNF-α, Granzyme-B, and Perforin antibodies respectively, incubate at room temperature for 30 minutes, then add 1 mL of BD Perm / Wash Buffer (1x), and wash twice at 300g for 5 minutes.
[0068] ⑧On-machine detection: Use Beckman's CytoFlex flow cytometer to detect cytokine secretion.
[0069] result:
[0070] Test results such as Figure 1 shown.
[0071] ① Determine the door position based on the unstain tube and ISO tube. (Unstain: 95.00%~100.00%, ISO: 95.00%~100.00%)
[0072] ②As shown in the figure, after stimulation by SW480 cells, the four cytokines were secreted at a high level (INF-γ+: 14.92%, TNF-α: 24.07%, Granzyme B+: 62.12%, perforin+: 24.51), proving the stimulating effect of SW480 on them.
[0073] As can be seen from the above embodiments, the present invention provides a culture method for enhancing the cytokine secretion ability of DC-CIK, comprising the following steps: (1) culturing colon cancer cells to obtain cultured colon cancer cells; (2) mixing the cultured colon cancer cells and DC-CIK cells and co-culturing to obtain a co-culture product; (3) adding BrefeldinA solution and monensin solution to the co-culture product for blocking to obtain a blocking product; (4) centrifuging the blocking product, resuspending the precipitate and sealing to obtain DC-CIK with enhanced cytokine secretion ability. The present invention utilizes the stimulating effect of colon cancer cells on DC-CIK cells, and co-stimulates through specific culture conditions to obtain DC-CIK cells with enhanced cytokine secretion ability. The cytokine secretion ability is enhanced, the killing effect on tumor cells is significantly improved, and the immune memory effect is enhanced.
[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 enhancing the cytokine secretion ability of DC-CIK, characterized in that: The steps include: (1) culturing colon cancer cells to obtain cultured colon cancer cells; (2) mixing the cultured colon cancer cells and DC-CIK cells and co-culturing them to obtain a co-culture product; (3) adding Brefeldin A solution and monensin solution to the co-culture product for blocking to obtain a blocking product; (4) The blocking product is centrifuged, and the precipitate is resuspended and then blocked to obtain DC-CIK with enhanced cytokine secretion ability.
2. The culture method according to claim 1, characterized in that The colon cancer cells described in step (1) are SW480 colon cancer cells.
3. The culture method according to claim 2, characterized in that The colon cancer cells described in step (1) need to be pre-cultured before culturing; The pre-cultivation method comprises the following steps: A. Place colon cancer cells at 35-39°C and 4-6% CO2 for 44-52 hours, and replace the culture medium with 1640 culture medium containing 8-12% FBS; B. When the cell confluence reaches 75-85%, cell passage is performed; C. Remove the culture supernatant, wash the cells with PBS and then digest with trypsin; D. Add 1640 culture medium containing 8-12% FBS, centrifuge and resuspend to obtain pre-cultured SW480 colon cancer cells; The centrifugal conditions are: 300-500g centrifugation for 5-10 min.
4. The culture method according to claim 3, characterized in that The culture conditions in step (1) are 35-39° C. and 4-6% CO 2 , and the culture time is 22-26 hours.
5. The culture method according to claim 4, characterized in that The ratio of the number of colon cancer cells and DC-CIK cells after culture in step (2) is 1:0.8-1.
2.
6. The culture method according to claim 5, characterized in that: The co-culture conditions in step (2) are 35-39° C. and 4-6% CO 2 , and the culture time is 18-22 hours.
7. The culture method according to claim 6, characterized in that: In step (3), the final concentration of the Brefeldin A solution is 3-5 mg / mL, the final concentration of the monensin solution is 3-5 mg / mL, and the blocking time is 4-5 h.
8. The culture method according to claim 7, characterized in that: The centrifugation conditions in step (4) are: 280-320 g, 4-6 min, and FBS is used for resuspension.
9. The culture method according to any one of claims 1 to 8, characterized in that: In step (4), Human BD Fc Block is used for blocking, and the blocking time is 8 to 12 minutes.