In-vitro culture method of T cells

Abstract

The invention provides an in-vitro culture method of T cells, and belongs to the technical field of genetic engineering.The culture method comprises the steps that immune cell serum-free culture media are added into peripheral blood mononuclear cells, and activated culture is conducted in the presence of cell factors to obtain activated T cells; the cell factor comprises an IL-2 monoclonal antibody and a CD3 monoclonal antibody, and further comprises two or three of an iCAM1 protein, a CCL21 protein and OK432. The T cells obtained through activation and multiplication culture are relatively stable in cell phenotype, relatively large in amplification multiple, relatively high in cell viability and relatively high in tumor killing efficiency. T cells are obtained after activation is performed for 24 hours and then multiplication culture is performed for 14 days, and the number of the cells is 6.2-8.9 * 10 < 9 >; the killing rate on Hela cells is 51.9 to 74.1 percent, and the killing rate on LOVO cells is 55.7 to 80.5 percent.

Description

Technical Field

[0001] The invention relates to an in vitro culture method of T cells, belonging to the technical field of genetic engineering. Background Art

[0002] The main member of lymphocytes in the human body is T cells, which have multiple biological functions, such as directly killing target cells, assisting or inhibiting B cells to produce antibodies, responding to specific antigens and mitogens, and producing cytokines. They are the "fighters" in the body against disease infection and tumor formation.

[0003] At present, the main methods of tumor treatment include surgery, radiotherapy, chemotherapy, and traditional therapy. Radiotherapy and chemotherapy can kill both tumor cells and lymphocytes in the body. If T lymphocytes can be supplemented in time, it will strengthen the damaged immune system, help restore the number and function of T cells, and reduce the side effects of radiotherapy and chemotherapy.

[0004] At present, the methods for culturing immune cells are mainly CIK cells and NK cells. Patent CN118773134A is an improvement on the culture medium for CIK cells, mainly adding three of anti-human CD3 monoclonal antibodies, anti-human CD28 antibodies, IFN-γ, GM-CSF, IL-2, IL-15, IL-7, IL-21, and Nicotinamide to increase the number of CIK cells. Patent CN118406649A developed a serum-free culture medium for CIK cells, adding glucose, L-alanyl-L-glutamine, N-acetylcysteine, PMA, ionomycin, and 2-mercaptoethanol, which increased the CD3 in CIK cells. + CD56 + The CN118440894A patent provides a NK cell in vitro expansion culture medium, the added factors include IL-15, IL-21, IL-18, autologous plasma, IL-2, vitamin B1, vitamin B6.

[0005] During the process of culturing T cells, the applicant discovered that conventional immune cell culture methods have problems such as slow T cell growth, poor proliferation effect, and weak cytotoxicity to tumor cells, which is not conducive to the high purity and quantity requirements of immune T cells in clinical treatment. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides an in vitro culture method for T cells to achieve the following invention objectives: to increase the proliferation multiples of T cells and enhance the cytotoxicity to tumor cells.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: An in vitro culture method for T cells comprises adding immune cell serum-free culture medium to peripheral blood mononuclear cells, and performing activation culture in the presence of cytokines to obtain activated T cells; the cytokines include IL-2, CD3 monoclonal antibody, and two or three of iCAM1 protein, CCL21 protein, and OK432.

[0008] The final concentration of the IL-2 is 490-510 IU / mL, the final concentration of the CD3 monoclonal antibody is 48-52 ng / mL, and the final concentration of the OK432 is 8-12 ng / mL.

[0009] The iCAM1 protein is added to the immune cell serum-free culture medium or coated on the culture flask; when the iCAM1 protein is added to the immune cell serum-free culture medium, the final concentration of the iCAM1 protein is 4.8-5.2 ng / mL; when the iCAM1 protein is coated on the culture flask, the culture flask is coated with 0.48-0.52 μg / mL of iCAM1 protein for 1 day.

[0010] The CCL21 protein is added to the immune cell serum-free culture medium or coated on the culture flask; when the CCL21 protein is added to the immune cell serum-free culture medium, the final concentration of the CCL21 protein is 14-16 ng / mL; when the CCL21 protein is coated on the culture flask, the culture flask is coated with 1.4-1.6 μg / mL of CCL21 protein for 1 day.

[0011] When iCAM1 protein and CCL21 protein are coated on culture flasks at the same time, the culture flasks are first coated with 0.48-0.52 μg / mL iCAM1 protein for 1 day, the coating solution is removed, the flasks are washed, and then coated with 1.4-1.6 μg / mL CCL21 protein for 1 day.

[0012] The activation culture time is 22-26 hours.

[0013] The activated T cells were expanded and cultured in an immune cell serum-free medium containing IL-2 at a final concentration of 490-510 IU / mL.

[0014] Compared with the prior art, the present invention achieves the following beneficial effects: The T cells obtained by activation and amplification culture of the present invention have a relatively stable cell phenotype, a large amplification multiple, a high cell viability, and a high tumor killing efficiency.

[0015] The T cells obtained by the present invention after activation for 24 hours and then amplification and culture for 14 days have a cell number of 6.2-8.9×10 9The cell viability was 96.18-98.25%, and the CD3+ ratio was 94.3-97.6%. The killing rate against Hela cells was 51.9-74.1%, and the killing rate against LOVO cells was 55.7-80.5%. DETAILED DESCRIPTION

[0016] Example 1 A method for culturing T cells in vitro 50 mL of peripheral blood donated by volunteers was collected and PBMC (peripheral blood mononuclear cells) was isolated using TBD sample density separation solution (purchased from Tianjin Haoyang Huake Biotechnology Co., Ltd.). The isolated PBMC cells were counted and the number of cells was calculated as 1×10 6 cells / mL, add immune cell serum-free medium, add IL-2 (purchased from Shenyang Sansheng Pharmaceutical) with a final concentration of 500 IU / mL, 50 ng / mL CD3 monoclonal antibody (purchased from Tongli Haiyuan), 5 ng / mL iCAM1 protein (purchased from MCE, catalog number: HY-P72621), 15 ng / mL CCL21 protein (purchased from MCE, catalog number: HY-P72875) at a final concentration, and place in an incubator at 37°C and 5% CO2 for activation and culture for 24 h to obtain activated T cells.

[0017] Immune cell serum-free culture medium containing 500 IU / mL (final concentration) of IL-2 was added in multiple ratios every 3 days, and the expansion culture was continued for 14 days to obtain expanded T cells.

[0018] The immune cell serum-free culture medium was purchased from Youkang Biotechnology (Beijing) Co., Ltd.

[0019] Example 2 A method for culturing T cells in vitro The cytokines added in the activation culture stage in this example are different from those in Example 1, and the remaining steps are the same as those in Example 1.

[0020] In this example, the cytokines added in the activation culture stage were IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody with a final concentration of 50 ng / mL, OK432 with a final concentration of 10 ng / mL (purchased from Tongli Haiyuan), and CCL21 protein with a final concentration of 15 ng / mL.

[0021] Example 3 A method for culturing T cells in vitro The cytokines added in the activation culture stage in this example are different from those in Example 1, and the remaining steps are the same as those in Example 1.

[0022] In this example, the cytokines added in the activation culture stage are IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody with a final concentration of 50 ng / mL, OK432 with a final concentration of 10 ng / mL, and iCAM1 protein with a final concentration of 5 ng / mL.

[0023] Example 4 A method for culturing T cells in vitro The cytokines added in the activation culture stage in this example are different from those in Example 1, and the remaining steps are the same as those in Example 1.

[0024] In this example, the cytokines added in the activation culture stage are IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody with a final concentration of 50 ng / mL, iCAM1 protein with a final concentration of 5 ng / mL, CCL21 protein with a final concentration of 15 ng / mL, and OK432 with a final concentration of 10 ng / mL.

[0025] Example 5 A method for culturing T cells in vitro In the activation culture stage of this embodiment, the types of cytokines used are the same as those in Example 4, the only difference is that CCL21 protein is not added to the immune cell serum-free culture medium, but the culture flask is coated, and the remaining steps are the same as those in Example 4.

[0026] In the activation culture stage of this embodiment, IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody of 50 ng / mL, OK432 of 10 ng / mL, and iCAM1 protein of 5 ng / mL were added. Before culturing cells, the culture flask was coated with CCL21 protein containing 1.5 μg / mL for 1 day, and the coating solution was removed before use, and the flask was washed once with PBS.

[0027] Example 6 A method for culturing T cells in vitro In the activation culture stage of this embodiment, the types of cytokines used are the same as those in Example 4, the only difference is that the iCAM1 protein is not added to the immune cell serum-free culture medium, but the culture flask is coated, and the remaining steps are the same as those in Example 4.

[0028] In the activation culture stage of this embodiment, IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody of 50 ng / mL, OK432 of 10 ng / mL, and CCL21 protein of 15 ng / mL were added. Before cell culture, the culture flask was coated with 0.5 μg / mL iCAM1 protein for 1 day, and the coating solution was removed before use, and the flask was washed once with PBS.

[0029] Example 7 A method for culturing T cells in vitro In the activation culture stage of this embodiment, the types of cytokines used are the same as those in Example 4, the only difference is that iCAM1 protein and CCL21 protein are not added to the immune cell serum-free culture medium, but the culture flask is coated, and the remaining steps are the same as those in Example 4.

[0030] In the activation culture stage of this embodiment, IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody with a concentration of 50 ng / mL, and OK432 with a concentration of 10 ng / mL were added. Before cell culture, the culture flask was first coated with 0.5 μg / mL iCAM1 protein for 1 day, the coating solution was removed, and the flask was washed with PBS, and then 1.5 μg / mL CCL21 protein was added for coating for 1 day, the coating solution was removed before use, and the flask was washed with PBS.

[0031] Comparative Example 1: In the activation culture stage of this example, IL-2 with a final concentration of 500 IU / mL and CD3 monoclonal antibody with a concentration of 50 ng / mL were added, and the remaining steps were the same as those in Example 1.

[0032] Comparative Example 2: In the activation culture stage of this example, IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody with a final concentration of 50 ng / mL, and iCAM1 protein with a final concentration of 5 ng / mL were added, and the remaining steps were the same as those in Example 1.

[0033] Comparative Example 3: In the activation culture stage of this example, IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody with a concentration of 50 ng / mL, and CCL21 protein with a concentration of 15 ng / mL were added, and the remaining steps were the same as those in Example 1.

[0034] Comparative Example 4: In the activation culture stage of this embodiment, IL-2 with a final concentration of 500 IU / mL, CD3 monoclonal antibody with a concentration of 50 ng / mL, and OK432 with a concentration of 10 ng / mL were added, and the remaining steps were the same as those in Example 1.

[0035] After 14 days of activation and expansion, samples were taken from the T cells of Examples 1-7 and Comparative Examples 1-4, and the cell count and viability were tested. At the same time, a portion of the samples were taken for flow cytometry testing of CD3+ indicators. The results are shown in Table 1.

[0036] Table 1 Changes of cell indexes in Examples and Comparative Examples

[0037] The results in Table 1 show that the number of cells in Examples 1-3 is significantly greater than that in Comparative Examples 2-4. In Example 1-3, two of iCAM1 protein, CCL21 protein, and OK432 were added during the activation culture stage, while Comparative Example 2-4 only added one of iCAM1 protein, CCL21 protein, and OK432, indicating that any two of iCAM1 protein, CCL21 protein, and OK432 have a synergistic effect in increasing the number of T cells. The number of cells in Example 4-7 is significantly greater than that in Example 1-3. In Example 4-7, T cells are activated in an environment containing iCAM1 protein, CCL21 protein, and OK432 at the same time, indicating that iCAM1 protein, CCL21 protein, and OK432 have a synergistic effect in increasing the number of T cells.

[0038] As for the indicators of cell viability and CD3+ positive rate, the results of the embodiment and the comparative example are not much different.

[0039] The same amount of cells were taken from the T cells activated and expanded for 14 days in the examples and comparative examples for in vitro killing efficiency detection. Hela cells and LOVO cells labeled with luciferase were used for in vitro killing of tumor cells detection. 4 The target cells were plated in 96-well plates and the in vitro killing experiment was performed at an effector-target ratio of 5:1. The results are shown in Table 2.

[0040] Table 2 In vitro cell killing efficiency of Examples and Comparative Examples

[0041] As can be seen from Table 2, when the effect-target ratio is 5:1, the killing efficiency of T cells cultured in Examples 1 to 7 on tumor cells is all above 50%, while the killing efficiency of T cells cultured in the comparative example on tumor cells is less than 40%. This indicates that the culture medium of Examples 1 to 7 has the function of activating T cells and improving their tumor killing rate. The killing rate of target cells in Examples 1-3 is significantly higher than that in Comparative Examples 2-4, indicating that any two of the iCAM1 protein, CCL21 protein, and OK432 have a synergistic effect in improving the killing rate of T cells on target cells. The killing rate of target cells in Examples 4-7 is significantly higher than that in Examples 1-3, indicating that the three of iCAM1 protein, CCL21 protein, and OK432 have a synergistic effect in improving the killing rate of T cells on target cells.

Claims

1. A method for culturing T cells in vitro, characterized in that: The culture method comprises adding immune cell serum-free culture medium to peripheral blood mononuclear cells, and performing activation culture in the presence of cytokines to obtain activated T cells; the cytokines include IL-2, CD3 monoclonal antibody, and two or three of iCAM1 protein, CCL21 protein, and OK432.

2. The in vitro culture method of T cells according to claim 1, characterized in that: The final concentration of the IL-2 is 490-510 IU / mL, the final concentration of the CD3 monoclonal antibody is 48-52 ng / mL, and the final concentration of the OK432 is 8-12 ng / mL.

3. The in vitro culture method of T cells according to claim 1, characterized in that: The iCAM1 protein is added to the immune cell serum-free culture medium or coated on the culture flask; when the iCAM1 protein is added to the immune cell serum-free culture medium, the final concentration of the iCAM1 protein is 4.8-5.2 ng / mL; when the iCAM1 protein is coated on the culture flask, the culture flask is coated with 0.48-0.52 μg / mL of iCAM1 protein for 1 day.

4. The in vitro culture method of T cells according to claim 3, characterized in that: The CCL21 protein is added to the immune cell serum-free culture medium or coated on the culture flask; when the CCL21 protein is added to the immune cell serum-free culture medium, the final concentration of the CCL21 protein is 14-16 ng / mL; when the CCL21 protein is coated on the culture flask, the culture flask is coated with 1.4-1.6 μg / mL of CCL21 protein for 1 day.

5. The in vitro culture method of T cells according to claim 4, characterized in that: When iCAM1 protein and CCL21 protein are coated on culture flasks at the same time, the culture flasks are first coated with 0.48-0.52 μg / mL iCAM1 protein for 1 day, the coating solution is removed, the flasks are washed, and then coated with 1.4-1.6 μg / mL CCL21 protein for 1 day.

6. The in vitro culture method of T cells according to claim 1, characterized in that: The activation culture time is 22-26 hours.

7. The in vitro culture method of T cells according to claim 1, characterized in that: The activated T cells were expanded and cultured in an immune cell serum-free medium containing IL-2 at a final concentration of 490-510 IU / mL.

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