Culture medium for gamma delta T cells and culture method of gamma delta T cells

Abstract

The invention provides a culture medium for gamma delta T cells and a culture method of the gamma delta T cells, and belongs to the technical field of cell culture. According to the culture medium of the gamma delta T cells, OpTmizer serves as a basic culture medium, and the OpTmizer culture medium further contains IL-2 with the concentration being 150-250 IU / mL, IL-6 with the concentration being 40-60 [mu] mol / L, vitamin C with the concentration being 40-60 [mu] mol / L, vitamin B3 with the concentration being 8-12 [mu] mol / L and coenzyme Q10 with the concentration being 3-7 [mu] mol / L. The prepared gamma delta T cell culture medium is used for culturing gamma delta T cells with improved killing activity, and the killing rate of the cultured gamma delta T cells on A549 cells and HepG2 cells can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a culture medium for γδT cells and a method for culturing γδT cells. Background Art

[0002] γδT cells are a type of T cell that performs innate immune functions and are considered to be a special type of immune cell between natural immunity and acquired immunity. Most γδT cells do not express CD4 and CD8 molecules, have the function of specifically recognizing antigens without MHC restrictions, and play an important role in the body's tumor immunity and infection immunity.

[0003] The prior art shows that there is a certain correlation between the killing activity of γδT cells against tumor cells and the components of the culture medium used. For example, CN 201510270659.3 discloses a method and application of efficiently inducing γδT cells, by including some components that can promote the specific proliferation and differentiation of γδT cells in a culture medium dedicated to culturing γδT cells, thereby being able to obtain more γδT cells with higher purity and stronger killing activity. Therefore, it is necessary to provide a culture medium for γδT cells and a method for culturing γδT cells, so as to provide a new method for developing γδT cells that efficiently kill tumor cells. Summary of the invention

[0004] The purpose of the present invention is to provide a culture medium for γδT cells and a culture method for γδT cells, so as to provide a theoretical basis for developing γδT cells with high efficiency in killing tumor cells.

[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 culture medium for γδT cells, using OpTmizer as a basic culture medium, wherein the OpTmizer culture medium further contains: IL-2 with a concentration of 150 to 250 IU / mL, IL-6 with a concentration of 40 to 60 μmol / L, vitamin C with a concentration of 40 to 60 μmol / L, vitamin B3 with a concentration of 8 to 12 μmol / L, and coenzyme Q10 with a concentration of 3 to 7 μmol / L.

[0007] Preferably, the OpTmizer culture medium further contains fetal bovine serum at a volume concentration of 2-8%.

[0008] The present invention provides an application of the culture medium in culturing γδT cells.

[0009] The invention provides a method for culturing γδT cells with improved killing activity. Peripheral blood mononuclear cells are cultured with the culture medium to obtain γδT cells with improved killing activity.

[0010] Preferably, the initial concentration of γδT cells during the culture is 1×10 5 ~1×10 7 Pieces / mL.

[0011] Preferably, the culture is carried out at a temperature of 36-38°C and a CO2 concentration of 4-6%.

[0012] Preferably, the cell medium is replaced every 2 to 4 days of culture.

[0013] Preferably, the total culture time is 10 to 20 days.

[0014] The present invention also provides a γδT cell cultured according to the method.

[0015] The present invention further provides an application of the γδT cells in the preparation of products for preventing or treating tumor cell killing.

[0016] The present invention provides a culture medium for γδT cells, which uses OpTmizer as a basic culture medium and is supplemented with IL-2, IL-6, vitamin C, vitamin B3 and coenzyme Q10. The present invention also uses the prepared culture medium for γδT cells for culturing γδT cells with improved killing activity, which can significantly improve the killing rate of the cultured γδT cells on A549 cells and HepG2 cells. DETAILED DESCRIPTION

[0017] The present invention provides a culture medium for γδT cells, using OpTmizer as a basic culture medium, wherein the OpTmizer culture medium further contains: IL-2 with a concentration of 150 to 250 IU / mL, IL-6 with a concentration of 40 to 60 μmol / L, vitamin C with a concentration of 40 to 60 μmol / L, vitamin B3 with a concentration of 8 to 12 μmol / L, and coenzyme Q10 with a concentration of 3 to 7 μmol / L.

[0018] In the present invention, the OpTmizer culture medium preferably also contains: IL-2 at a concentration of 180 to 220 IU / mL, IL-6 at a concentration of 45 to 55 μmol / L, vitamin C at a concentration of 45 to 55 μmol / L, vitamin B3 at a concentration of 9 to 11 μmol / L, and coenzyme Q10 at a concentration of 4 to 6 μmol / L; and further preferably, it also contains: IL-2 at a concentration of 200 IU / mL, IL-6 at a concentration of 50 μmol / L, vitamin C at a concentration of 50 μmol / L, vitamin B3 at a concentration of 10 μmol / L, and coenzyme Q10 at a concentration of 5 μmol / L.

[0019] In the present invention, the OpTmizer culture medium further contains fetal bovine serum at a volume concentration of 2-8%, preferably 3-7%, further preferably 4-6%, and more preferably 5%.

[0020] The present invention provides an application of the culture medium in culturing γδT cells.

[0021] The invention provides a method for culturing γδT cells with improved killing activity. Peripheral blood mononuclear cells are cultured with the culture medium to obtain γδT cells with improved killing activity.

[0022] In the present invention, the initial concentration of γδT cells during culture is 1×10 5 ~1×10 7 / mL, preferably 5×10 5 ~5×10 6 / mL, more preferably 1×10 6 Pieces / mL.

[0023] In the present invention, the culture is carried out at a temperature of 36 to 38° C. and a CO 2 concentration of 4 to 6%, preferably at a temperature of 37° C. and a CO 2 concentration of 5%.

[0024] In the present invention, the cell culture medium is replaced once every 2 to 4 days, preferably once every 3 days.

[0025] In the present invention, the total culture time is 10 to 20 days, preferably 12 to 16 days, more preferably 13 to 15 days, and even more preferably 14 days.

[0026] The present invention also provides a γδT cell cultured according to the method.

[0027] The present invention further provides an application of the γδT cells in the preparation of products for preventing or treating tumor cell killing.

[0028] In the present invention, the tumor cells include liver cancer cells and lung cancer cells.

[0029] 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.

[0030] Example 1

[0031] A method for culturing γδT cells, comprising the following steps:

[0032] (1) Mix peripheral blood with an equal volume of PBS and slowly add it to a centrifuge tube containing cell separation solution (Ficoll), centrifuge at 2000 rpm for 20 min, pipette peripheral blood mononuclear cells and add them to a centrifuge tube containing PBS, centrifuge at 8500 rpm for 10 min, remove the supernatant, resuspend the precipitate with PBS and centrifuge at 2000 rpm for 5 min. The resulting precipitate is peripheral blood mononuclear cells (PBMC);

[0033] (2) The PBMC cells prepared in step (1) were resuspended in the culture medium of γδT cells and the concentration of γδT cells was adjusted to 1×10 6 / mL, cultured at a temperature of 37°C and 5% CO2, changing the medium every 3 days, and culturing for a total of 14 days to obtain γδT cells; wherein the culture medium of the γδT cells is a culture medium using OpTmizer as a basic culture medium and supplemented with 150 IU / mL of IL-2, 40 μmol / L of IL-6, 40 μmol / L of vitamin C, 8 μmol / L of vitamin B3, and 3 μmol / L of coenzyme Q10.

[0034] Example 2

[0035] The difference between this embodiment and embodiment 1 is that the amounts of the components in the culture medium for γδT cells used are different. The culture medium for γδT cells described in this embodiment uses OpTmizer as the basic culture medium, and is supplemented with 250 IU / mL of IL-2, 60 μmol / L of IL-6, 60 μmol / L of vitamin C, 12 μmol / L of vitamin B3, and 7 μmol / L of coenzyme Q10. The remaining steps are the same as those in embodiment 1.

[0036] Example 3

[0037] The difference between this embodiment and embodiment 1 is that the amounts of the components in the culture medium for γδT cells used are different. The culture medium for γδT cells described in this embodiment uses OpTmizer as the basic culture medium, and is supplemented with 200 IU / mL IL-2, 50 μmol / L IL-6, 50 μmol / L vitamin C, 10 μmol / L vitamin B3 and 5 μmol / L coenzyme Q10. The remaining steps are the same as in embodiment 1.

[0038] Example 4

[0039] The difference between this embodiment and embodiment 3 is that fetal bovine serum with a volume concentration of 5% is also added to the culture medium of γδT cells used in this embodiment, and the remaining steps are the same as those in embodiment 3.

[0040] Comparative Example 1

[0041] The difference between this comparative example and Example 3 is that different basic culture medium is used. This comparative example uses RPMI-1640 as the basic culture medium, and the remaining steps are the same as Example 3.

[0042] Comparative Example 2

[0043] The difference between this comparative example and Example 3 is that the culture medium of γδT cells used is different. Vitamin B3 is not added to the culture medium of γδT cells in this comparative example. The remaining steps are the same as those in Example 3.

[0044] Comparative Example 3

[0045] The difference between this comparative example and Example 3 is that the amounts of the components in the culture medium for γδT cells used are different. The culture medium for γδT cells in this comparative example uses OpTmizer as the basic culture medium, and is supplemented with 200 IU / mL IL-2, 100 μmol / L IL-6, 50 μmol / L vitamin C, 20 μmol / L vitamin B3 and 10 μmol / L coenzyme Q10. The remaining steps are the same as in Example 3.

[0046] Test example: Determination of γδT cell killing activity

[0047] A549 cells and HepG2 cells in the logarithmic growth phase were used as target cells, and the γδT cells obtained by culture in Examples 1 to 4 and Comparative Examples 1 to 3 were added at an effector-target ratio of 1:10, 1:20 and 1:50, respectively. Three replicates were set for each group. After culturing for 24 hours at 37°C and 5% CO2, the supernatant was discarded, and CCK-8 was added to continue culturing for 4 hours. After the culture was completed, the absorbance (A) was measured at 450nm with an enzyme marker, and the killing rate of each γδT cell on A549 cells and HepG2 cells was calculated. The results are shown in Tables 1 and 2.

[0048] Killing rate (%) = 1 - {(experimental well A590 - effector well A590) / target cell well A590} × 100%

[0049] Table 1 Detection results of the killing rate of γδT cells on A549 cells (%)

[0050] Group 1：10 1：20 1：50 Example 1 61.3 73.2 91.7 Example 2 62.2 70.9 91.4 Example 3 62.3 74.1 93.6 Example 4 63.0 74.6 94.2 Comparative Example 1 59.7 70.2 88.6 Comparative Example 2 57.5 68.5 85.9 Comparative Example 3 60.1 70.4 89.3

[0051] Table 2 Detection results of killing rate of γδT cells on HepG2 cells (%)

[0052] Group 1：10 1：20 1：50 Example 1 49.7 61.3 79.4 Example 2 48.2 60.8 78.8 Example 3 49.9 61.7 81.3 Example 4 50.3 62.0 81.7 Comparative Example 1 49.2 61.1 79.8 Comparative Example 2 43.5 56.9 71.4 Comparative Example 3 46.8 60.6 78.3

[0053] It can be seen from Table 1 and Table 2 that, compared with Comparative Examples 1 to 3, the γδT cells cultured in Examples 1 to 4 of the present invention have a good killing rate for A549 cells and HepG2 cells, indicating that the components added to the culture medium of the γδT cells provided by the present invention have the effect of synergistically improving the killing activity of γδT cells.

[0054] 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 culture medium for γδT cells, characterized in that OpTmizer is used as the basic culture medium, and the OpTmizer culture medium also contains: IL-2 at a concentration of 150 to 250 IU / mL, IL-6 at a concentration of 40 to 60 μmol / L, vitamin C at a concentration of 40 to 60 μmol / L, vitamin B3 at a concentration of 8 to 12 μmol / L, and coenzyme Q10 at a concentration of 3 to 7 μmol / L.

2. The culture medium according to claim 1, characterized in that The OpTmizer culture medium also contains fetal bovine serum with a volume concentration of 2-8%.

3. Use of the culture medium according to claim 1 or 2 in culturing γδT cells.

4. A method for culturing γδT cells with enhanced cytotoxic activity, characterized in that: Peripheral blood mononuclear cells are cultured in the culture medium according to claim 1 or 2 to obtain γδT cells with enhanced cytotoxic activity.

5. The method according to claim 4, characterized in that The initial concentration of γδT cells during the culture was 1×10 5 ~1×10 7 Pieces / mL.

6. The method according to claim 5, characterized in that The culture is carried out at a temperature of 36-38°C and a CO2 concentration of 4-6%.

7. The method according to any one of claims 4 to 6, characterized in that: The cell culture medium was changed every 2 to 4 days.

8. The method according to claim 7, characterized in that The total culture time is 10 to 20 days.

9. A γδT cell cultured according to the method of any one of claims 4 to 8.

10. Use of the γδT cells according to claim 9 in preparing a product for preventing or treating killing tumor cells.