Gamma delta T cell, proliferation culture method thereof and application of gamma delta T cell as medical preparation

Abstract

The invention provides a gamma delta T cell, a proliferation culture method thereof and application of the gamma delta T cell as a medical preparation, and belongs to the technical field of medical preparations. The proliferation culture method for gamma delta T cells provided by the invention comprises the following steps: (1) separating mononuclear cells in peripheral blood, inoculating the mononuclear cells into a culture medium containing a chemical stimulant and cytokines, and culturing the mononuclear cells; (2) supplementing cell factors on the 3th day of culture, and continuing to culture until the multiplication culture is finished; the chemical stimulant is isopentenyl pyrophosphate, and the cell factor is IL-2 (Interleukin-2). According to the proliferation culture method of the gamma delta T cells, provided by the invention, the proliferation efficiency of the gamma delta T cells and the killing activity of the gamma delta T cells on lung cancer cells are remarkably improved, and when culture is finished, the proliferation multiple of the gamma delta T cells is 10000 times, and the killing efficiency of the gamma delta T cells on the lung cancer cells is 70%, which are remarkably higher than those in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of medical preparations, and in particular to a γδT cell and a proliferation and culture method thereof, and an application thereof as a medical preparation. Background Art

[0002] γδ T cells are a type of immune cell with anti-tumor activity, but their proportion in human peripheral blood is low and their expansion efficiency is limited. Therefore, developing an effective γδ T cell culture protocol is of great significance for cancer immunotherapy.

[0003] CN112300990A A culture medium for in vitro expansion and activation of γδ T cells discloses a scheme for improving the in vitro expansion efficiency and activity of γδ T cells. The scheme mainly uses a culture medium containing autologous dendritic cell exosomes to proliferate and culture γδ T cells. At the end of the 12th day of culture, the cell expansion multiple was measured to be 6236 times, the toxicity efficiency to Daudi was 78.3%, and the toxicity efficiency to A549 cells was 58.9%.

[0004] CN117050939A discloses a method for in vitro expansion of peripheral blood-derived γδ T cells, and also discloses a method for in vitro expansion of γδ T cells, which mainly uses dendritic cells as feeder cells to culture γδ T cells in vitro. The results show that after 14 days of culture, the average expansion multiple is 1878.595±495.005 times, and it has a certain killing effect on the human lung cancer cell line A549-Luciferase, and the specific killing efficiency is not disclosed.

[0005] It can be seen that although there are methods for the proliferation and cultivation of γδ T cells in the prior art, the amplification efficiency is low and the killing activity against lung cancer cells is also low. Summary of the invention

[0006] The object of the present invention is to provide an optimized proliferation and culture method of γδT cells to promote their in vitro expansion and anti-tumor activity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for culturing γδT cells, comprising the following steps: (1) Isolate mononuclear cells from peripheral blood and inoculate them into culture medium containing chemical stimulants and cytokines for culture; (2) Add cytokines on the third day of culture and continue culturing until the proliferation culture ends; The chemical irritants are isopentenyl pyrophosphate and saperin, and the cytokine is IL-2.

[0008] Preferably, the number of mononuclear cells inoculated into the culture medium is (0.5-1)×10 6 / mL; the culture medium is AIM-V serum-free culture medium.

[0009] Preferably, the concentration of isopentenyl pyrophosphate in the culture medium is 2±0.2 μg / mL, and the concentration of saperyl in the culture medium is 10±1 ng / mL.

[0010] Preferably, the concentration of IL-2 in the culture medium is 90-110 IU / mL.

[0011] Preferably, the cytokine added in step (2) is IL-2, which is added to a concentration of 900-1100 IU / mL.

[0012] Preferably, the culture conditions in steps (1) and (2) are 37°C±1°C, 5%±0.5% CO2; and the proliferation culture is carried out for 14 days.

[0013] Preferably, the γδT cells are Vγ9δ2 T cells.

[0014] The present invention also provides γδT cells obtained by the proliferation and culture method.

[0015] The present invention also provides an application of the γδT cells in preparing a medical preparation for killing tumors.

[0016] Preferably, the tumor is lung cancer cells.

[0017] The γδT cell proliferation and culture method provided by the present invention significantly improves the proliferation efficiency of γδT cells and the killing activity against lung cancer cells. The present invention cultured until the 14th day, at which time the cell proliferation multiple was 10,000 times, and the killing efficiency against lung cancer cells was 70%, which was significantly higher than the prior art.

[0018] The culture scheme of the present invention can efficiently expand γδ T cells, especially Vγ9δ2 T cells, while maintaining their anti-tumor activity. The scheme not only improves the expansion efficiency and activity of γδ T cells, but also reduces the culture cost, providing an economical and effective source of cell medical preparations for cancer immunotherapy. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1 1. Isolation of mononuclear cells: a) Mononuclear cells (including lymphocytes and monocytes) were isolated from peripheral blood of healthy donors by density gradient centrifugation.

[0021] b) Use Ficoll-Paque to separate mononuclear cells.

[0022] 2. Proliferation and culture of Vγ9δ2 T cells a) Use AIM-V serum-free medium as the basal medium and resuspend the mononuclear cells to a concentration of 1×10 6 Then, isopentenyl pyrophosphate (IPP) was added to a final concentration of 2 μg / mL, saperin was added to a final concentration of 10 ng / mL, and IL-2 cytokine was added to a final concentration of 100 IU / mL, and cultured in a cell culture incubator at 37°C and 5% CO2, which was recorded as day 0.

[0023] b) On the third day of culture, the number of cells reached 5×10 6 Pieces / mL.

[0024] c) On the fifth day of culture, IL-2 was added to increase the IL-2 to 1000 U / ml, and the number of cells proliferated was measured to reach 20×10 6 Pieces / mL.

[0025] d) On the 7th day of culture, the number of Vγ9δ2 T cells expanded to 100×10 6 Pieces / mL.

[0026] e) On the 9th day of culture, the number of Vγ9δ2 T cells expanded to 500×10 6 Pieces / mL.

[0027] f) On the 12th day of culture, the number of Vγ9δ2 T cells reached 2500×10 6 Pieces / mL.

[0028] g) On the 14th day of culture, the number of Vγ9δ2 T cells expanded to 10000×10 6 Pieces / mL.

[0029] It can be seen that the amplification factor is from 1×10 6 cells and reached 10,000×10 on day 14. 6 cells, then the actual fold expansion = final cell number / initial cell number = 10,000 × 10 6 / 1×10 6 = 10,000 times. Therefore, using the culture scheme of the present invention, the proliferation multiple of Vγ9δ2 T cells can reach 10,000 times.

[0030] Example 2 1. Analyze the proportion and phenotype of the Vγ9δ2 T cell subsets cultured in Example 1 using flow cytometry a) First, cultured Vγ9δ2 T cells were stained with specific surface marker antibodies that recognize different γδ T cell subsets.

[0031] b) Commonly used surface markers include CD3, CD4, CD8, Vδ1 and Vδ2.

[0032] c) The stained cells are analyzed by flow cytometry, which can detect and quantify the fluorescent signals on the cell surface.

[0033] d) Flow cytometry data were analyzed using specialized software to determine the proportions and phenotypes of different subpopulations.

[0034] The results show: CD3+ T cells: 90% CD4-, CD8- (double negative): 30% Vδ1+ T cells: 15% Vδ2+ T cells: 85% 2. Evaluation of the anti-tumor activity of γδ T cells by in vitro killing assay The Vγ9δ2 T cells cultured in Example 1 were co-cultured with a lung cancer cell line (A549), with the ratio of effector cells to target cells being 10:1.

[0035] Calcein AM / PI staining was used to assess tumor cell survival.

[0036] The results showed: A549 tumor cell survival rate: 30%, killing efficiency: 70%.

[0037] As can be seen from the above examples, γδ T cells expanded 10,000 times in 14 days. Flow cytometry analysis showed that the cultured cells were mainly CD3+, double negative, Vδ2+ T cells. In vitro killing experiments showed that these cells had significant killing ability against lung cancer cells, with a killing efficiency of 70%.

[0038] 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 the proliferation and cultivation of γδ T cells, characterized in that: The steps include: (1) Isolate mononuclear cells from peripheral blood and inoculate them into culture medium containing chemical stimulants and cytokines for culture; (2) Add cytokines on the third day of culture and continue culturing until the proliferation culture ends; The chemical irritants are isopentenyl pyrophosphate and saperin, and the cytokine is IL-2.

2. A method for the proliferation and cultivation of γδ T cells according to claim 1, characterized in that: The number of cells inoculated into the culture medium is (0.5-1)×10 6 / mL; the culture medium is AIM-V serum-free culture medium.

3. A method for the proliferation and cultivation of γδ T cells according to claim 2, characterized in that: The concentration of the isopentenyl pyrophosphate in the culture medium is 2±0.2 μg / mL, and the concentration of the saperyl in the culture medium is 10±1 ng / mL.

4. A method for the proliferation and cultivation of γδ T cells according to claim 3, characterized in that: The concentration of IL-2 in the culture medium is 90-110 IU / mL.

5. A method for the proliferation and cultivation of γδT cells according to claim 4, characterized in that: The cytokine added in step (2) is IL-2, which is added to a concentration of 900~1100 IU / mL.

6. A method for the proliferation and cultivation of γδ T cells according to claim 5, characterized in that: The culture conditions in steps (1) and (2) are 37°C ± 1°C and 5% ± 0.5% CO2; the proliferation culture is carried out for 14 days.

7. A method for culturing γδ T cells according to any one of claims 1 to 6, characterized in that: The γδ T cells are Vγ9δ2 T cells.

8. A γδ T cell obtained by the proliferation and culture method according to any one of claims 1 to 7.

9. Use of the γδ T cells according to claim 8 in preparing a medical preparation for killing tumors.

10. The use according to claim 9, characterized in that The tumor is lung cancer cells.