A γδ T cell and its proliferation culture method and use as a medical preparation

Abstract

The present invention provides a γδ T cell and its proliferation and culture method and application as a medical preparation, belonging to the technical field of medical preparations. The proliferation and culture method for γδ T cells provided by the present invention comprises the following steps: (1) isolating mononuclear cells from peripheral blood and inoculating them into a culture medium containing a chemical stimulant and a cytokine for culture; (2) adding cytokines on the third day of culture and continuing to culture until the proliferation culture is completed; the chemical stimulant isopentenyl pyrophosphate and the cytokine is IL-2. The proliferation and culture method for γδ T cells provided by the present invention significantly improves the proliferation efficiency of γδ T cells and the killing activity against lung cancer cells. At the end of the culture of the present invention, the proliferation multiple of γδ T cells is 10,000 times, and the killing efficiency against lung cancer cells is 70%, which is significantly higher than the existing technology.

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 discloses a culture medium for in vitro expansion and activation of γδ T cells, and 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 culture on the 12th day, the cell expansion multiple was measured to be 6236 times, the cytotoxicity against Daudi was 78.3%, and the cytotoxicity against A549 cells was 58.9%.

[0004] CN117050939A discloses a method for in vitro expansion of peripheral blood-derived γδ T cells. The method mainly involves using 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 the cells have a certain killing effect on the human lung cancer cell line A549-Luciferase. 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 expansion efficiency is low and the killing activity against lung cancer cells is also low. Summary of the Invention

[0006] The purpose of the present invention is to provide an optimized method for the proliferation and culture 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:

[0008] The present invention provides a method for culturing γδT cells, comprising the following steps:

[0009] (1) Isolate mononuclear cells from peripheral blood and inoculate them into culture medium containing chemical stimulants and cytokines for culture;

[0010] (2) Add cytokines on the third day of culture and continue culturing until the proliferation culture ends;

[0011] The chemical irritants are isopentenyl pyrophosphate and saperin, and the cytokine is IL-2.

[0012] Preferably, the number of mononuclear cells inoculated into the culture medium is (0.5-1)×10 6 cells / mL; the culture medium is AIM-V serum-free culture medium.

[0013] Preferably, the concentration of isopentenyl pyrophosphate in the culture medium is 2±0.2 μg / mL, and the concentration of sapylin in the culture medium is 10±1 ng / mL.

[0014] Preferably, the concentration of IL-2 in the culture medium is 90-110 IU / mL.

[0015] Preferably, the cytokine added in step (2) is IL-2, which is added to a concentration of 900-1100 IU / mL.

[0016] 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.

[0017] Preferably, the γδT cells are Vγ9δ2 T cells.

[0018] The present invention also provides γδT cells obtained by the proliferation and culture method.

[0019] The present invention also provides an application of the γδT cells in preparing a medical preparation for killing tumors.

[0020] Preferably, the tumor is lung cancer cells.

[0021] The γδT cell proliferation and culture method provided by the present invention significantly improves the proliferation efficiency of γδT cells and their killing activity against lung cancer cells. When the culture ends on the 14th day, the cell proliferation multiple is 10,000 times, and the killing efficiency against lung cancer cells is 70%, which is significantly higher than the existing technology.

[0022] The culture protocol of the present invention efficiently expands γδ T cells, particularly Vγ9δ2 T cells, while maintaining their anti-tumor activity. This protocol not only improves the expansion efficiency and activity of γδ T cells but also reduces culture costs, providing a cost-effective source of cell-based medical preparations for cancer immunotherapy. DETAILED DESCRIPTION

[0023] The technical solutions provided by the present invention are 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.

[0024] Example 1

[0025] 1. Isolation of mononuclear cells:

[0026] a) Mononuclear cells (including lymphocytes and monocytes) were isolated from peripheral blood of healthy donors by density gradient centrifugation.

[0027] b) Mononuclear cells were isolated using Ficoll-Paque.

[0028] 2. Proliferation and culture of Vγ9δ2 T cells

[0029] a) Use AIM-V serum-free medium as the basal medium and resuspend the mononuclear cells to a concentration of 1×10 6 / mL. Then, isopentenyl pyrophosphate (IPP) was added to a final concentration of 2 μg / mL, sapyrin was added to a final concentration of 10 ng / mL, and IL-2 cytokine was added to a final concentration of 100 IU / mL. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. This was recorded as day 0.

[0030] b) On the third day of culture, the cell proliferation was measured to reach 5×10 6 pieces / mL.

[0031] c) On the fifth day of culture, IL-2 was added to increase the IL-2 to 1000 U / ml, and the cell proliferation number was measured to reach 20×10 6 pieces / mL.

[0032] d) On the 7th day of culture, the number of expanded Vγ9δ2 T cells reached 100×10 6 pieces / mL.

[0033] e) On the 9th day of culture, the number of expanded Vγ9δ2 T cells reached 500×10 6 pieces / mL.

[0034] f) On the 12th day of culture, the number of expanded Vγ9δ2 T cells reached 2500×10 6 pieces / mL.

[0035] g) On the 14th day of culture, the number of expanded Vγ9δ2 T cells reached 10000×10 6 pieces / mL.

[0036] It can be seen that the amplification factor is from 1×10 6 cells and reached 10,000×10 6 cells, then the actual fold expansion = final cell number / initial cell number = 10,000 × 106 / 1×10 6 =10,000-fold. Therefore, using the culture protocol of the present invention, the proliferation fold of Vγ9δ2 T cells can reach 10,000-fold.

[0037] Example 2

[0038] 1. Flow cytometry analysis of the proportion and phenotype of the Vγ9δ2 T cell subsets cultured in Example 1

[0039] a) First, cultured Vγ9δ2 T cells were stained with specific surface marker antibodies that recognize different γδ T cell subsets.

[0040] b) Commonly used surface markers include CD3, CD4, CD8, Vδ1 and Vδ2.

[0041] c) The stained cells are analyzed by flow cytometry, which can detect and quantify the fluorescent signals on the cell surface.

[0042] d) Flow cytometry data were analyzed using specialized software to determine the proportions and phenotypes of different subpopulations.

[0043] The results show:

[0044] CD3+ T cells: 90%

[0045] CD4-, CD8- (double negative): 30%

[0046] Vδ1+ T cells: 15%

[0047] Vδ2+ T cells: 85%

[0048] 2. Evaluation of the anti-tumor activity of γδ T cells through in vitro killing assays

[0049] The Vγ9δ2 T cells cultured in Example 1 were co-cultured with a lung cancer cell line (A549), with a ratio of effector cells to target cells of 10:1.

[0050] Calcein AM / PI staining was used to assess tumor cell survival.

[0051] The results showed that the survival rate of A549 tumor cells was 30% and the killing efficiency was 70%.

[0052] As shown in the above examples, γδ T cells expanded 10,000-fold within 14 days. Flow cytometry analysis showed that the cultured cells were primarily CD3+, double-negative, Vδ2+ T cells. In vitro cytotoxicity experiments demonstrated that these cells had a significant ability to kill lung cancer cells, with a killing efficiency of 70%.

[0053] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for the proliferation and culture of γδT cells, characterized in that: The steps include: (1) Isolate mononuclear cells from peripheral blood and inoculate them into a culture medium containing chemical stimulants and cytokines for culture; (2) Cytokines were added on the third day of culture and the culture was continued until the proliferation culture was completed; The chemical irritants are isopentenyl pyrophosphate and saperin, and the cytokine is IL-2; The concentration of isopentenyl pyrophosphate in the culture medium is 2±0.2 μg / mL, the concentration of saperyl in the culture medium is 10±1 ng / mL; the concentration of IL-2 in the culture medium is 90-110 IU / mL; The γδT cells are Vγ9δ2T cells.

2. The method for culturing γδT cells according to claim 1, wherein: The number of cells inoculated into the culture medium is (0.5-1)×10 6 cells / mL; the culture medium is AIM-V serum-free culture medium.

3. The method for culturing γδT cells according to claim 2, wherein: The cytokine added in step (2) is IL-2, which is added to a concentration of 900 to 1100 IU / mL.

4. The method for culturing γδT cells according to claim 3, wherein: The culture conditions in steps (1) and (2) are 37°C ± 1°C, 5% ± 0.5% CO2; the proliferation culture is carried out for 14 days.