Method for amplifying killing activity gamma-delta T cell by induction in vitro
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A technology of killing activity and cells, applied in the direction of animal cells, vertebrate cells, cell culture active agents, etc., to meet the needs of clinical applications, simple operation steps, and strong anti-tumor activity
Active Publication Date: 2018-12-07
吉林省吉恩致合生物治疗技术有限公司
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[0004] The purpose of the present invention is to solve the problem of how to expand a large number of γδT cells with high killing activity in the prior art, and to provide a method for inducing and expanding γδT cells with high killing activity in vitro
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Embodiment 1
[0026] A method for inducing and expanding γδT cells in vitro, comprising the steps of:
[0027] (1) Preparation of PBMCs:
[0028] A. Use heparin anticoagulated vacuum blood collection tube to extract 50mL peripheral blood from a healthy volunteer, centrifuge at 3000rpm room temperature for 10min, put the plasma into a new 50mL centrifuge tube, and place it in a constant temperature incubator at 56°C for 30min to inactivate complement.
[0029] B. Take 30mL of lymphocyte separation solution and add it evenly to the lymphocyte separation tubes, 15mL in each tube.
[0030] C. Add 40mL of PBS to the centrifuged cell pellet, mix well, slowly add the diluted blood cells into the lymphocyte separation tube containing 15mL of lymphocyte separation medium, and centrifuge at 1800rpm for 20min at room temperature.
[0031] D. After centrifugation, gently remove the buffy coat cells in the middle of the lymphocyte separation tube to two new 50mL centrifuge tubes, then add 10 times the...
Embodiment 2
[0044] A method for inducing and expanding γδT cells in vitro, comprising the steps of:
[0045] (1) Preparation of PBMCs:
[0046] A. Use a heparin anticoagulated vacuum blood collection tube to extract 50mL peripheral blood from another healthy volunteer, centrifuge at 3000rpm room temperature for 10min, put the plasma into a new 50mL centrifuge tube, and place it in a constant temperature incubator at 56°C 30min to inactivate complement.
[0047] B. Take 30mL of lymphocyte separation solution and add it evenly to the lymphocyte separation tubes, 15mL in each tube.
[0048] C. Add 40mL of PBS to the centrifuged cell pellet, mix well, slowly add the diluted blood cells into the lymphocyte separation tube containing 15mL of lymphocyte separation medium, and centrifuge at 1800rpm for 20min at room temperature.
[0049] D. After centrifugation, gently remove the buffy coat cells in the middle of the lymphocyte separation tube to two new 50mL centrifuge tubes, then add 10 times...
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Abstract
The invention relates to a method for amplifying a gamma-delta T cell by induction in vitro, and belongs to the technical field of biology. The method comprises the following steps: acquiring peripheral blood mononuclear cells (PBMCs) through a lymphocyte separation medium; performing stimulation culturing for 3 days by utilizing a serum-free medium containing zoledronic acid, IL-2, IL-7 and IL-15; performing stimulation amplifying culturing with a serum-free medium containing IL-2, IL-7 and IL-15; and adding nicotinamide when culturing is performed for 7-9 days to improve the anti-tumor activity of the gamma-delta T cells. According to the method, the PBMCs do not needed to be purified, and the gamma-delta T cells having the purity of 90 percent or more can be obtained when culturing is performed for 14 days; and the anti-tumor activity of the gamma-delta T cells can be remarkably improved by adding the nicotinamide. The gamma-delta T cells can be amplified for 1500 times or more, which can meet the requirement for the number of gamma-delta T cells in clinical application. The method is simple in operation steps and strong in operability, and has a good popularization value.
Description
technical field [0001] The invention relates to a method for inducing and expanding gamma delta T cells with high killing activity in vitro, belonging to the field of biotechnology. Background technique [0002] γδT cells are T cells that perform innate immune functions and are a type of innate immune cells, and their TCR is composed of γ and δ chains. Its TCR lacks diversity and can directly recognize some complete polypeptide antigens. The types of antigens recognized by γδT cells are limited: ① HSP; ② lipid antigens extracted from CD1 molecules on the surface of infected cells; ③ certain viral proteins or viral proteins expressed on the surface of infected cells; ④ phosphorylated antigens in bacterial lysates. Such T cells are mainly distributed in mucosal and subcutaneous tissues such as the intestinal respiratory tract and genitourinary tract, and only account for CD3 cells in peripheral blood. + 0.5%-1% of T cells, although the peripheral blood T lymphocytes are rela...
Claims
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