A method for inducing and expanding γδt cells with high killing activity in vitro

Abstract

The invention relates to a method for inducing and expanding γδT cells in vitro, which belongs to the field of biotechnology. The method first obtains peripheral blood mononuclear cells (PBMCs) through lymphocyte separation fluid, and then utilizes serum-free medium containing zoledronic acid, IL-2, IL-7 and IL-15 to stimulate culture for 3 days; Serum-free medium containing IL-2, IL-7 and IL-15 was used for stimulation and expansion culture; when cultured to 7-9 days, Nicotinamide was added to improve the anti-tumor activity of γδT cells. The invention does not need to purify PBMCs, and can obtain γδT cells with a purity of more than 90% after 14 days of culture; the addition of nicotinamide can obviously improve the anti-tumor activity of γδT cells. The invention can expand the γδT cells by more than 1500 times, and can meet the clinical requirement for the number of γδT cells. The invention has simple operation steps, strong operability and 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...

Examples

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...

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...

Examples

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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