Efficient in-vitro amplification method of human natural killer cells
A natural killer cell and high-efficiency technology, applied in the field of biomedicine, can solve the problems of high price, hidden safety hazards, low amplification efficiency, etc., achieve high production efficiency and ensure the effect of aseptic operation
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Embodiment 1
[0048] A method for high-efficiency expansion of human natural killer cells in vitro, comprising the following steps:
[0049] a. Prepare complete cell culture solution for use as a spare, to provide NK cells with the nutrients needed for their survival and expansion; the complete culture solution contains 2mM Gluta-MAX, 100μg / ml penicillin and streptomycin, and the rest is GMPCellGro R A mixture of DCSerum-freeMedium and fetal bovine serum; where GMPCellGro R The molar ratio of DCSerum-freeMedium to fetal bovine serum is 19:1;
[0050] b. Take 50ml of lymphocyte separation liquid at room temperature, pour 200ml of spare human peripheral blood onto the surface of the lymphocyte separation liquid, and centrifuge for the first time at 2500rpm for 15mins to form cell stratification;
[0051] c. Absorb the buffy coat cells in the cell layer obtained in step b into a new centrifuge tube, wash with phosphate buffer, and centrifuge for the second time at 2000 rpm for 5 mins to obtai...
Embodiment 2
[0059] A method for high-efficiency expansion of human natural killer cells in vitro, comprising the following steps:
[0060] a. Prepare complete cell culture solution for use as a spare, to provide NK cells with the nutrients needed for their survival and expansion; the complete culture solution contains 2mM Gluta-MAX, 100μg / ml penicillin and streptomycin, and the rest is GMPCellGro R A mixture of DCSerum-freeMedium and fetal bovine serum; where GMPCellGro R The molar ratio of DCSerum-freeMedium to fetal bovine serum is 4:1;
[0061] b. Take 300ml of lymphocyte separation liquid at room temperature, pour 200ml of spare human peripheral blood onto the surface of the lymphocyte separation liquid, and centrifuge for the first time at 1200rpm for 25mins to form cell stratification;
[0062] c. Absorb the buffy coat cells in the cell stratification obtained in step b into a new centrifuge tube, wash with phosphate buffer, and centrifuge for the second time at 800 rpm for 15 mins...
Embodiment 3
[0070] A method for high-efficiency expansion of human natural killer cells in vitro, comprising the following steps:
[0071] a. Prepare complete cell culture solution for use as a spare, to provide NK cells with the nutrients needed for their survival and expansion; the complete culture solution contains 2mM Gluta-MAX, 100μg / ml penicillin and streptomycin, and the rest is GMPCellGro R DCSerum-freeMedium and autologous serum mixture, wherein autologous serum accounts for 1% of the volume of the complete culture medium;
[0072] b. Take 100ml of lymphocyte separation liquid at room temperature, pour 200ml of spare human peripheral blood on the surface of the lymphocyte separation liquid, and centrifuge for the first time at 1800rpm for 20mins to form cell stratification;
[0073] c. Absorb the buffy coat cells in the cell stratification obtained in step b into a new centrifuge tube, wash with phosphate buffer, and centrifuge for the second time, the centrifugation condition is...
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