A method for inactivating feeder cells combined with cytokines to stimulate NK cell expansion
A feeder cell and cytokine technology, applied in the field of cell culture, can solve the problems of incomplete inactivation, high test cost and high requirements, and achieve the effects of good targeted killing of tumor cells, high killing activity and fast proliferation rate.
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Embodiment 1
[0024] 1 Isolation of PBMC: Collect 50ml of peripheral blood from tumor patients, anticoagulated with heparin, and centrifuge the whole blood at 700g for 20min. The upper layer is the plasma layer, the middle layer is the buffy coat, and the lower layer is the red blood cells. The plasma layer was collected, inactivated at 56°C for 30min, then stood at 4°C for 15min, 1800rpm / 800g, 4°C, 25min, and the supernatant was used as autologous plasma for later use. Collect the intermediate buffy coat layer, add it to 20ml PBS, mix well, and then slowly add the centrifuge tube to which the lymphatic separation medium has been added at a ratio of 4:3, 1800rpm / 800g, room temperature, 20min, no-break, wash PBMC; After centrifugation, the white PBMC layer in the middle was sucked out by a Steer pipette, and about 30 ml of PBS was added to a new 50 ml centrifuge tube for washing, 1500 rpm, 8 min, washed once with culture medium, and counted.
[0025] 2. Induction and expansion of NK cells: ...
Embodiment 2
[0031] 1 Isolate PBMCs
[0032]50ml of peripheral blood was collected from tumor patients, anticoagulated with heparin, and whole blood was centrifuged at 700g for 20min. The upper layer is the plasma layer, the middle layer is the buffy coat, and the lower layer is the red blood cells. The plasma layer was collected, inactivated at 56°C for 30min, then stood at 4°C for 15min, 1800rpm / 800g, 4°C, 25min, and the supernatant was used as autologous plasma for later use. Collect the intermediate buffy coat layer, add it to 20ml PBS, mix well, and then slowly add the centrifuge tube to which the lymphatic separation medium has been added at a ratio of 4:3, 1800rpm / 800g, room temperature, 20min, no-break, wash PBMC; After centrifugation, the white PBMC layer in the middle was sucked out by a Steer pipette, and about 30 ml of PBS was added to a new 50 ml centrifuge tube to wash, 1500 rpm, 8 min, washed once with medium, and counted.
[0033] 2 Induction and expansion of NK cells
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