Ex Vivo Expansion of Natural Killer Cells from Peripheral Blood Mononuclear Cells

Wash the peripheral blood mononuclear cells, or PBMCs, and 100 gamma-irradiated 221-mIL-21 cells separately by centrifugation at 400 g for 5 minutes with 10 milliliters of R-10 media.

After centrifugation, save 1 x 10⁶ PBMCs for flow cytometry, and mix 5 x 10⁶ PBMCs with 10 x 10⁶ 100 gamma-irradiated 221-mIL-21 cells in a special 6-well plate. Add 30 milliliters of R-10 media supplemented with human interleukin-2 and human interleukin-15 to the same 6-well plate, and incubate the plate at 37 degrees Celsius with 5% carbon dioxide with replacing the media every three to four days.

While on incubation, record the total peripheral blood natural killer or PBNK cell number, viability, and perform flow cytometry every three to four days to calculate the NK cell expansion rate.