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

Natural killer, NK, cells are large granular lymphocytes that eliminate tumors and microbial infections.

For ex vivo NK cell expansion, take a suspension of peripheral blood mononuclear cells, PBMCs, containing a heterogenous population of lymphocytes, including NK cells. Add irradiated feeder cells — non-proliferating lymphoblastoid cells — which express membrane-bound interleukin 21, mIL-21, required to activate and expand NK cells.

Add the co-culture in a specialized culture well with a gas-permeable membrane at the bottom, allowing gas exchange. The cells settle down and proliferate for an extended period without requiring passaging.

Add interleukin 2, IL-2, and interleukin 15, IL-15 — cytokines for NK cell proliferation. Incubate for an adequate period in physiological conditions.

mIL-21 on the feeder cells binds to a specific heterodimeric receptor on the NK cells. Exogenous IL-15 and IL-2 bind to their respective receptors. The binding induces different downstream signaling pathways — activating gene expression — limiting cellular senescence and leading to prolonged NK cell proliferation.

Using flow cytometry, assess the NK cell expansion at regular time intervals.

Plot the recorded data to analyze the expansion rate of the cells and their purity — selective growth of the target NK cell type. The manifold expansion over the incubation period indicates a sustained proliferation while maintaining high purity.

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.