Serum- and feeder-free culture expansion of human peripheral blood NK cells

Abstract Natural killer (NK) cells are critical effectors of innate immunity that secrete proinflammatory cytokines and kill tumor cells and virus-infected cells. The ability to expand human NK cells in culture provides a ready source of cells for disease modeling, development of immunotherapies, and basic research. We have developed a culture system that supports the expansion of primary NK cells in the absence of feeder cells and serum. NK cells were enriched from peripheral blood using EasySep™ negative selection and cultured in serum-free medium with interleukin-2 in plates coated with a NK cell ‘activation coating matrix’ for 14 days. Cells were passaged with medium changes on days 7 and 11, and were harvested on day 14 for assessment or functional characterization. The average frequency of CD56+CD3− NK cells was 92% (range 80 – 97%, n=16) with an average expansion of 121-fold (range 10 – 274). On average 79% (48–92%) of expanded NK cells expressed CD16. The ability of expanded NK cells to produce interferon-γ (IFN-γ) and to degranulate were tested after stimulation with phorbol 12-myristate 13-acetate (PMA) + ionomycin or co-culture with K562 cells. As detected by intracellular flow cytometry, the average frequency of IFN-γ+ NK cells was 56% (range 41 – 63%, n = 4) and 40% (range 12 – 55%) when stimulated by PMA/ionomycin or K562 cells, respectively. Similarly, the average frequency of degranulated NK cells as detected by surface expression of CD107a, a lysosomal-associated membrane protein, was 69% (range 35 – 84%, n = 4) and 53% (range 18 – 75%), respectively. These results show that NK cells can be expanded and stimulated under serum- and feeder-free conditions to generate large numbers of functional NK cells for basic and translational research.