Ex vivo expansion of natural killer cells using cryopreserved irradiated feeder cells

Currently, feeder cells are γ-irradiated immediately before use for the ex vivo expansion of natural killer (NK) cells from human peripheral blood. Storing irradiated feeder cells by cryopreserving them in multiple vials would be more convenient than irradiating cells each time they are needed. We compared NK cell expansion using cryopreserved-irradiated feeder cells (cryopreserved group) and freshly-irradiated feeder cells (fresh group). To expand NK cells, peripheral blood mononuclear cells were isolated and co-cultured with-100 Gy-irradiated K562 leukemia cells that had been modified to express 4-1BB ligand and membrane-bound (mb) interleukin (IL)-15 (K562-mb15-41BBL cells) for three weeks in the presence of IL-2 and IL-15. Fresh and cryopreserved K562-mb15-41BBL feeder cells expressed similar levels of 4-1BB ligand, whereas membrane-bound IL-15 expression was lower in the cryopreserved cells than in the fresh cells. The NK cell expansion rate did not differ between the two groups (980-fold vs. 1058-fold, respectively), although the mean NK cell purity was higher in the fresh-group than in the cryopreserved-group at day 14 (94.1% vs. 92.5%, respectively) and day 21 (97.1% vs. 95.4%, respectively). The NK cells from the two feeder cell groups did not differ in cytotoxicity against various malignant cell lines at effector-to-target ratios of 4:1, 2:1, and 1:1, or in the expression pattern of NK cell receptors [cluster of differentiation (CD)-16, natural killer group-2, member D (NKG2D), CD69, NKp30, NKp44, NKp46, and CD158b] and level of interferon-γ secretion. Our results demonstrate that cryopreserved irradiated feeder cells can be used for the ex vivo expansion of human NK cells and provide a convenient improvement on current methods.