Optimized feeder-free ex vivo expansion enabled robust cell production from cryopreserved primary human NK cells. (A) The expansion protocol consists of two phases, starting with the thawing of cryopreserved NK cells. Expansion rates and NK cell purity in different culture media and IL-2 dosages were analyzed. (B) Expansion rates in Phase I in four different media plus 1,000 U/ml IL-2. (C) Mean percentages and representative flow cytometry plots of CD56⁺ CD3⁻ cells on day 14. (D) NK cells were nucleofected with CD96-targeting Cas9 RNP using the reference condition (Rautela et al., 2018Preprint) on day 14 to determine cell viability and CD96⁻ cells for quality controls. (E) Expansion rates in Phase II in NK MACS and EL837 media supplemented with two dosages of IL-2. (F) Mean percentages and representative flow cytometry plots of CD56⁺ CD3⁻ cells on day 28. (G) Cell viability and CD96⁻ cells on day 28. Expansion data are shown as mean ± SD of five donors (n = 5). CD96 KO data are shown as mean ± SD of three donors (n = 3). Two-tailed Welch's unequal variances t test was used to test for statistical significance. *, P ≤ 0.05; **, P ≤ 0.01. ns, not significant; std, standard for comparison.