Off-the-shelf FLT3 CAR_sIL-15 cord blood NK cells retain transgene expression and cytolytic functions after 1 cycle of freeze-thaw. (A) sIL-15 NK and FLT3 CAR_sIL-15 NK cell numbers before freezing and 1 to 6 hours after thawing were compared. NK cells were thawed in the freezing medium at room temperature and the number of cells was determined using a Muse Cell Analyzer at the times indicated (n = 3 donors). (B) The transduction efficiency of sIL-15 NK or FLT3 CAR_sIL-15 NK cells before and after 1 week of freezing assessed by the expression of tEGFR (n = 3 donors). (C) Immunophenotypes of FLT3 CAR_sIL-15 NK cells before and after 1 week of freezing (n = 3 donors). Median mean fluorescence intensity (MFI) for each of the indicated cell surface markers is shown as a bar graph. (D) FLT3 CAR_sIL-15 NK cells after being frozen for 1 week were defrosted. The defrosted and freshly expanded FLT3 CAR_sIL-15 NK cells were cocultured with a FLT3⁺ AML cell line (EOL1 or MOLM-13) for 24 hours at the indicated E:T ratios, followed by cytolytic function testing by a flow cytometry-based assay (n = 3 donors). Data are shown as mean ± SEM. There are no statistically significant differences for any of the measurements taken between fresh cells and frozen cells transduced with the same construct in panels A-D.