A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

Abstract

Functional testing of cytotoxic lymphocytes is essential for research and quality control (QC), but most assays require freshly prepared target cells and extensive handling. A ready-to-thaw, no-wash, flow cytometry-based cytotoxicity assay was developed using pre-labeled K562 targets cryopreserved in STEM-CELLBANKER(R) EX (SCB) as suitably sized aliquots. SCB tolerability was evaluated in K562, NK-92, and primary natural killer (NK) cells; post-cryopreservation label stability of CellTrace Violet (CTV) and carboxyfluorescein succinimidyl ester (CFSE) was assessed; freezing and thawing conditions were optimized; and wash versus no-wash workflows were compared using viability-based and absolute-count readouts, across effector-to-target (E:T) ratios with NK donors and NK-92 cells. Effector viability remained high at SCB concentrations up to 10%, and 5% SCB was selected for assay design. After cryopreservation, CTV labeling remained stable over the tested storage period, whereas CFSE showed substantial signal loss. Warm-medium thawing performed comparably to water-bath thawing, and the consolidated protocol (SCB plus fetal calf serum and thermal buffering) maintained high post-thaw target viability and recovery. In killing assays, lysis increased with increasing E:T ratios; omission of the post-thaw wash had minimal impact, and 5% SCB did not impair cytotoxic function. This ready-to-thaw workflow reduces hands-on time and sample manipulation, while improving standardization for reproducible results and enabling high-throughput functional testing and QC.