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bioRxiv · 10.64898/2026.01.21.700863

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

Storm, J.·Kusch, N.·Guettler, M.·Fode, C.·Breuer, L.·Bartling, J.·Knabbe, C.·Kaltschmidt, B.·Kaltschmidt, C.
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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.

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Storm, J., Kusch, N., Guettler, M., Fode, C., Breuer, L., Bartling, J., Knabbe, C., Kaltschmidt, B., Kaltschmidt, C.. 2026-01-23. A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps. https://doi.org/10.64898/2026.01.21.700863

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