To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional ⁵¹Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.

Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line. Intra- and inter-assay reproducibility of these assays and their correlation with ⁵¹Cr release assay were analyzed.

After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range. Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1. BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity. Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency. All of the four assays, especially BATDA and CAM assays, showed good intra- and inter-assay reproducibility. Among these assays, BATDA assay showed the best correlation with the traditional ⁵¹Cr release assay. BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.

Compared with the other three assays, BATDA assay shows the best linear correlation with ⁵¹Cr release assay and has the advantages of time-saving and good reproducibility. Besides, it is a better assay for detecting the cytotoxicity of adherent cells. BATDA assay is a promising substitute for ⁵¹Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.

NK cell; Cytotoxicity; BATDA; CAM; CytoTox-Glo; PKH