Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development

Abstract Cell viability is one of the critical quality attributes (CQAs) for analyzing and characterizing cellular therapeutic products. The characterization of cell viability is essential to ensure quality, safety and efficacy during cell and gene therapy development. In this work, we characterized and compared two commonly used viability dual-staining methods, acridine orange/propidium iodide (AO/PI) and acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI) using the Cellaca MX high-throughput cell counter. In general, the AO/DAPI method provided higher viability results than AO/PI, with the measured viability difference between these two methods ranging from 0% to 24%. To identify the sources of the measured viability difference, we tested a list of factors including cell dying process, staining time and initial viability levels. First, we observed a larger viability difference from naturally-dying Jurkat cells compared to the result from heat-killed cells, suggesting that the differences may depend on the cell dying process. Second, we found that AO/PI provided more consistent cell count and viability than AO/DAPI during the first 30 min of staining time. Finally, we showed that measured viability difference might be larger for low-viability, naturally-dying Jurkat cells, as compared to healthy samples. The results from our characterization studies may benefit the selection of viability methods that are fit-for-purpose for analyzing cellular therapeutic products.