Abstract 5332: Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development
Authors: Huang Yongyang , Patel Samir , Pierce Mackenzie , Nitta Carolina Franco , Lin Bo , Chan Leo 
Published: April 2023

Abstract
Cell viability is one of the critical quality attributes (CQAs) for cell and gene therapy products. Characterization of cell viability, in addition to cell identity, cell count and other CQAs, are essential to ensure the quality, safety and efficacy throughout various developmental stages of cell and gene therapies. To determine cell viability, a variety of viability detection methods have been used, such as trypan blue (TB) exclusion assay, and fluorescence-based dyes including acridine orange/propidium iodide (AO/PI), acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI), all of which have been employed on flow or image-based cytometry systems. In this work, we employed the Cellaca MX high-throughput cell counter to characterize and compare AO/PI and AO/DAPI viability detection methods. First, we measured the viabilities of different ratios of viable and non-viable Jurkat cells created via two cell dying processes. Next, viabilities of the stained cells were measured multiple times within a 30 min staining time for each method in order to monitor the trend of viability changes. The viability results showed that the AO/DAPI method were generally higher than AO/PI method with differences ranging from 0% to 24%. We also observed the discrepancy of the measured viability differences between naturally-dying and heat-killed Jurkat cells, suggesting that the viability difference might be dependent on the cell dying process. In the time-dependent viability characterization, results from AO/DAPI method showed increasing dead cell counts leading to decreasing viability, revealing an on-going staining process. In contrast, results from AO/PI method showed more consistent cell count and viability measurements, indicating a more instantaneous staining process. Lastly, we conducted a cell proliferation assay as an orthogonal method to confirm replicability of the low viability cell samples. The proliferation assay results showed no significant increase in cell concentrations within the 4-day time frame. The viability results before the culture were approximately 0% for both naturally-dying and heat-killed Jurkat cells, using the AO/PI staining method, and 12% and 9% using the AO/DAPI respectively. Furthermore, spiked samples of natural-dying and heat-killed cells with healthy cells (&gt;90% viability) were generated and measured, which showed exponential growth. The viability results of these spiked samples before the culture were 11% and 9% by AO/PI and 17% and 18% by AO/DAPI, respectively. Taken together, these results suggest that cell viability methods need to be optimized for different fluorescent stains in order to generate reliable, robust, and fit-for-purpose results for the downstream assays for cell and gene therapy products..
Citation Format: Yongyang Huang, Samir Patel, Mackenzie Pierce, Carolina Franco Nitta, Bo Lin, Leo Chan. Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5332.