Abstract 6291: CellPyAbility: an integrated toolkit for high-throughput cell viability assays and automated analysis

Abstract Assessing cell viability and proliferation in response to chemical gradients is fundamental to cancer biology research. We provide a cell viability “toolkit” that includes detailed protocols for high-throughput cell viability assays in response to a single agent or two agents in combination at varying concentrations with several advantages over other methods. Additionally, we developed automated software (CellPyAbility) that seamlessly integrates with the provided protocols, outputting detailed tabular and graphical analyses of experiments from bulk raw images. This combined approach significantly lowers the barriers to implementing cell viability assays by simplifying experimental setup and accelerating data analysis. Our lab often uses short-term, nuclei-based growth delay assays (GDAs) to assess the selectivity of drugs against specific genetic alterations found in tumors. A GDA is conducted using a single 96-well plate with the innermost 60 wells containing two cell lines, a vehicle control, and a log-based gradient of nine concentrations in technical triplicate. Nuclei counting provides several advantages over other common methods. Compared to the commonly used methylthiazol tetrazolium (MTT; reduction-based) or CellTiter-Glo (ATP-based) assays, GDAs provide single-cell resolution of survival; are insensitive to metabolic variability within a cell or between cell lines; are compatible with redox-altering drugs; require simpler methodology; and could be used on live cells using non-toxic nuclear dyes like Hoechst. A disadvantage of the GDA is the computational and temporal cost of the required image analysis. We present CellPyAbility, which rapidly generates dose-response metrics and publication-ready graphics from a folder of unedited, whole-well GDA images in approximately one minute on commodity hardware. Similarly, a researcher may want to assess how two drugs act in combination with one another across a wide range of concentrations. Our lab uses modified GDAs with simultaneous vertical and horizontal drug gradients, referred to as synergy assays. Synergy assays use three 96-well plates to assess 59 unique drug concentration combinations and a vehicle control in technical triplicate. CellPyAbility includes a synergy analysis module which generates dose-response metrics for each condition and a 3D surface map of cell viability and Bliss independence, a mathematical model for synergy in cellular systems, in approximately three minutes on commodity hardware. By making these tools open-access, we aim to empower cancer researchers with a streamlined, user-friendly platform for the rapid assessment of single- and dual-agent therapies in cell line models. Citation Format: James L. Elia, Sam L. Friedman, Ranjit S. Bindra. CellPyAbility: an integrated toolkit for high-throughput cell viability assays and automated analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6291.