Cytotoxicity assays for cancer drug screening: methodological insights and considerations for reliable assessment in drug discovery

The importance of cytotoxicity assays in in vitro drug discovery investigations has led to their rising profile. Drugs and other substances can disrupt cell membranes, limit protein synthesis, and bind irreversibly to receptors, all of which lead to cell death in cancer cells. To precisely measure the cell death resulting from these damages, one must choose a cytotoxicity test that meets specific criteria. A systematic search strategy was used to gather grey literature from 2001 to 2024, utilizing databases such as PubMed and Google Scholar. Specific keywords related to colorimetric, fluorometric, and dye exclusion assays, as well as "cytotoxicity," were employed. Here, we only focus on screening drug cytotoxicity for cancer cells. This review discusses various cytotoxicity assays, such as "dye exclusion assays," "colorimetric assays," and "fluorometric assays." It is crucial to prioritize safety, speed, reliability, efficiency, and cost-effectiveness, while also ensuring minimal interference with the test compound. Commonly used in toxicology and pharmacology, cytotoxicity assays are based on several biological processes. Selecting the correct assay method requires considerations such as assay specificity and sensitivity, detection mechanism, test drug properties, and laboratory availability. This review aims to assist researchers in performing reliable cytotoxicity assessments by providing insights into assay choices.