Methodological Variations in Cell Viability Assays: A Systematic Review and Experimental Evaluation Using a Caco-2 Cell Model

Cell viability assays are essential tools for evaluating chemical safety. However, a lack of methodological standardization continues to compromise data reproducibility and comparability. In this study, we focused on the Caco-2 cell line and a combination of three cell viability assays: alamar blue (AB), 5-carboxyfluorescein diacetate acetoxy methyl ester (CFDA-AM), and neutral red (NR). A systematic literature review revealed substantial heterogeneity in the application of these assays. We identified prevailing experimental practices and evaluated their impact on cell viability outcomes. Seeding density significantly affected the outcomes of all three assays, with lower densities consistently producing lower viability measurements at several toxicant concentrations. The incubation medium (DMEM versus DPBS) significantly influenced AB and NR (not CFDA-AM) outcomes, although its effect was less pronounced. Assay incubation time (30 min, 1 or 2 h), evaluated for AB and CFDA-AM assays, had no statistically significant impact on the outcomes. However, its potential influence on subsequent NR measurements remains inconclusive. These findings demonstrate that routinely applied experimental conditions can substantially influence cell viability measurements. Standardization and transparent reporting of methodological parameters, together with appropriate positive controls, are therefore essential to improve the reliability, reproducibility, comparability, and interpretation of cell viability data.