Smaller scale, same impact: replicating high-throughput phenotypic profiling in a medium-throughput lab for use in chemical risk assessment.

Cell Painting visualizes toxicity-induced morphological changes by staining cellular structures with fluorescent dyes. Coupled with high-content imaging and analysis software, Cell Painting allows high-throughput phenotypic profiling (HTPP) to quantify phenotypic changes and estimate points of departure for toxicity assessments. Regulatory agencies have applied HTPP in 384-well plates for chemical hazard screening. In this study, established protocols for 384-well plates were adapted for use in 96-well plates to increase accessibility for laboratories with lower throughput. U-2 OS human osteosarcoma cells in 96-well plates were exposed to 12 phenotypic reference compounds for 24 h before fixation and staining with fluorescent dyes (golgi apparatus, endoplasmic reticulum, nucleic acids, cytoskeleton, mitochondria). Four independent chemical exposures across eight concentrations generated four biological replicates. Stained cells were imaged on an Opera Phenix, a high-content imaging system, and the Columbus analysis software extracted numerical values for 1300 morphological features. Features were normalized to control cells, followed by principal component analysis and a calculation of Mahalanobis for each treatment concentration. Mahalanobis distances were modeled to calculate benchmark concentrations (BMC) for chemicals. Most BMCs differed by less than one order of magnitude across experiments, demonstrating intra-laboratory consistency. Compared to published BMCs, ten compounds had comparable BMCs in both plate formats. In addition, we observed a significant inverse relationship between seeding density and Mahalanobis distances, suggesting that experimental factors like cell density may influence BMCs. Overall, we demonstrate that Cell Painting is adaptable across formats and laboratories, supporting efforts to develop and validate it as a complementary new approach methodology to existing toxicity tests.