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Cell painting in HepaRG cells: an interlaboratory reproducibility study.

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Bibliographic Details
Title:	Cell painting in HepaRG cells: an interlaboratory reproducibility study.
Authors:	Sherer, Eric¹ (AUTHOR), Chen, Wei¹ (AUTHOR), Aberasturi, Dillon² (AUTHOR), Sagar, Ksheera¹ (AUTHOR), Li, Yang¹ (AUTHOR), Sutake, Zachary¹ (AUTHOR), Xu, Jiaqi³ (AUTHOR), Harris, Felix R⁴ (AUTHOR), Harrill, Joshua A⁵ (AUTHOR), LaRocca, Jessica¹ (AUTHOR)
Source:	Toxicological Sciences. Dec2025, Vol. 208 Issue 2, p301-316. 16p.
Subjects:	In vitro toxicity testing, Liver cells, Reproducible research, Phenotypes, In vitro studies, Xenobiotics, Toxicity testing
Abstract:	Traditional toxicological safety assessment relies heavily on the use of animals, and animal-free new approach methodologies (NAMs) are therefore critical for increasing the efficiency and human relevance of chemical hazard screening. Cell Painting, a high-content imaging assay that quantifies phenotypic changes at the cellular level, is an approach that has been widely used for pharmacological discovery purposes. In the present study, Cell Painting methodologies were adapted to the human liver cell line, HepaRG, given that the liver is a common target organ in standard repeat-dose toxicological studies. The HepaRG cell line was selected for its expression of phase I and II enzymes and ability to metabolize xenobiotic chemicals, which are critical features for an in vitro toxicological assay to assess chemicals that could be extensively metabolized in vivo. An interlaboratory reproducibility assessment was conducted to optimize culture conditions, image acquisition, computational workflows for image segmentation and feature extraction, and derivation of phenotype-altering concentrations (PACs). Two laboratories, the US EPA Center for Computational Toxicology and Exposure (site 1) and Corteva Agriscience (site 2), tested a set of 20 phenotypic reference chemicals in the HepaRG Cell Painting assay for derivation of PACs. The results from site 1 and site 2 were highly concordant both in terms of PAC estimates and the profiles of phenotypic effects observed for the test chemicals. These results support the reproducibility and robustness of the Cell Painting assay in the metabolically competent HepaRG cell line, thereby providing a NAM with the potential to predict in vivo toxicity. [ABSTRACT FROM AUTHOR]
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Database:	Engineering Source

Description
Abstract:	Traditional toxicological safety assessment relies heavily on the use of animals, and animal-free new approach methodologies (NAMs) are therefore critical for increasing the efficiency and human relevance of chemical hazard screening. Cell Painting, a high-content imaging assay that quantifies phenotypic changes at the cellular level, is an approach that has been widely used for pharmacological discovery purposes. In the present study, Cell Painting methodologies were adapted to the human liver cell line, HepaRG, given that the liver is a common target organ in standard repeat-dose toxicological studies. The HepaRG cell line was selected for its expression of phase I and II enzymes and ability to metabolize xenobiotic chemicals, which are critical features for an in vitro toxicological assay to assess chemicals that could be extensively metabolized in vivo. An interlaboratory reproducibility assessment was conducted to optimize culture conditions, image acquisition, computational workflows for image segmentation and feature extraction, and derivation of phenotype-altering concentrations (PACs). Two laboratories, the US EPA Center for Computational Toxicology and Exposure (site 1) and Corteva Agriscience (site 2), tested a set of 20 phenotypic reference chemicals in the HepaRG Cell Painting assay for derivation of PACs. The results from site 1 and site 2 were highly concordant both in terms of PAC estimates and the profiles of phenotypic effects observed for the test chemicals. These results support the reproducibility and robustness of the Cell Painting assay in the metabolically competent HepaRG cell line, thereby providing a NAM with the potential to predict in vivo toxicity. [ABSTRACT FROM AUTHOR]
ISSN:	10966080
DOI:	10.1093/toxsci/kfaf139