Cell painting in HepaRG cells: an interlaboratory reproducibility study

Area of Science:

• Toxicology
• Cellular Imaging
• In Vitro Assays

Background:

• Traditional toxicological assessments heavily rely on animal testing.
• New Approach Methodologies (NAMs) are crucial for enhancing chemical hazard screening efficiency and human relevance.
• Cell Painting, a high-content imaging assay, quantifies cellular phenotypic changes and is used in pharmacological discovery.

Purpose of the Study:

• To adapt Cell Painting methodologies for use with the HepaRG human liver cell line for toxicological safety assessment.
• To evaluate the interlaboratory reproducibility of the Cell Painting assay in HepaRG cells.
• To establish the assay's potential as a NAM for predicting in vivo toxicity.

Main Methods:

• The Cell Painting assay was applied to the HepaRG cell line, chosen for its metabolic capabilities.
• An interlaboratory reproducibility assessment optimized culture conditions, image acquisition, and computational analysis.
• Two laboratories (US EPA and Corteva Agriscience) tested 20 reference chemicals to derive phenotype-altering concentrations (PACs).

Main Results:

• The Cell Painting assay in HepaRG cells showed high concordance between the two participating laboratories.
• PAC estimates and observed phenotypic effect profiles were consistent across sites.
• The assay demonstrated robustness and reproducibility in a metabolically competent cell line.

Conclusions:

• The Cell Painting assay in HepaRG cells is a reproducible and robust animal-free method.
• This validated NAM holds significant potential for predicting chemical toxicity in vivo.
• The findings support the advancement of in vitro methods for chemical safety assessment.