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

Area of Science:

• Cellular imaging and analysis
• Toxicology and hazard assessment
• High-content screening

Background:

• Cell Painting enables high-throughput phenotypic profiling (HTPP) for toxicity assessment using fluorescent dyes and high-content imaging.
• Current HTPP protocols are often optimized for 384-well plates, limiting accessibility for lower-throughput laboratories.
• Regulatory agencies utilize HTPP for chemical hazard screening.

Purpose of the Study:

• To adapt established Cell Painting protocols from 384-well to 96-well plates.
• To assess the consistency and comparability of toxicity data generated in the 96-well format.
• To evaluate the adaptability of Cell Painting across different laboratory formats.

Main Methods:

• U-2 OS cells were cultured and treated with 12 reference compounds in 96-well plates.
• Cells were stained for five cellular compartments (Golgi, ER, nucleic acids, cytoskeleton, mitochondria) and imaged.
• Morphological features were extracted, normalized, and analyzed using principal component analysis and Mahalanobis distance to calculate benchmark concentrations (BMC).

Main Results:

• The adapted 96-well plate protocol demonstrated intra-laboratory consistency, with most BMCs differing by less than one order of magnitude.
• BMC values obtained in the 96-well format were comparable to published BMCs for ten out of twelve compounds.
• An inverse relationship between cell seeding density and Mahalanobis distances was observed, indicating potential influence of experimental factors.

Conclusions:

• Cell Painting is adaptable to 96-well plate formats, enhancing its accessibility for toxicity testing.
• The 96-well Cell Painting protocol provides consistent and comparable results to established methods.
• This adaptation supports the validation of Cell Painting as a complementary new approach methodology for toxicity testing.