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High Content Screening: Cell-based Assays for In Vitro Toxicology Prediction

High Content Screening (HCS), also known as High Content Analysis (HCA) or High Content Imaging (HCI), is a powerful tool in preclinical drug discovery. Depending on the type of analysis needed, HCS can be used to predict toxicity, confirm or identify targets or pathways for lead compounds, or even elucidate possible mechanism of action for orphan compounds.

High Content Screening uses automated microscopy to evaluate cellular responses to xenobiotics, typically by using fluorescent dyes or fluorescently labelled antibodies to mark specific phenotypic features of a cell, like proteins or organelles. Multiple dyes known to bind to specific cell sites can be employed at the same time because each has a specific light wavelength that causes them to fluoresce. This can provide a rich set of data that may show multiple mechanisms of toxicity from a single compound within individual cells.

Like standard microscopes, HCS instruments employ one of three types of light sources that cause excitation of the fluorescent dyes; lamps, lasers or light emitting diodes (LEDs), each with their own advantages and disadvantages. The excitation responses are recorded as images, and after acquisition is complete, the images gathered are analysed by specialised software programmed to quantify phenotypic differences in a given cell type. It is often within the software provided that the greatest differences in HCS platforms are evident. Despite the fact that the instruments themselves vary in many ways, such as light source, environmental control capabilities and objective type, they serve the same purpose: to aggregate images. The ability to accurately analyse these images lies in the software. Building analysis algorithms and statistical models should occur during assay development whenever possible. As a cell-based assay is validated, phenotypic response can and should be used to define criteria for analysis, thus reducing false positives and false negatives, so the ability to customise and extend a platform to fit the goals of an HCS program is critical.

Phenotypic screening on HCS platforms can provide a wealth of cellular response information. Depending on the objectives, these data can be used to assess biological responses and identify potential therapeutic targets, or suggest possible mechanistic pathways.

Cyprotex have recently authored a publication entitled ‘High-Content Screening : Understanding and Managing Mechanistic Data to Better Predict Toxicity’. The book chapter by Walker et al., (2015) was published in Computational Systems Toxicology (Editors, Hoeng J and Peitsch M). The chapter covers HCS approaches used to determine drug-induced cellular toxicity.

More information on HCS can be found on