Contemporary high-content screening approaches to assess the biological impact of single compounds and complex mixtures in vitro
High-content screening (HCS) is an automated image-based screening technology that allows analysing macroscopically the biological impact of single compounds or complex mixtures on cells growing in multi-well culture dishes. The technology involves fluorescence microscopy that is paired with high-throughput image acquisition and a series of algorithms and software tools for image processing and analysis. Taking advantage of fluorescent labeling using different dyes, multiplexing to simultaneously measure various cellular parameters can be achieved. To run a comprehensive analysis of a cytotoxic impact, HCS, through a battery of different assays, usually analyses structural or quantitative changes in cellular compartments or individual biomolecules such as nuclear size, DNA structure, mitochondrial mass, mitochondrial membrane potential, cytochrome C release and cellular membrane permeability. In addition, changes in subcellular localization of biomolecules upon cytotoxic impacts can be monitored. Resulting from the development of confocal versions of HCS readers, assays previously established for two-dimensional (2D) cellular models may also being applied for HCS analysis of reconstituted human three-dimensional (3D) organotypic cell models. These models more closely mimic the physiology of the corresponding tissue counterpart in humans and therefore provide enhanced translatability of results. HCS may be combined with omics technologies to expand the assessment to the systems level and allow to investigate more readily the underlying mechanisms of toxicity.
This presentation intends to provide a general overview on HCS and how it can be used in in vitro respiratory toxicology of next generation tobacco and nicotine products, illustrated by examples of testing aerosol fractions and individual constituents measured in 2D cell culture, as well as applications of HCS to 3D organotypic cell models.