Singapore, Feb 25, 2010: Cell-based in vitro assays are a key tool for the assessment of compound-induced toxicity in early drug development before entering regulatory animal testing. Given the dramatically increasing costs for drug development at later stages, it is of high importance to select the most efficacious and safe drugs for animal and human testing at an early stage in vitro.

To date, cellular assays to a large extent consist of endpoint assays where the drug effect on the cell is measured after a defined experimental duration time, and in most cases cells are lysed or treated with an invasive dye or antibody. Some effects remain undetected under such experimental paradigms because they may develop either before or after the endpoint measurement is performed, and thus may lead to a wrong assessment of the compound’s toxic potential. To overcome this problem, large series of experiments with different durations would be needed. This increases costs, hands on- and turnaround-time.

A tool which allows measuring cellular behavior upon drug treatment over the entire length of the experiment therefore bears the potential of generating in vitro toxicity data of different quality and higher relevancy as compared to conventional assay technologies. Such a non-invasive and “label-free” tool, which allows continuous monitoring of cells in culture is provided by the xCELLigence Real-Time Cell Analyzer (RTCA) System. Co-developed by Roche and ACEA Biosciences, xCELLigence is based on measuring electric impedance generated by adherent cells, which interact with a microelectrode biosensor at the bottom of the well of a cell culture plate. The readout, called ‘cell index’ (CI), measures morphological alterations of cells caused by e.g. drug-treatment. The technique allows hands-free dynamic monitoring of cellular events, such as proliferation, cell death, adhesion, spreading and shrinking in a 96-well format over an extended period of time.

We have explored the applicability of such a tool for assaying drug candidates in pre-clinical development. For example, dynamic RTCA analysis and conventional biochemical endpoint assays were performed in parallel to study drug-induced toxicity in liver cells and to compare both experimental assays with each other. The comprehensive information obtained from impedance readouts over the entire length of the experiment allowed timepoint-decisions for the experimental set-up: optimal time of administration of compound or when to apply down-stream analyses such as biochemical assays and gene expression. In many cases, CI proved to be a highly sensitive indicator for drug-induced heaptotoxicity at low concentrations and at earlier timepoints as compared to established standard measurements such as Glutathione depletion, ATP content, or release of Lactate dehydrogenase.

Other tests included cardiac cells, fibroblasts or epithelial cells to address different modes of toxicities relevant for preclinical testing: Commercially available rapidly dividing cell types appeared to be well suited model systems to study compound-induced inhibitory effects on cell growth, i.e. cell cycle and cell division, while pro-proliferative effects could be studied in otherwise slowly growing cell types such as the medullary carcinoma cell-line “TT”. Fully differentiated, non-dividing cell types such as primary cardiomyocytes were used to study morphological alterations induced by drug candidates. In this case, xCELLigence was able to detect subtle changes in the shape of heart cells grown in a subconfluent culture and this may help prioritizing compound series in the future. Likewise, pro-inflammatory markers which are known to alter the barrier function of the endothelium were used in vitro to characterize effects on endothelial cells. These measurements gave insight into the mode of action of known toxicants and may be used to classify development compounds with unknown properties.

The xCELLigence System may significantly improve the quality of toxicological in vitro tests as it appeared to be more sensitive when compared to standard endpoints. Each compound generated characteristic concentration-dependent kinetic patterns, and the shape of the impedance profiles might hold information regarding the type of toxicity. As the impedance measurement is non-invasive, the xCELLigence system may be coupled to other readouts such as analysis of medium supernatant, image-based monitoring of cells as well as biochemical or molecular analysis of cell lysates at the end of the assay.

Impedance measurements therefore have the potential to improve the quality of in vitro assays widely used in preclinical development and further applications are currently being explored, such as assaying stem cell-derived in vitro models or additional primary cell types otherwise difficult to handle in an in vitro setting.

Dr. Adrian Roth is a Senior Scientist and Group Head Mechanistic Toxicology Non-Clinical Safety, Pharma Research, at Hoffmann-La Roche in Basel, Switzerland. After graduating in Molecular Biology and a PhD in Experimental Pharmacology, he joined the group of Prof. Urs A. Meyer in 2001 at the Biocenter in Basel for postdoctoral studies on nuclear receptors within the Genome Biology program. He then joined Roche to become a Laboratory Head for Toxicogenomics in 2005. During this time he also explored various other technologies for use in toxicology beyond microarrays. Since January 2009 he leads the Mechanistic Toxicology group within Non-clinical Safety at Hoffmann-La Roche which supports both early as well as late stage drug development projects and establishes novel technologies and tools for pharmaceutical research