Technological advances in cytotoxicity analysis have now made it possible to obtain real time data on changes in cell growth, morphology and cell death. This type of testing has a great potential for reducing and refining traditional in vivo toxicology tests. By monitoring the dynamic response profile of living cells via the xCELLigence real-time cell analyzer for high-throughput (RTCA HT) system, cellular changes including cell number (cell index, CI) are recorded and analyzed. A special scaled index defined as normalized cell index (NCI) is used in the analysis which reduces the influence of inter-experimental variations. To assess the extent of exposure of the tested chemicals, a two-exponent model is presented to describe rate of cell growth and death. This model is embodied in the time and concentration-dependent cellular response curves, and the parameters k 1 and k 2 in this model are used to describe the rate of cell growth and death. Based on calculated k 2 values and the corresponding concentrations, a concentration–response curve is fitted. As a result, a cytotoxicity assessment named KC 50 is calculated. The validation of the proposed method is demonstrated by exposing six cell lines to 14 chemical compounds. Our findings suggest that the proposed KC 50-based toxicity assay can be an alternative to the traditional single time-point assay such as LC 50 (the concentration at which 50% of the cells are killed). The proposed index has a potential for routine evaluation of cytotoxicities. Another advantage of the proposed index is that it extracts cytotoxicity information when CI fails to detect the low toxicity. 
In vitro cytotoxicity assessment based on KC 50 with real-time cell analyzer (RTCA) assay