The role of time-lapse fluorescent microscopy in the characterization of toxic effects in cell populations cultivated in vitro.

Responses of cell populations in vitro to toxic substances are very dynamic and exceed hours or even days. Toxicologists realise that cell-based dynamic assays can acquire important additional information about toxic responses of individual cell within a treated population. In the past, this type of cellular dynamics could have been monitored only with the help of time-lapse microcinematography. In spite of several advantages, the time-lapse approach has been used relatively infrequently in the routine in vitro cytotoxicity assessment. The main reasons were demanding time and work schedules, problems with quantification of visual information, and lack of mechanistic data. Recently, the situation has changed dramatically. The progress in digital imaging technology coincides with enormous development in the field of fluorescence microscopy. With the help of specific fluorochromes or fluorescent proteins we can now analyse practically all sub-cellular and cellular events. Some producers developed large-scale high-throughput systems for live cell imaging. Nevertheless, there has been significant progress in small-scale approach as well. New versions of motorised microscopes are fulfilling principal demands for in vitro assessment of toxic effects: ease of performance, high-throughput of data, quantitative cell-based analysis, simultaneous assessment of several parameters, and control of the environment conditions for cultivation of cells. In this paper, we present our experience with two of these systems designed for long-lasting observation of living cells in phase contrast and fluorescence. Our aim is to draw attention to the suitability of small-scale cell-based assays for determination of cytotoxicity in vitro. We believe that these methods could be considered as a further step towards the replacement of animal testing.