application I

It is well known that this reagent leads to a depletion of extracellular Ca2+ which in turn causes a disassembly of tight junction [1, 2]. The latter is reflected by a significant drop in the TER readings. Subsequent replacement of the EGTA containing medium by standard medium led to a regeneration of the tight junctions network as revealed by increasing TER readings. For validation of the temporary break down of the barrier function two cell cultures were fixed just before removal of EGTA. Samples were then stained for immunofluorescent analysis of cell nuclei and ZO-1 proteins. Imaging by Confocal Laser Scanning Microscopy and comparison with the untreated reference cell culture clearly revealed the disintegration of the tight junctions network induced by EGTA exposure. These findings are in excellent agreement with the TER results and demonstrate the benefits of using a label-free and noninvasive technique as implemented in the cellZscope.

Cell layer formation

Application of impedance spectroscopy for cell analysis is not limited to short-term following of dynamic processes but also allows continuous monitoring of cell cultures for several days or even weeks. The fact that various factors such as seeding density, type of growth medium, concentration of serum, type of substrate coating and temperature have a strong influence on cell layer formation calls for a means to monitor growth continuously while maintaining physiological conditions. Both readout parameters of the cellZscope, the transepithelial / -endothelial electric resistance (TER) and the cell layers’ capacity (Ccl) provide valuable information about the current state of cells as they grow confluent and differentiate. Caco-2 is a well-established cell line derived from human colon adenocarcinoma which is commonly used for drug-transport studies. Precise knowledge of the cell layer’s growth state is mandatory for use as an intestinal permeability model. In the experiment depicted below the cellZscope was employed to follow layer formation and differentiation of Caco-2 cells, with automated data recording beginning right after seeding and continuing for a total time span of more than three weeks.

Except for media exchange at regular time intervals no manual intervention was necessary for recording the data. Thus optimal physiological conditions were maintained throughout the experiment. The full time course reveals distinct differences in the growth behavior of the two different Caco-2 cell lines. Hence, the cellZscope provides quality control for cell layers and allows to have well-defined starting conditions for subsequent experiments such as permeability studies.