Impedance spectroscopy for in vitro toxicology

The impedance of biological material changes with frequency, a phenomenon that has been discovered more than 100 years ago. It is due to the fact that the cell membrane acts as a capacitor which filters out currents at low frequency and lets them pass at high frequency. This fundamental knowledge about biological dielectrics has incompletely been exploited to detect and distinguish toxicity effects on cell cultures, although impedance measurements have been used for long in this field. In this thesis, it was found that low frequency impedance signals are linked to initial stress responses of cells within cell populations when exposed to a toxin whereas high frequency measurements inform about major cell damage as is indicated by intracellular conductivity changes. In addition, when cells gain resistance to a toxin, they experience a higher cell stiffness which is expressed by an increased low frequency impedance. The study of impedance changes as a function of frequency and drug concentrations lead to the creation of an impedimetric concentration-response map which distinguishes cell responses within four concentration ranges without the use of any label. Although being inherently non-specific, this measurement method was shown to report on distinct toxicity effects, an important prerequisite when studying drug action on cancer cells where stimulating and lethal effects need to be distinguished rigorously. This thesis further encompasses the subject of three-dimensional impedance measurements, i.e. the screening of the entire depth of a three-dimensional tissue culture. Given the success of impedance measurements on cell monolayers, one would expect this development to continue with 3D cultures since the complex structure of in vivo tissues is mimicked more closely and, above all, since rapid and inexpensive techniques which are able to probe thick tissue samples are currently inexistent. Nevertheless, few studies have been carried out in this field. Here, the requirements of three-dimensional impedance sensors are discussed and challenged by the fabrication of a corresponding device, involving the development of so-called gel electrodes through a novel 2-step-soft-lithography process. Their specific design allows for the decrease of leak currents, a common problem when performing three-dimensional impedance measurements. The simultaneous measurement of multiple samples in parallel is an an essential condition when performing high throughput drug toxicity screening. Electrode switch systems are necessary which ultimately lead to setup complexity and signal noises. In this thesis, a method is introduced, enabling the simultaneous implementation of impedance measurements of multiple tissue samples with one electrode pair only. This is simply achieved by exploiting the frequency domain and finally contributed to reducing setup complexity.