Ralf Ehret

Monitoring of cellular behaviour by impedance measurements on interdigitated electrode structures

A new method for on-line and real-time monitoring of concentration, growth and physiological state of cells in culture is described. This biosensor is based on impedance measurements of adherently growing cells on interdigitated electrode structures (IDES). The measurements can be performed for several days as there is no detectable electrical influence on the cells. The versatility of this new sensor is shown with some exemplary experiments. Cell density, growth and long-term behaviour of cells on the electrodes clearly change the impedance of the IDES. Both, the global influence of serum components (deprivation of foetal bovine serum) and the toxic effects of heavy metal ions (cadmium) result in changes of the sensor signal and can be visualized.

On-line control of cellular adhesion with impedance measurements using interdigitated electrode structures

Critical parameters to be assessed in cell culture are the number of viable cells and cell viability. Growth, product formation, toxicity effects and the overall success of cell culture can depend largely on these. With interdigitated electrode structures (IDES) adherent cells are cultured directly on a pair of interdigitated electrodes, and the impedance of the system gives insight into the adhesive behaviour of the cells. The signal is influenced by the changes in number, growth and morphological behaviour of adherently growing cells, mainly owing to the insulating effects of the cell membranes. Five different cell lines are used, and their divergent behaviour is monitored over a period of four days, from inoculation of the cells to killing of the cells at the end of the experiments. Even when the cells form close monolayers, great fluctuations in the impedance signal can be observed. Nevertheless, for a more complete description of cellular systems, other parameters, such as acidification and respiration, have to be included in the measuring system.

Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET.

In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.

Monitoring of cellular signalling and metabolism with modular sensor-technique: the PhysioControl-Microsystem (PCM).

Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. The outstanding benefit lies in the passive behaviour of continuously working transducers, which in turn allows the dynamic recording of function-specific cellular processes. The microsensor system presented in this paper is a modular arrangement of various planar and non-planar sensor elements surrounding small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it proved to be an effective device both for toxicological and environmental research.

Non-invasive measurement of cell membrane associated proton gradients by ion-sensitive field effect transistor arrays for microphysiological and bioelectronical applications.

The pH in the cellular microenvironment (pH(M)) is an important regulator of cell-to-cell and cell-to-host interactions. Additionally the extracellular acidification rate of a cell culture is an important indicator of global cellular metabolism. In a new approach a biocompatible ion-sensitive field effect transistor (ISFET)-array was developed to measure the pH(M) close to a surface and the global extracellular acidification rate at the same time. This ISFET-array is part of a new multiparametric microsensor chip. The paper highlights some basic applications of this method for in-vitro measurements. Using a fluid perfusion system for cell culture media, it is possible to measure the pH(M) of few (five to ten) adherent tumor cells in a distance of 10-100 nm from the cell plasma membrane. Experiments showed a pH(M)-value of 6.68 +/- 0.06 pH. Further experiments suggest that both the low pH, and the extracellular acidification rate of the examined tumor cell line are mainly built up by glycolysis.

Approach to a multiparametric sensor-chip-based tumor chemosensitivity assay.

Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinicians decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

Biofunctional hybrid structures—cell–silicon hybrids for applications in biomedicine and bioinformatics

Abstract Cell–silicon hybrids represent miniaturized analytical systems which are qualified by means of the cellular signal processing network to identify biomolecules and biophysical signals in a highly specific and quantitative manner. Such biofunctional hybrid structures are also appropriate tools for a number of experimental approaches where specific reaction patterns and signal transducing processes of living cells are tested in response to defined environmental stimuli. The paper describes how cellular systems can function as multi-potent signal discriminators and interfaces to a variety of physical detection arrays. The analysis is based on recent results regarding the understanding of the intracellular signal transduction network. Our systems analytical approach gives an idea how the biological component of a cellular biosensor works and facilitates the directed design of new families of biofunctional hybrid structures. After a brief review of the construction lines of cell–silicon hybrids, novel improvements of their design and their applicability in tumor biology will be discussed.

Microsensor-Aided Measurements of Cellular Signalling and Metabolism on Tumor Cells

Microsensors provide instruments particularly suited for the rapid, noninvasive and on-line analysis of cell and tissue cultures. The microsensor system presented in this paper is a modular arrangement of various planar and nonplanar sensor elements for the measurement of physiological parameters of cell cultures. An optic access to the cultures (e.g. for light microscopy and spectrophotometric techniques) is also provided for a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy (predicative chemotherapy assays) and pharmacology but it turned out to be also an effective tool for toxicological and environmental research.

Mikrosensorische Systeme in der zellbiologischen Grundlagenforschung und der medizinischen Diagnostik

The development of an integrated sensor system, called the physiocontrol microsystem, is presented. It is suited for microscopy, and works with both adherent cell types and cultures growing in suspension, as well as with tissue biopsies. The central part, a miniaturized culture chamber equipped with differently constructed microsensors, allows continuous observation of important physiological parameters even in the course of long-lasting experiments. Besides a description of the physical components, the study provides a summary of selected applications of the physiocontrol microsystem in basic cellular research and biomedical diagnostics.

The Physiocontrol-Microsystem (PCM): Analysis of Cellular Behaviour for Biomedical Research

Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. Their outstanding benefit is the passive behaviour of continuously working transducers, which allows the dynamic recording of function-specific cellular processes. The microsensor system presented is a modular arrangement of various planar and non-planar sensor elements arranged in small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it turned out to be an effective device for toxicological and environmental research as well.