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The intracellular protease calpain, abundant in endothelial cells (EC), is assumed to be inactive under physiological conditions but may account for Ca2+ -linked pathophysiological events. However, nonstimulated EC contained autolyzed, activated calpain. Adding 12-48 microM calpain inhibitor I (CI) or 0.5-1 microM of the novel, membrane-permeable conjugate of calpastatin peptide-penetratin (CPP) caused rapid rounding and retraction of cultured EC (phase contrast, capacitance) and translocation of Syk, Rac, and Rho to the membrane, signifying activation upon inhibition of calpain. Isolated hearts (guinea pig) perfused with 12 microM CI or 0.5 muM CPP developed coronary leak. We conclude that calpain is constitutively active in EC and regulates vascular permeability by governing cell-cell attachment.

Chemotherapeutic drugs affect the metabolism of tumor cells regardless of the specific target of action. Basic parameters of cell metabolism are extrusion of acids into the microenvironment and oxygen consumption. To analyze these changes on living cells in real-time, a test system based on multiparametric chips with an array of sensors for monitoring pH and O(2) as well as electric impedance has been developed. Cells are cultivated on these chips and supplied with medium by a fluid perfusion set-up which mimics microphysiological conditions and allows for drug addition and removal. Human colon carcinoma cells LS174T were used as a model to test the effect of drugs. Cells growing on chips were monitored for 24 h and longer. Untreated cells showed a continuous increase in the rate of acidification, while the rate of respiration remained fairly constant. Addition of chloroacetaldehyde (50 microM) rapidly attenuated O(2) consumption with a gradual decrease in acidification following. In contrast, with cisplatin (16.7 microM) a delayed and gradual decrease in both the rates of acidification and respiration effect occurred over 2-3 days. These results provide insights to the mechanisms of action of these drugs, which are coherent with those already known. Thus, multiparametric sensor chips provide elementary information on drug action.

Based on the experience with multiparametric bioelectronic sensor chips for the monitoring of living cells, we have combined the established multi-well-format with the advantages of microelectronic sensors. The result is a 24-well-plate where the bottom of the wells is replaced by glass-based chips with integrated IDES (interdigital electrode structure). By using IDES it is possible to detect adhesion and morphological changes of adherent growing cell cultures. Up to now these measurements were inaccessible in conjunction with multi-well-plates, especially in high throughput applications. If microscopic monitoring of the cell culture is required, the IDES can be fabricated using transparent conductor materials like ITO (indium tin oxide). Both the transparent material for the sensors and the sensor-carrier make the multi-well-plate also applicable for all kinds of fluorescence and luminescence biological tests. In addition to the impedance-sensors optical read-out sensor layers for pH and pO2 can be integrated. For this reason there are many possible fields of application in biological and biomedical areas such as drug screening, chemosensitive testing and environmental toxicology as well as in biosensing for biological and chemical warfare. Especially the possibility of using this multi-well-plate together with automated imaging-systems has the great advantage of combining optical and sensory monitoring accessible to high-throughput-applications.

An alternative assay for replacing animal experiments should serve the specific microphysiological needs of the cells and be endowed with multiparametric signal monitoring. These requirements are provided by a test system in which the key elements are biocompatible electronic sensor-chips. It is also connected to a medium perfusion set-up, which allows to control the supply of nutrients and test compounds, and the removal of culture medium. The chips are equipped with sensors that continuously monitor basic metabolic parameters and membrane-associated changes of living cells: pH-ISFETs for extracellular acidification, amperometric O2-sensors for oxygen consumption, and IDES for electrical impedance of the cell layer. Experiments with LS174T colon carcinoma cells in culture show the metabolic and electrical changes upon incubation with Zytochalasin B and chloroacetaldehyde. The signal patterns vary and indicate different mechanisms of action for these test compounds. With this test system it is possible to detect effects of unknown substances and mixtures, and to analyse the cellular probe for prolonged times.

A constraint in the reliability of predictive chemosensitivity assays is linked to the fact that they analyze only a single cellular or biochemical parameter. A multiparametric test system using microsensor chips has been developed which can detect online microphysiological changes in living cells. Tumor cells were grown directly on glass- or silicon-based electronic sensor chips. Changes in extracellular pH and pO(2), reflecting metabolic activities, and changes in impedance, reflecting morphological properties, were monitored. In this study, colon and breast cancer cells as well as doxorubicin-sensitive and doxorubicin-resistant sarcoma cell lines were exposed to cytochalasin B, chloroacetaldehyde, or doxorubicin. Results show (1) reduction in medium acidification, (2) marked and rapid changes in O(2) consumption, and (3) modulations in impedance correlating with morphological changes observed in the microscope. Drug-resistant cells do not show these changes. Therefore, this microphysiological monitoring is a versatile tool for chemosensitivity testing of tumor cells.

Many different assays have been developed for testing the chemosensitivity of tumor cells in vitro, usually based on a single biochemical or cellular parameter. A multiparametric test system has been developed that accommodates on a single chip numerous sensors for metabolic parameters, deltapH and deltapO2, as well as for morphological changes. The cells grow directly on the chips and can be continuously monitored online up to several days. The effects of various chemotherapeutic drugs on the metabolic profile of several tumor cell lines have been investigated. In colon carcinoma-derived LS174T cells, cytochalasin B markedly increased oxygen consumption while decreasing the rate of extracellular acidification. These effects, which reflect the biochemical action of cytochalasin B, were reversible on drug removal. In contrast, chloroacetaldehyde markedly reduced respiration, which recovered when the drug was removed. Primary breast cancer cells also responded to chloroacetaldehyde with a marked reduction in deltapO2, followed by a reduced rate of acidification. Comparing the metabolism of doxorubicin-sensitive and -resistant mouse sarcoma S180 cells, the rates of acidification and respiration were inhibited by doxorubicin only in the sensitive cells, whereas in the resistant cells oxygen consumption even increased. These examples demonstrate that this chip-based test system provides rapid and important information for assessing chemosensitivity of tumor cells.