We describe here the use of the xCELLigence system for label-free and real-time monitoring of cell -viability. The xCELLigence system uses specially designed microtiter plates containing interdigitated gold microelectrodes to noninvasively monitor the viability of cultured cells using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system makes it possible to distinguish between different perturbations of cell viability, such as senescence, cell toxicity (cell death), and reduced proliferation (cell cycle arrest). In addition, the time resolution of the xCELLigence system allows for the determination of optimal time points to perform standard cell viability assays as well as other end-point assays to understand the mode of action. We have used the WST-1 assay (end-point viability readout), the cell index determination (continuous monitoring of viability by xCELLigence), and the DNA fragmentation assay (end-point apoptosis assay) to systematically examine cytotoxic effects triggered by two cytotoxic compounds with different cell-killing kinetics. Good correlation was observed for viability readouts between WST-1 and cell index. The significance of time resolution by xCELLigence readout is exemplified by its ability to pinpoint the optimal time points for conducting end point viability and apoptosis assays.

A real-time cell electronic sensing (RT-CES) system was used for label-free, dynamic measurement of cell responses to cytotoxicants. Cells were grown onto the surfaces of microelectronic sensors, which are comprised of circle-on-line electrode arrays and are integrated into the bottom surfaces of the microtiter plate. Changes in cell status such as cell number, viability, morphology, and adherence were monitored and quantified by detecting sensor electrical impedance. For cell quantification and viability measurement, the data generated on the RT-CES system correlated well with those from the colorimetric (MTT) assay. For cytotoxicity assessment, cells growing on microelectronic sensors were treated with different cytotoxicants, such as arsenic, mercury, and sodium dichromate. The dynamic responses of the cells to the toxicants were continuously monitored by the RT-CES system. On the basis of the IC50 values, the RT-CES system displays an equal sensitivity to the neutral red uptake assay at specific time points. Furthermore, because the RT-CES system provides real-time information regarding the state of cell morphology and adhesion in addition to cell number, we were able to discern a previously unreported effect of arsenic on NIH 3T3 cells prior to cell death. Also, using the RT-CES system, we were able to monitor cytotoxicity effects that occur within a minute of compound addition. Taken together, the RT-CES system allows for real-time, continuous monitoring and quantitative recording of the whole assay process and provides new insight into the cell-toxicant interaction.

Summary We describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This non-invasive readout allows continuous sampling of cellular responses to biologically active compounds and the ensuing kinetic profile provides information regarding the temporal interaction of compounds with cells. The utility of this approach was tested by screening a library containing FDA approved drugs, experimental compounds and nature compounds. Compounds with similar activity produced similar impedance-based time-dependent cell response profiles (TCRP). The compounds were clustered based on TCRP similarity. We identified novel mechanisms for existing drugs, confirmed previously reported calcium modulating activity for COX-2 inhibitor, celecoxib and identified an additional mechanism for the experimental compound, monastrol. We also identified and characterized a new anti-mitotic agent. Our findings indicate TCRP approach provides predictive mechanistic information for small molecule compounds.

G protein-coupled receptors (GPCRs) constitute important targets for drug discovery against a wide range of ailments including cancer, inflammatory, and cardiovascular diseases. Efforts are underway to screen selective modulators of GPCRs and also to deorphanize GPCRs with unidentified natural ligands. Most GPCR-based cellular screens depend on labeling or recombinant expression of receptor or reporter proteins, which may not capture the true physiology or pharmacology of the GPCRs. In this paper, we describe a noninvasive and label-free assay for GPCRs that can be used with both engineered and nonengineered cell lines. The assay is based on using cell-electrode impedance to measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation. We have used this technology to assay the functional activation of GPCRs coupled to different signaling pathways and have compared it to standard assays. We have used pharmacological modulators of GPCR signaling pathways to demonstrate the specificity of impedance-based measurements. Our data indicate that cell-electrode impedance measurements offer a convenient, sensitive, and quantitative method for assessing GPCR function. Moreover, the noninvasive nature of the readout offers the added advantage of performing multiple treatments in the same well to study events such as desensitization and receptor cross-talk.

Label-free detection emerges as a new approach in the development of technologies for cell-based screening assays. Unlike the classic detection methods that use fluorescence, radioisotope, luminescence, or light absorption, label-free detection directly measures the cell function without using a labeled molecule. The advantages of label-free detection include a simple homogeneous assay format, noninvasive measurement, less interference with normal cell function, kinetic measurement, and reduced time for assay development. Here, we have applied the electrical impedance detection method in a real-time cell electronic sensing (RT-CES trade mark ) system for cell-based assays. The cell growth rate measured by this RT-CES system was comparable to actual cell number counted manually. In addition, cell proliferation, cytotoxicity, cytoprotection, cell growth inhibition, and apoptosis data generated by this RT-CES system correlated with those determined by the classic methods. The conclusion is that the RT-CES system is a useful tool for label-free detection of certain cell-based parameters.

Aortic dissection is a major aortic catastrophe with a high morbidity and mortality risk caused by the formation of a tear in the aortic wall. The development of a second blood filled region defined as the “false lumen” causes highly disturbed flow patterns and creates local hemodynamic conditions likely to promote the formation of thrombus in the false lumen. Previous research has shown that patient prognosis is influenced by the level of thrombosis in the false lumen, with false lumen patency and partial thrombosis being associated with late complications and complete thrombosis of the false lumen having beneficial effects on patient outcomes. In this paper, a new hemodynamics-based model is proposed to predict the formation of thrombus in Type B dissection. Shear rates, fluid residence time, and platelet distribution are employed to evaluate the likelihood for thrombosis and to simulate the growth of thrombus and its effects on blood flow over time. The model is applied to different idealized aortic dissections to investigate the effect of geometric features on thrombus formation. Our results are in qualitative agreement with in-vivo observations, and show the potential applicability of such a modeling approach to predict the progression of aortic dissection in anatomically realistic geometries.

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Cellular interaction with and adhesion on different biological surfaces is a dynamic and integrated process requiring the participation of specialized cell surface receptors, structural proteins, signaling proteins, and the cellular cytoskeleton. In this report, the authors describe a label-free and real-time method for measuring and monitoring cell adhesion on special microplates integrated with electronic cell sensor arrays. These plates were used in conjunction with the real-time cell electronic sensing (RT-CES™) system to dynamically and quantitatively monitor the specific interaction of fibroblasts with extracellular matrix (ECM) proteins and compared with standard adhesion techniques. Cell adhesion on ECM-coated cell sensor arrays is dependent on the concentration of ECM proteins coated and is inhibited by agents that disrupt the interaction of ECM with cell surface receptors. Furthermore, the authors demonstrate that the integrity of the actin cytoskeleton is required for productive cell adhesion and spreading on ECM-coated microelectronic sensors. Confirming earlier results, it is shown that interfering with Src expression or activity, via siRNA or small molecule, results in the disruption of adhesion and spreading of BxPC3 cells. The results indicate that the RT-CES system offers a convenient and quantitative means of assessing the kinetics of cell adhesion in a high-throughput manner. (Journal of Biomolecular Screening 2005:795-805)