Real-time monitoring and quantification of drug induced changes in endothelial cytoskeleton filaments using a cellular impedance biosensor

by 1982- Nandakumar, Vijay

The analysis of electrical signals originating from biological cells and tissues have yielded a large amount of useful information over the past years. Technologies have been developed, wherein we can monitor the health of the biological material of interest using these electrical signals. Instruments for the study of living cells have historically been of significant importance for such things as basic neuroscience, cell biology, pharmaceutical screening, environmental monitoring, and toxin detection. One of the practical realizations of these methods was successfully implemented by Giaver and Keese with their Electric Cell-Substrate Impedance Sensing (ECIS) system. The purpose of this Masters Thesis is to showcase the use of a Cellular Impedance Biosensor setup based on the ECIS system in real-time monitoring of endothelial barrier function and quantify the dynamic changes of the cytoskeleton filaments induced by different drugs. The filaments of the endothelial cell cytoskeleton play a critical role in cellular micro motion and the inflammatory response. The fact that the cellular cytoskeleton is the essential force behind all the motile activities of the cell is used in drug discovery methods to design ways so as to kill tumor cells and developing cell-based therapies for different cardiovascular pathologies. Cytochalasin D was chosen to study the actin filament response as the drug specifically inhibits the polymerization of actin filaments, which play a crucial role in the cellular mechanical strength and micromotion. Nocodazole was iv chosen to study microtubules, which play a pivotal part in the cellular mitotic activities and locomotion. These drugs were chosen as they have extreme effects on the cytoskeleton and would be ideal to showcase the use of the biosensor in tracking the intracellular dynamics. The biosensor system provides a simple interface for monitoring the electrical activity and impedance characteristics of populations of cultured cells over extended periods. The cell - sensor interface is created as cells attach to the gold electrode surface pre-coated with fibronectin. Using these impedance measurement techniques based on a simple cellular geometric model, we have been able to successfully monitor cellular adhesion, motility, proliferation and changes in the cellular cytoskeleton induced by different drugs as mentioned above. The kinetic response of the cellular cytoskeleton to different doses of these drugs is translated as changes in the impedance measured by the biosensor setup. The data obtained from the setup were quantified by correlating them with the images obtained by confocal microscopy. A wavelet transformation algorithm is applied to the acquired data in an attempt to capture the fluctuations and compare the cellular behavior before and after the addition of drugs. Study of the disruption of these filaments by toxins and pharmacological agents holds lot of promise to provide a model for studying their role in endothelial cell biomechanics and the pathology of cardiovascular, pulmonary, and renal disease. This thesis presents a cellular impedance biosensor setup that can carry out this study in real time with high sensitivity and reproducibility. Using these impedance measurement techniques, we have v been able to successfully monitor cellular adhesion, motility, proliferation of different type of cells and their response to external stimuli. vi