Impedance-Based Real-Time Monitoring of Mammalian Cells upon Introduction of Xenobiotics into the Cytoplasm by In Situ Electroporation

Whole-cell biosensors are irreplaceable tool for studies of cellular mechanisms and behavior of the cell as a smallest living unit. Their development have progressed rapidly over past decades and nowadays we have powerful tools to study cell-based assays and to examine behavior of the cells exposed to different kinds of stimuli and challenges. Limited and selective permeability of the plasma membrane prevents the introduction of hydrophilic xenomolecules into the cytoplasm of mammalian cells. However, it is essential for many fields of cell biology, biomedicine or biotechnology to allow transport of such molecules (e.g. nucleic acids, antibodies, peptides or drugs) across the cell membrane. An ultimate goal of this thesis was to establish proof-of-principle assays for delivery of various bioactive molecules into adherent cells by in situ electroporation, and to monitor how these compounds influence cellular behavior, once they are internalized within the cell cytosol. Studies of in situ electroporation (ISE) were conducted using different types of mammalian cells (BAEC, CHO-K1/CHO-GFP, HaCaT, NRK and NIH-3T3) grown to confluence on small planar gold film electrodes. For every cell line individually, electric pulse parameters were optimized to achieve maximal loading efficiency, while keeping the invasiveness of the operation as low as possible. Impedance monitoring of in situ electroporation conducted with high time resolution showed biphasic changes of impedance signal after pulse application, indicating (i) fast recovery of the cell membrane integrity and (ii) relatively slow process of cell recovery after changes induced by membrane permeabilization. For the first time, release of intracellular material from the cells by ISE was studied using ECIS setup. Direct time-resolved imaging of NRK cells showed measurable efflux of fluorescence-labeled probes upon multiply applied electric pulses. In situ electroporation allowed transfer of second messenger (8-OH-)cAMP in the cell monolayers. Subsequent changes in impedance signal were in agreement with those observed after stimulation of the cells with membrane-permeable compounds CPT-cAMP and forskolin, as a consequence of triggering of the corresponding signaling cascades. Transport of nucleic acids into cytoplasm and nuclei of NRK and CHO-GFP cells was conducted in a highly-efficient manner. Besides fluorescent DNA aptamers, various types of siRNA molecules were successfully delivered into cells by in situ electroporation and their long-term sequence-specific silencing effect on cells was demonstrated and quantified by using microscopy and/ or impedance analysis. Transfection performance of ISE was compared with conventional and widespread delivery transfection method. In conclusion, this thesis demonstrated that in situ electroporation allows for highly efficient delivery of emerging types of molecules into monolayers of various types of cells