Electric manipulation and impedance analysis of adherent cells on gold-film electrodes

This work addresses the electric manipulation, i.e. electroporation or electrofusion, of anchorage-dependent mammalian cells and the subsequent analysis of the cellular response using a well-established impedimetric biosensor (ECIS). Cells were grown to confluence on planar gold-film electrodes, which were used for the application of invasive electric AC pulses as well as for the non-invasive impedimetric monitoring of the cell layer response. The first part of this work deals with the electroporation-assisted delivery of different classes of membrane-impermeable bioactive probes into various types of anchorage-dependent mammalian cells and the analysis of cell morphology changes by non-invasive impedance measurements. Optimal pulse parameters for electroporation of different adherent cell lines (NRK-52E, HEK-293, Hep G2, CHO, NIH-3T3) were obtained from dye loading studies with high molecular weight FITC-dextran in combination with impedance monitoring of cell layer recovery kinetics. The cell lines showed an individual impedance profile after application of the electroporation pulse. Impedance measurements with a high time resolution and timelapse CLSM recordings revealed that within ~ 5 s after electroporation the membrane is resealed while the uptake of 250 kDa FITC-dextran is completed after ~ 3 s. Post-pulse impedance changes recorded over about 1 h after electroporation have been ascribed to changes in cell morphology. Electroporation of NRK cells in presence of membrane-impermeable cytotoxic molecules (azide, bleomycin, cytochrome c) changed the cellular impedance response profile, depending on the individual mechanism of cytotoxicity. Accompanying live/dead assays confirmed the impedimetric readout. The experimental setup for electric manipulation and sensing was further optimized to allow for economic use of costly bioactive macromolecules, like enzymes, antibodies or nucleic acids in electroporation-assisted delivery. The miniaturized setup requires only sample volumes of less than 30 μl without influencing the loading efficiency or the quality of the ECIS measurement. After optimization of the electroporation parameters for different cell types the miniaturized microelectrode setup was used to monitor the cell layer response after loading with cytochrome c, DNase I and RNase A. Intracellular delivery of antibodies, nucleic acids and nanoparticles was proven by fluorescence microscopy. NRK and Hep G2 cells were loaded with antibodies that specifically bound to their intracellular epitopes ß-catenin, occludin and ZO-1, respectively. The in situ electroporation of adherent cells (NRK, Hep G2, HEK-293) in presence of plasmid DNA or a linear DNA fragment encoding fluorescent reporter genes resulted in cell type-dependent transfection efficiencies between 10 % and 70 %. Moreover, NRK cells were efficiently loaded with PEGylated core-shell CdSe/ZnS quantum dots, resulting in a homogeneous cytoplasmic distribution of the probe. The second part of this work was devoted to the use of substrate-integrated planar gold-film electrodes to induce and detect cell fusion. The electroporation of HEK-293 cells in presence of polyethyleneimine (PEI) coated iron oxide particles designed for transfection (PolyMAG) led to a considerable cell fusion and the formation of polynucleate giant cells on the ECIS electrode. Multi-cell fusion was proven by specific cytochemical stainings as well as by fusing genetically engineered cell lines expressing fluorescent cytoplasmic (HEK-EGFP, HEK-ECFP) or membrane proteins (HEK-EYFP/pAbcg2). The process of multi-cell fusion was monitored electrically by ECIS readings at 4 kHz with a time resolution down to 0.6 min. Cell fusion was dependent on the HEK cell line. The experimentally obtained impedance spectra correlated well with those from theoretical simulations and provided an estimate for the average number of fused cells. Since the average number of fused cells is only an estimate for fusion efficiency, the impedance magnitude at 4 kHz was taken to evaluate different parameters, like electric pulse parameters and fusogenic additives, for their impact on cell fusion. Electric pulsing of cells in presence of negatively charged polymers and polystyrene particles did not induce fusion. In contrast, cationic soluble polymers like PEI and poly-L-lysine were shown to induce multi-cell fusion after electroporation, whereas amine modified cationic polystyrene nanoparticles had no fusogenic effect. Taken together, the fusogenic effect of PolyMAG particles in combination with electric pulsing has been ascribed to its polycationic and flexible PEI coat