Gene electrotransfer is one of the most widely used non-viral methods for delivering plasmid DNA into cells. It is based on the phenomenon of electroporation, in which exposure of cells to a pulsed electric field causes a transient increase in cell membrane permeability, thereby enabling the entry of nucleic acids into the cell. Although the method is well established, classical electroporation requires relatively high voltages, which often leads to reduced cell viability. Devices with nanostructured geometries, such as nanochannels and nanopores, allow spatial localization of the electric field to small regions of the cell membrane, which can significantly enhance transfection efficiency while preserving cell viability. In this thesis, we developed a system for localized electroporation based on nanoporous substrates designed for cell culture and research. The development proceeded in several steps. We designed a printed circuit board (PCBs) to deliver electrical pulses to the cells. In parallel, numerical simulations were performed to determine the optimal electrode spacing, ensuring a sufficiently homogeneous voltage disribution across the surface of the porous substrate. Based on the selected geometric parameters, a mechanical holder with slots for the PCBs and porous substrate inserts was designed using computer-aided design (CAD). The developed system was experimentally tested through electrical resistance measurements, with results compared against numerical model predictions. Transfection efficiency and cell viability were assessed by fluorescence microscopy, using EGFP as a reporter gene and propidium iodide to monitor cell viability. To accelerate the analysis of microscopy images, an algorithm for automated cell segmentation and counting was developed in Python. The developed system represents a low-cost and reproducible platform for localized electroporation that does not require high-voltage pulse generators, making it accessible to a broader range of research laboratories. The results suggest that this approach holds considerable promise for the further development of efficient and cell-friendly methods for gene electrotransfer.
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