Gene electrotransfer (GET) represents a highly promising non-viral tool for targeted gene therapy; however, its successful clinical translation to live tissue is hindered by the challenge of precisely verifying local electric field thresholds required for efficient plasmid transfection and cell survival. To bridge the gap between experimental outcomes and complex numerical models, we developed a robust, automated image processing framework to evaluate electroporation dynamics within 3D collagen hydrogel tissue models, which mimic the structural complexity of the natural extracellular matrix better than the 2D cell cultures on glass or plastic. By computationally aligning high-resolution experimental fluorescence microscopy images with 3D numerical electric field distributions modeled in COMSOL Multiphysics, our custom pipeline successfully mapped individual cell centroids and bulk fluorescence intensities directly to their exact local electric field strengths. Using a multi-parametric fluorescent staining approach, we quantitatively determined exact empirical thresholds for reversible membrane permeabilization (YO-PRO-1), irreversible membrane damage (propidium iodide), and overall cell viability (Calcein AM) across varying pulse parameters and needle geometries. High-throughput location-dependant analysis of cell response further revealed a significant, polarity-dependent asymmetry: cells located near the cathode (negative electrode) required consistently lower localized electric field intensities to achieve both successful permeabilization and cell death compared to those situated near the anode (positive electrode). Ultimately, this assay framework clearly defines the narrow therapeutic window for GET and provides a highly reproducible method for bridging experimental observations with numerical simulations, offering a powerful approach to optimize pulse parameters and electrode designs for future applications in complex tissue environments.
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