This study combines microdosimetry techniques and computational models to investigate the effects of ultrashort pulsed electric fields (PEFs) on cellular membranes. It focuses on identifying optimal stimulation protocols to meet RISEUP project goals, where an implantable electro pulsed bio-hybrid (EPB) device is under development for spinal cord injury neurogenesis. The EPB employs PEFs stimulation to modulate intracellular calcium fluxes, promoting stem cell proliferation and differentiation, by targeting plasma and endoplasmic reticulum (ER) membranes via electroporation. This approach integrates cutting-edge research to advance neurogenesis using mesenchymal stem cells (MSCs) and induced neuronal stem cells (iNSCs). In this work, starting from high-resolution confocal microscopy images, a semi-automatic reconstruction procedure is employed to generate 3D virtual digital twins of iNSCs and MSCs, incorporating their subcellular structures. Microdosimetric simulations are conducted to model the effects of various bipolar pulse intensities (9, 12, 15 V) and durations (10, 100, 1000 µs) on a mixture of virtual stem cells within the EPB device. At 12 V, a 10 µs-bipolar pulse is estimated able to porate plasma membranes, whereas increasing the pulse duration to 1000 µs results in ER electropermeabilization, showing that, at given pulse intensities, adjusting the pulse duration allows poration of both plasma and ER membranes. This strategy is particularly important when voltage cannot be increased, such as in RISEUP, where the fixed onboard power source limits voltage modulation. In such cases, pulse duration becomes a key parameter for achieving the desired membrane poration effects. Furthermore, advanced 3D virtual cells are undeniable in microdosimetry to optimize innovative stimulation protocols aimed at targeting specific cellular compartments. © 2026, © The Author(s)

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