Electron microscopy characterization of ultrastructural alterations induced by reversible and irreversible electroporation

Abstract

Abstract

Electroporation produces reversible or irreversible plasma membrane permeabilization depending on pulse parameters and biological context. However, direct comparisons of membrane and organelle ultrastructure after reversible electroporation (RE) and irreversible electroporation (IRE) remain limited. This study characterized their multilevel structural effects in tumor cells and porcine tissues. KPC pancreatic cancer cells and Hepa1-6 hepatocellular carcinoma cells were treated with 60 pulses of 90 μs at 750 V/cm (operational RE) or 1,250 V/cm (operational IRE). Transmission electron microscopy (TEM) was used to examine mitochondrial and endoplasmic reticulum morphology, whereas scanning electron microscopy (SEM) and free-fluorescein isothiocyanate uptake were used to assess cell surface morphology and membrane permeability. Candidate nanopores were operationally identified using control-derived diameter thresholds and analyzed by Gaussian mixture modeling. Porcine liver and pancreas from animals were examined 24 h after IRE. RE was associated with mitochondrial contraction, increased matrix density, reduced cristae, and limited endoplasmic reticulum dilation. IRE caused extensive mitochondrial and endoplasmic reticulum swelling, cristae disruption, membrane fragmentation, increased FITC uptake, and numerous membrane surface depressions. IRE shifted the median depression diameter from 28 to 48 nm in KPC cells and from 30 to 111 nm in Hepa1-6 cells (Holm-adjusted P < 0.001). Candidate-nanopore diameters showed cell line-specific non-unimodal distributions, with fitted components centered at approximately 91 and 262 nm in KPC cells and 101, 199, and 369 nm in Hepa1-6 cells. Related tissue surface alterations and pore-like erythrocyte membrane discontinuities were observed in porcine liver and pancreas. Microsecond electroporation produced a graded ultrastructural response, ranging from limited organelle alterations after RE to extensive membrane and organelle disruption after IRE. These findings establish a structural framework for future mechanistic studies and pulse-parameter optimization.