Electroporation of spheroids using an electric field gradient: a tool to study intensity-dependent permeabilization

Electroporation (EPN) is the process by which cell membranes become transiently or permanently permeable upon exposure to pulsed electric fields of suitable intensity and duration. Depending on the pulse parameters, permeabilization can be reversible or irreversible, enabling a wide range of biomedical applications. To improve our understanding of EPN effects on tissues and select efficient treatments and parameters, relevant <i>in vitro</i> tumour models are required. Three-dimensional (3D) cell spheroids have emerged as valuable systems, as they more accurately replicate tumour microenvironment and cell-to-cell interactions than conventional 2D cultures. In this work, we present a new microdevice designed for the culture and gradual electroporation of a population of several hundred uniformly sized spheroids, allowing the systematic study of electroporation over a wide range of electric field intensities within a single experiment. Gradual permeabilization of HT-29 colorectal cancer cell spheroids was performed using a standard electrochemotherapy protocol, and electroporation efficiency was assessed by analysing propidium iodide (PI) uptake. Spheroids were treated with electric fields ranging from 800 V cm^-1 to 3400 V cm^-1. <i>In toto</i> analysis of PI distribution within spheroids by confocal microscopy revealed highly heterogeneous permeabilization patterns throughout the spheroid volume, for all intensities investigated, even at the highest one of 3400 V cm^-1. This study introduces a robust 3D <i>in vitro</i> assay for the systematic evaluation of electroporation-based treatments, providing new insights into the influence of electric field heterogeneity, electrical protocol, and estimation of molecular uptake in the spheroid volume.