A programmable electroculture platform for direction-dependent alternating electric field delivery in cancer cell models

OBJECTIVE: Alternating electric fields (AEFs) at intermediate frequencies have been shown to disrupt mitosis and inhibit the proliferation of rapidly dividing tumor cells. Here, we developed a programmable in vitro AEF exposure system (0-500 kHz, 1-3 V/cm) featuring an electroculture platform compatible with standard 12-well tissue culture plates. The system enables investigation of cellular responses to fixed orthogonal field orientations and periodic polarization switching (PPS), defined as sequential alternation of the field direction between x- and y-axes at user-configurable intervals of 10-300 s Approach: Exposure conditions were characterized using a fully three-dimensional (3D) finite-element model to evaluate electric field distribution and specific absorption rate (SAR), while thermal simulations and experimental temperature measurements were used to assess thermal stability and non-thermal operation. Human glioblastoma (U-118 MG), neuroblastoma (SH-SY5Y), and non-malignant endothelial (HUVEC) cells were treated with AEFs (200 kHz, 3 V/cm) for 24-72 h. Cytotoxic effects were assessed using MTT assay, Live/Dead fluorescence staining, and Annexin V/PI imaging, followed by quantitative image analysis. Main results: AEF treatment induced significant, time-dependent reductions in cancer cell viability, reaching an approximately 80% reduction relative to controls in U-118 and SH-SY5Y cells after 72 h. Fluorescence imaging supported pronounced cytotoxic effects in malignant cells, whereas endothelial cells showed comparatively lower sensitivity. In the PPS mode evaluated after 24 h with a 300 s switching interval, viability in both cancer cell lines was further reduced by approximately 15% relative to the corresponding fixed-axis configurations. Significance: These findings show that, under the tested AEF exposure parameters, periodically switched field orientations can produce stronger cellular responses than fixed-orientation AEF delivery, including the first demonstration in SH-SY5Y cells. By integrating programmable directional switching into a standard 12-well electroculture format, the platform provides a configurable framework for future studies of field orientation and switching interval modulation. .