Electrophysiological profiling of hiPSC-derived neurospheres using a novel NeuroMPS with integrated electrodes

Despite advances in microphysiological systems, in vitro platforms for neuronal models remain limited by insufficient electrophysiological resolution, poor structural compatibility with 3D tissue architecture, and an inability to capture functional network dynamics alongside morphological and metabolic readouts. Here, we develop a neuromicrophysiological system (NeuroMPS) that pairs human iPSC-derived neurospheres, comprising neurons and glial cells, with tailored microelectrode arrays (MEAs) for non-invasive, high-resolution monitoring of neuronal network dynamics and functional maturation in vitro. The NeuroMPS combines a custom MEA with capped electrodes optimized for neurite-level signal detection and a glass microwell module that provides structural confinement and optical compatibility for imaging. Importantly, this configuration enables stable, longitudinal electrophysiological recordings from 3D neural constructs while supporting multimodal analyses. Following exposure to pharmacological modulators (PTX, TTX, bicuculline, CNQX, and 4-AP) and the neurotoxin rotenone, NeuroMPS detects alterations in network activity within minutes, even at the lowest concentrations tested, whereas morphological and metabolic changes emerge only at higher doses and later time points. This work provides a physiologically relevant, scalable, non-invasive platform that integrates high-sensitivity electrophysiological readouts with morphological and metabolic profiling to enable early prediction of compound-induced effects in human iPSC-derived 3D neural networks, with applications in neuropharmacology, neurotoxicology, and disease modeling.