A Water Compartment Cell Culture Lid Enables Stable Longitudinal Recording of Neuronal Networks In Vitro

ABSTRACT Longitudinal electrophysiological recordings of neuronal networks are essential for studying network maturation, plasticity, and pharmacological responses. Yet, current microelectrode array (MEA) approaches are limited by evaporation‐induced drift in culture conditions, exacerbated by heat dissipation from active recording electronics on CMOS‐based high‐density MEAs. We present a cell culture lid featuring a water compartment at its interface that eliminates evaporation whilst maintaining gas exchange. Combined with a custom incubator that uses independent temperature control of the MEA to prevent condensation, the system enables stable, uninterrupted recordings for weeks. We show that perturbations in firing rate and functional connectivity following medium exchange are significantly reduced by suppressing evaporation. We demonstrate continuous 35‐day recordings of patterned human iPSC‐derived neuronal networks with a single half‐medium exchange, revealing multiple spatiotemporal firing patterns that are held concurrently and evolve over weeks. All design files are provided, enabling adoption across culturing platforms for longitudinal studies of development within individual cultures, and establishing the conditions required for studies of plasticity and chronic pharmacology.