Multilayered microelectrode array for monitoring electrophysiological signals of 3d neural networks in cerebral organoid

Microelectrode arrays (MEAs) are widely used platforms for monitoring neuronal activity in biological systems. The advent of brain organoid technology-three-dimensional (3D) neural tissue models derived from human stem cells-has opened unprecedented opportunities to study network connectivity and developmental processes of brain-like tissues in vitro. However, most commercial MEAs are restricted to planar, two-dimensional configurations, limiting recordings to a single surface and failing to capture the multilayered architecture of organoids. To overcome this limitation, we develop a multilayered MEA (MLMEA) capable of interfacing with multiple depths within brain organoids. The MLMEA features customizable interlayer spacing achieved through the stacking of mesh-type electrode layers separated by mechanically compliant PDMS spacers. This design allows simultaneous, spatially resolved electrophysiological recordings across layers along the vertical axis, enabling detailed characterization of interlayer connectivity and network dynamics. Noninvasive integration of the MLMEA with cerebral organoid sustained for over four weeks demonstrates the stable, increasing neural activities with layer-specific electrophysiological metrics. Our findings demonstrate that MLMEA can reliably capture heterogeneous, depth-dependent neuronal activity, establishing it as a powerful tool for advancing 3D organoid-based neuroscience research and high-content drug screening.