HydroMEA: A 3D Hydrogel‐Based Microfluidic Device to Study Electrophysiology for Myelinated Nerve‐on‐Chip

Engineered in vitro platforms are powerful systems to study information flow in the nervous system. While existing polydimethylsiloxane (PDMS)-based microfluidic platforms offer precise architectures, the cultured neurons grow on two-dimensional (2D) planar multielectrode arrays (MEA). To mimic the native microenvironment, where neurons grow in three-dimensional (3D) extracellular matrices (ECM), 3D hydrogels can be designed to encapsulate cells and enable physiologically mimicked behaviors. Here, we describe 'hydroMEA,' a 3D platform fabricated by placing PDMS microstructures on a high-density MEA and filled with a desired hydrogel, to offer controlled topologies, physiologically relevant microenvironments, and real-time electrophysiological measurements. First, we developed a gelatin methacryloyl (GelMA) hydrogel with incorporated ECM components and tuned the mechanical properties to match those of nerve tissue. The hydrogel was able to support: (1) the growth of iPSC-derived sensory neurons (hSNs) for >100 days; (2) co-cultures of hSN with human embryonic stem cell-derived Schwann cells (hSCs), to enable reliable 3D myelination. Next, hydroMEA were prepared for topologically defined 3D growth and myelination in designated compartments. Finally, electrophysiological evaluation of hSN-hSCs co-cultures revealed increased conduction speeds indicating functional myelin. This platform is a promising tool to study cell-cell interactions and to functionally evaluate the effect of pharmacological compounds in a more translational manner.