Protocol for tailored in vitro neuronal networks on high-density microelectrode arrays with polydimethylsiloxane microstructures

Complementary metal-oxide-semiconductor (CMOS)-based high-density microelectrode arrays (HD-MEAs) enable neuronal recordings with high spatiotemporal resolution. However, integrating polydimethylsiloxane (PDMS) microstructures onto HD-MEA surfaces to control network architecture is currently challenging and platform specific. Here, we present a protocol for PDMS fabrication, HD-MEA chip preparation, PDMS-HD-MEA microstructure alignment, and cell culture, including alternatives. Our results show reproducible formation of modular networks with characteristic activity patterns across different systems. This protocol supports engineering of defined neuronal architectures while maintaining compatibility with various HD-MEA systems. For complete details on the use and execution of this protocol, please refer to Sato et al.1