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

## TL;DR

This paper provides a detailed protocol for creating modular neuronal networks using PDMS microstructures on HD-MEAs, enabling consistent and reproducible results across different systems.

## Contribution

A universal protocol for integrating PDMS microstructures on HD-MEAs to engineer defined neuronal architectures.

## Key findings

- Reproducible formation of modular networks with characteristic activity patterns across different systems.
- The protocol maintains compatibility with various HD-MEA systems while enabling precise control of network architecture.

## Abstract

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

•Protocol to generate PDMS-based modular networks for different HD-MEA chip manufacturers•Steps for PDMS fabrication, coating, and attachment onto HD-MEA surfaces•Handling and cleaning protocols to reuse and maximize the lifespan of HD-MEA chips•Details on optimization of the protocol, limitations, and possible solutions

Protocol to generate PDMS-based modular networks for different HD-MEA chip manufacturers

Steps for PDMS fabrication, coating, and attachment onto HD-MEA surfaces

Handling and cleaning protocols to reuse and maximize the lifespan of HD-MEA chips

Details on optimization of the protocol, limitations, and possible solutions

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.