Protocol to generate PDMS topographical patterns for hiPSC-derived or rat primary neuronal cultures.

Soft lithography is a promising technique to fabricate tailored substrates on polydimethylsiloxane (PDMS) casts. Here, we present a protocol to generate PDMS topographical patterns for neuronal networks in vitro. The protocol outlines the development of a two-level resin master mold with soft lithography and the creation of a topographical PDMS cast from the mold. We then detail the procedures for culturing either human induced pluripotent stem cell (hiPSC)-derived or rat neurons on the PDMS cast with topographical patterns and for acquiring data using calcium imaging. This protocol provides neuronal networks with imprinted connectivity. For complete details on the use and execution of this protocol, please refer to Montalà-Flaquer et al.(1).