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In vitro cultured neuronal networks with defined connectivity are required to improve neuronal cell culture models. However, most protocols for their formation do not provide sufficient control of the direction and timing of neurite outgrowth with simultaneous access for analytical tools such as immunocytochemistry or patch-clamp recordings. Here, we present a proof-of-concept for the dynamic (i.e., time-gated) control of neurite outgrowth on a cell culture substrate based on 2D-micropatterned coatings of thermoresponsive polymers (TRP). The pattern consists of uncoated microstructures where neurons can readily adhere and neurites can extend along defined pathways. The surrounding regions are coated with TRP that does not facilitate cell or neurite growth at 33 °C. Increasing the ambient temperature to 37 °C renders the TRP coating cell adhesive and enables the crossing of gaps coated with TRP by neurites to contact neighboring cells. Here, we demonstrate the realization of this approach employing human neuronal SH-SY5Y cells and human induced neuronal cells. Our results suggest that this approach may help to establish a spatiotemporal control over the connectivity of multinodal neuronal networks.

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