Nanostructured superhydrophobic substrates trigger the development of 3D neuronal networks

The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano-patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration. A novel method for culturing primary mouse hippocampal neurons and neuron-astrocyte co-cultures using superhydrophobic substrates with characteristic lengths falling into the microscale range and surface patterning in the nano-range is developed. This new culture method fosters neuronal growth and differentiation in an open scaffold, by reducing the interactions with the substrate and maximizing the physiological processes of cell-cell adhesion and synapse formation.