Neuronal Cells Confinement by Micropatterned Cluster-Assembled Dots with Mechanotransductive Nanotopography

Artificially grown neuronal cultures of brain cells have been used for decades in the attempt to reproduce and study in vitro the complexity of brain circuits. It soon became evident that this alone was insufficient, because of the random architecture of these artificial networks. Important groundwork therefore resulted in the development of methods to confine neuronal adhesion at specific locations to match predefined network topologies and connectivity. Despite this notable progress in neural circuitry engineering, there is still need for micropatterned substrates that recapitulate better biophysical cues of the neuronal microenvironment, taking into account recent findings of their significance for neuronal differentiation and functioning. Here, we report the development and characterization of a novel approach that, by using supersonic cluster beam deposition of zirconia nanoparticles, allows the patterning of small nanostructured cell-adhesive areas according to predefined geometries onto elsewhere nonadhesive antifouling glass surfaces. As distinguishing features, compared to other micropatterning approaches in this context, the integrated nanostructured surfaces possess extracellular matrix-like nanotopographies of predetermined roughness; previously shown to be able to promote neuronal differentiation due to their impact on mechanotransductive processes, and can be used in their original state without any coating requirements. These micropatterned substrates were validated using (i) a neuron-like PC12 cell line and (ii) primary cultures of rat hippocampal neurons. After initial uniform plating, both neuronal cells types were found to converge and adhere specifically to the nanostructured regions. The cell-adhesive areas effectively confined cells, even when these were highly mobile and repeatedly attempted to cross boundaries. Inside these small permissive islands, cells grew and differentiated, in the case of the hippocampal neurons, up to the formation of mature, functionally active, and highly connected synaptic networks. In addition, when spontaneous instances of axon bridging between nearby dots occurred, a functional interdot communication between these subgroups of cells was observed.