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The central nervous system is a dense, layered, 3D interconnected network of populations of neurons, and thus recapitulating that complexity for  in vitro  CNS models requires methods that can create defined topologically-complex neuronal networks. Several three-dimensional patterning approaches have been developed but none have demonstrated the ability to control the connections between populations of neurons. Here we report a method using AC electrokinetic forces that can guide, accelerate, slow down and push up neurites in un-modified collagen scaffolds. We present a means to create  in vitro  neural networks of arbitrary complexity by using such forces to create 3D intersections of primary neuronal populations that are plated in a 2D plane. We report for the first time  in vitro  basic brain motifs that have been previously observed  in vivo  and show that their functional network is highly decorrelated to their structure. This platform can provide building blocks to reproduce  in vitro  the complexity of neural circuits and provide a minimalistic environment to study the structure-function relationship of the brain circuitry.

Microfluidic neurite guidance to study structure-function relationships in topologically-complex population-based neural networks