In this paper, we describe a technology platform to study the effect of nanocues on the cell growth direction in primary cortical cell culture. Topographical cues to cells are provided using nanoscale features created by Jet and Flash Imprint Lithography, coated with polyethylenimine. We investigated nanoscaffolds with periodicities ranging from 200 nm to 2000 nm, and found that the samples with a period between 400 nm and 600 nm and a height of 118 nm showed highly ordered regions of neurites in a newly formed network with a preferential alignment tendency for astrocytes. Live/dead staining results showed that different materials, such as silicon, glass, and imprinted resist are rendered biocompatible by coating with polyethylenimine. This coating therefore allows for a free choice of scaffold materials and promotes good cell-substrate adhesion. From our results we conclude particular length scales of nanoscaffold can impose a degree of order on cell spreading behavior in a complex cellular brain-on-a-chip network, which could thus be used to emulate ordered brain regions and their function in vitro. © 2014 Elsevier B.V. All rights reserved.