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Achieving alignment of cells is key to the success of regenerative strategies of neural tissue. We report a high-throughput method to investigate neural cell response to surface chemistry overlaid orthogonally onto a gradient of gradually changing groove widths. Using a bio-inspired approach wherein radial glial cells, which naturally guide neurons in the developing brain, enhance the attachment and directional outgrowth of neurons, we show the differences in the interaction and cellular response of glia, neurons and co-cultured cells. Radial glia were found to preferentially reside in grooves of width 6–35 μm with greater alignment to grooves <10 μm on the hydrophobic and hydrophilic extremes of chemistry. When neurons were sequentially cultured after radial glia, they showed enhanced alignment compared to when they were cultured alone, for all chemistries and groove widths. This is not dependent on co …