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After injury, regeneration of the central nervous system (CNS) is inhibited by the secretion of repulsive guidance cues that mediate axon growth and guidance. These repulsive cues are perceived as spatial gradients of neurons. Microfluidic systems can enable studies of neuron response to spatial gradients, by generating precise and reproducible surface gradients of guidance cues. Understanding neuron response to these gradients could provide valuable information for therapies seeking to promote regeneration in the CNS. Here we report on the successful use of a microfluidic gradient generator to deposit spatially-extended and uniform surface gradients of one repulsive guidance cue, chondroitin sulfate proteoglycans (CSPGs). The patterned substrates are compatible with high-quality culture of large numbers of primary neurons, and initial findings indicate that neuron growth and guidance are responsive to the deposited CSPGs.