Guiding neuron development with planar surface gradients of substrate cues deposited using microfluidic devices

Wiring the nervous system relies on the interplay of intrinsic and extrinsic signaling molecules that control neurite extension, neuronal polarity, process maturation and experience-dependent refinement. Extrinsic signals establish and enrich neuron-neuron interactions during development. Understanding how such extrinsic cues direct neurons to establish neural connections in vitro will facilitate the development of organized neural networks for investigating the development and function of nervous system networks. Producing ordered networks of neurons with defined connectivity in vitro presents special technical challenges because the results must be compliant with the biological requirements of rewiring neural networks. Here we demonstrate the ability to form stable, instructive surface-bound gradients of laminin that guide postnatal hippocampal neuron development in vitro. Our work uses a three-channel, interconnected microfluidic device that permits the production of adlayers of planar substrates through the combination of laminar flow, diffusion and physisorption. Through simple flow modifications, a variety of patterns and gradients of laminin (LN) and fluorescein isothiocyanate-conjugated poly-L-lysine (FITC-PLL) were deposited to present neurons with an instructive substratum to guide neuronal development. We present three variations in substrate design that produce distinct growth regimens for postnatal neurons in dispersed cell cultures. In the first approach, diffusion-mediated gradients of LN were formed on cover slips to guide neurons toward increasing LN concentrations. In the second approach, a combined gradient of LN and FITC-PLL was produced using aspiration-driven laminar flow to restrict neuronal growth to a 15 mu m wide growth zone at the center of the two superimposed gradients. The last approach demonstrates the capacity to combine binary lines of FITC-PLL in conjunction with surface gradients of LN and bovine serum albumin (BSA) to produce substrate adlayers that provide additional levels of control over growth. This work demonstrates the advantages of spatio-temporal fluid control for patterning surface-bound gradients using a simple microfluidics-based substrate deposition procedure. We anticipate that this microfluidics-based patterning approach will provide instructive patterns and surface-bound gradients to enable a new level of control in guiding neuron development and network formation.