Chemotactic and haptotactic cues guide neurite growth toward appropriate targets by eliciting attractive or repulsive responses from the neurite growth cones. Here we present an integrated system allowing both structural and molecular micropatterning in dual hydrogel 3D tissue culture constructs for directing in vitro neuronal growth via structural, immobilized, and soluble guidance cues. These tissue culture constructs were fabricated into specifiable geometries using UV light reflected from a digital micromirror device acting as a dynamic photomask, resulting in dual hydrogel constructs consisting of a cell growth-restrictive polyethylene glycol (PEG) boundary with a cell growth-permissive interior of photolabile α-carboxy-2-nitrobenzyl cysteine agarose (CNBC-A). This CNBC-A was irradiated in discrete areas and subsequently tagged with maleimide-conjugated biomolecules. Fluorescent microscopy showed biomolecule binding only at the sites of irradiation in CNBC-A, and confocal microscopy confirmed 3D binding through the depth of the construct. Neurite outgrowth studies showed contained growth throughout CNBC-A. The diffusion rate of soluble fluorescein-bovine serum albumin through the dual hydrogel construct was controlled by PEG concentration and the distance between the protein source and the agarose interior; the timescale for a transient protein gradient changed with these parameters. These findings suggest the dual hydrogel system is a usefulplatform for manipulating a 3D in vitro microenvironment with patterned structural and molecular guidance cues for modeling neural growth and guidance.

Immobilized NT-3 enhanced DRG neurite growth while Sema3A strongly repelled it, versus neutravidin controls, in a hydrogel choice point model.

The direct effect of guidance cues on developing and regenerating axons in vivo is not fully understood, as the process involves a multiplicity of attractive and repulsive signals, presented both as soluble and membrane-bound ligands. A better understanding of axon guidance is critical to functional recovery following injury to the nervous system through improved outgrowth and mapping of damaged nerves. Due to their implications as inhibitors to CNS regeneration, we investigated the repulsive properties of Semaphorin 6A and Ephrin-B3 on E15 rat dorsal root ganglion explants, as well as possible interactions with soluble gradients of chemoattractive nerve growth factor. We employed a 3D biomimetic in vitro choice point model, which enabled the simple and rapid preparation of patterned gel growth matrices with quantifiable presentation of guidance cues in a specifiable manner that resembles the in vivo presentation of soluble and/or immobilized ligands. Neurites demonstrated an inhibitory response to immobilized Sema6A by lumbosacral DRG explants, while no such repulsion was observed for immobilized Ephrin-B3 by explants at any spinal level. Interestingly, Sema6A inhibition could be partially attenuated in a concentration-dependent manner through the simultaneous presentation of soluble NGF gradients. The in vitro model described herein represents a versatile and valuable investigative tool in the quest for understanding developmental processes and improving regeneration following nervous system injury.

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