A microfabricated on-chip approach to the micropipette growth cone-turning assay

Abstract

Chemical gradients and physical contact with the surrounding microenvironment play an important role in regulating axonal pathfinding. Traditional experiments for chemically-induced growth coneturning in vitro involve micropipettes filled with a guidance-cue solution. The localized chemical gradient generated through gravity-induced or pulsatile pressure application is used to evaluate the response of a single growth cone to the guidance-cue solution. Thus, the micropipette growth coneturning assay has intrinsic application deficits that limit the duration of signaling. Moreover, it does not account for spatial reproducibility. To improve spatial resolution and expose multiple individual growth cones to identical environmental conditions, we developed a biochip system that incorporates geometric and chemical signaling to regulate axonal pathfinding. With this system, we were able to passively generate chemical gradients for long periods of time (>1 d) at continual flowrates of ∼40 pL s −1 . Over the course of 4 DIV, angular distributions for axonal pathfinding of individual laser cellpatterned CFNs showed statistically different frequencies when a chemical guidance gradient was generated from a localized source. The biochip described here can be used to systematically test various chemical guidance molecules with high repeatability and longer durations. Abbreviations CFN chick forebrain neuron PBS phosphate buffer saline PDMS polydimethylsiloxane PLL poly-L-lysine FITC-BSA fluorescein isothiocyanate-labeled bovine serum albumin RECEIVED