Quantitative Studies of Neuronal Chemotaxis in 3D

During development a variety of cell types are guided by molecular concentration gradients to form tissues and organ systems. In the nervous system, the migration and neuronal pathfinding that occurs during development is organized and driven by “guidance cues.” Some of these cues are substrate bound or nondiffusible, while many are diffusible and form gradients within the developing embryo to guide neurons and neurites to their appropriate destination. There have been many approaches used to discover and characterize the multitude of guidance cues, their cognate receptors, and how these cues and receptors are regulated to achieve the highly detailed connections found in the nervous system. Here we present a method for creating precisely controlled gradients of molecular factors within a three-dimensional culture environment. The method is based on a non contact mediated delivery of biomolecules to the surface of a collagen gel. The factors are printed in a pattern on the top of a gel containing the tissue or cell type of interest embedded in the gel. The formation of the gradient is dependent upon the diffusion of the printed molecule in the gel. The concentration of the factor within the gel becomes independent of depth rapidly, and the gradient becomes smooth on a similar time scale. The gradients formed can remain relatively stable for a day or more. Moreover, the steepness and molar concentration of tropic or trophic factors within the gradient can be controlled.