Neuronal cell culture models in vitro are often restricted to 2D surfaces. Engineering the complexity of the neuronal microenvironment in microfluidic systems can help to generate more tissue like cultures. We have developed a new neuronal cell culturing system based on a microfluidic device that can culture primary neurons in a 3D patterned hydro- gel based microenvironment. Perpendicular to the culture channel a chemical gradient was established to guide neurites. Neurons cultured under a 62.5 ng (ml mm)-1 nerve growth factor (NGF) gradient up to 9 days in vitro (DIV), extended their neurites through the hydrogel in the direction of the higher concentration.