Micropatterning of biological cues is important for the guided formation of neuronal outgrowth and neuronal differentiation. Nerve growth factor (NGF) was micropatterned in a three-dimensional collagen sponges by using micropatterned ice lines that were composed of collagen and NGF. The micropatterned ice lines were prepared by a dispersing machine. PC12 cells were cultured in the NGF-micropatterned collagen sponges and showed micropatterned neurite outgrowth. The neurite outgrowth followed the micropattern of NGF with more neurite outgrowth in the collagen/NGF lines than in the regions between the collagen/NGF lines. The micropattern of the NGF and the neurite network of the PC12 cells can be manipulated by controlling the micropattern of the NGF. The three-dimensional porous scaffolds prepared by this method will have a potential application for the regeneration and repair of the nervous system. (C) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012