Laser micromachining of hydrophobic gels into CAD/CAM patterns was used to develop differentially adherent surfaces and induce the attachment of B35 rat neuroblasts that would later form engineered nerve bundles. Narrow channels, 60-400 mu m wide, were micromachined in a 2% agarose gel using an ArF laser, and subsequently filled with an extracellular matrix gel. Upon the addition of I ml of a 2 x 104 cells/ml neuroblast suspension, the cells selectively adhered to the ECM-lined channels in a non-confluent manner and we monitored their growth at various time points. The adherent neuroblasts were fluorescently imaged with a propidium iodide live/dead assay, which revealed that the cells were alive within the channels. After 72 h growth, the neuroblasts grew, proliferated, and differentiated into nerve bundles. The fully grown I cm long nerve bundle organoids maintained an aspect ratio on the order of 100. The results presented in this paper provide the foundation for laser micromachining technique to develop bioactive substrates for development of three-dimensional tissues. Laser micromachining offers rapid prototyping of substrates, excellent resolution, control of pattern depth and dimensions, and ease of fabrication. (c) 2005 Elsevier B.V. All rights reserved.