by Sandra F. Lam, Venktesh S. Shirure, Yunli E. Chu, Alan G. Soetikno, Steven C. GeorgeMicrofluidic devices have been successfully used to recreate in vitro biological microenvironments, including disease states. However, one constant issue for replicating microenvironments is that atmospheric oxygen concentration (21% O2) does not mimic physiological values (often around 5% O2). We have created a microfluidic device that can control both the spatial and temporal variations in oxygen tensions that are characteristic of in vivo biology. Additionally, since the microcirculation is responsive to hypoxia, we used a 3D sprouting angiogenesis assay to confirm the biological relevance of the microfluidic platform. Our device consists of three parallel connected tissue chambers and an oxygen scavenger channel placed adjacent to these tissue chambers. Experimentally measured oxygen maps were constructed using phosphorescent lifetime imaging microscopy and compared with values from a computational model. The central chamber was loaded with endothelial and fibroblast cells to form a 3D vascular network. Four to six days later, fibroblasts were loaded into the side chambers, and a day later the oxygen scavenger (sodium sulfite) was flowed through the adjacent channel to induce a spatial and temporal oxygen gradient. Our results demonstrate that both constant chronic and intermittent hypoxia can bias vessel growth, with constant chronic hypoxia showing higher degrees of biased angiogenesis. Our simple design provides consistent control of spatial and temporal oxygen gradients in the tissue microenvironment and can be used to investigate important oxygen-dependent biological processes in conditions such as cancer and ischemic heart disease.