Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

The availability of oxygen (O2) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O2 tension during 3D cultures of HUVECs. Noninvasive measurements of DO levels during culture under atmospheric conditions revealed a profound decrease that reached as low as 2% O2 at the end of 24 h. After media replacement, DO levels rose rapidly and equilibrated at ∼15% O2, creating a reoxygenated environment. To accurately estimate DO gradients in 3D collagen gels, we developed a 3D mathematical model and determined the Michaelis-Menten parameters, Vmax and Km, of HUVECs in collagen gels. We detected an increase in ROS levels throughout the culture period. Using dip...