Confirmation of endothelial monolayer integrity.

The integrity of the endothelial monolayer was confirmed by both fluorescence imaging of the dextran distribution and confocal microscopy of fixed and labeled cells. An intact endothelial monolayer gives rise to an abrupt intensity drop between the channel and the gel region once the fluorescently-labeled dextran is introduced. Three hours after dextran injection, a sharp drop in fluorescence intensity is seen across the endothelial layer demonstrating its function as a barrier to macromolecules (a). Fluorescence intensity is quantified using Matlab (b). The dashed arrow in (a) the location and direction for the quantification.The intensity value drops to 15% of is peak value due to the barrier effect. The endothelial monolayer is located near the 400 µm point on the plot (shown with dashed line). Samples fixed on the third day after cell seeding and stained for VE-cadherin and nuclei (DAPI-blue) exhibit well-defined junctions with no apparent gaps in the confluent monolayer (c). The confocal image shows the front view of the microfluidic device.