Optimization of isolation and functional characterization of primary murine aortic endothelial cells

In this study the authors sought to improve the technique of isolating and culturing murine aortic endothelial cells (MAECs) for future use in studying endothelium-specific roles in complex pathologies such as atherosclerosis. The authors utilized a transgenic mouse expressing green fluorescent protein (GFP) under control of the endothelial specific Tie-2 promoter, allowing the tracking of endothelial cells throughout the isolation and purification. Briefly, aortas were harvested, the adventitia was removed, and strips were placed lumen side down into Matrigel. After 14 days, endothelial cells were isolated from the total cell population by fluorescence-activated cell sorting (FACS) using the GFP signal. The authors confirmed the endothelial phenotype of sorted cells based upon endothelial-specific Griffonia simplicifolia lectin staining, uptake of acetylated low-density lipoprotein (LDL), and von Willebrand factor (vWF) and VE-cadherin staining. The authors also confirmed the cells' ability to form tubes in Matrigel, and for cellular alignment with flow to occur following monolayer culture under unidirectional laminar shear stress but not following culture under oscillatory flow. Although GFP fluorescence--based vital sorting was used initially to optimize the isolation, the authors have verified that this method is applicable to the isolation of MAECs from other strains and backgrounds of mice through the use of other endothelial markers.