Assessment of Human Endothelial Permeability In Vitro: The Importance of Cell Seeding Density

In this study we have evaluated the importance of certain factors affecting the permeability of confluent monolayers of human umbilical vein endothelial cells (HUVEC) to albumin. Inclusion of serum in the assay medium and fibronectin-pretreatment of the membrane supports on which the monolayers were grown decreased basal permeability. Most importantly, the endothelial cell (EC) seeding density was demonstrated to be a crucial determinant of basal permeability. In this study, the lowest % clearance of albumin was obtained with a seeding density of 6.0 × 105 cells/cm2 (% clearance = 2.90 ± 0.08%). In contrast, lower (4.8 × 105 cells/cm2) or higher (9.91 × 105 cells/cm2) seeding densities resulted in significantly (P < 0.05) higher clearances. Permeability was increased on stimulation with tumour necrosis factor-α, interleukin-1α/β or interferon-γ and the fold increases were demonstrated to be dependent on the initial HUVEC seeding density. These results demonstrate the importance of assessing the effects of...