Albumin permeability and electrical resistance as means of assessing endothelial monolayer integrity in vitro

Abstract

Methods were developed to measure albumin permeability and electrical resistance of bovine aortic endothelial cell (BAEC) monolayers cultured on porous polycarbonate filters. Permeability to 1% bovine serum albumin (Pe) was quantified by measuring the flux of fluorescent-labeled albumin with an apparatus in which there were no transmural oncotic or hydrostatic pressure gradients. The effect of passage of BAEC monolayers in culture on permeability was studied using 60 BAEC monolayers of Passage 6 to 10. There was no significant difference in Pe between passages, and the mean Pe of all monolayers was 4.5 ± 0.5 (SEM) × 10−6 cm/s. Using these same BAEC monolayers, a fluorescent technique was developed to examine en face permeability patterns. Most BAEC monolayers demonstrated diffuse permeability across the monolayer, whereas others had focal regions of enhanced permeability despite similar Pe values. In those monolayers with punctate permeability, there were 5.4 ± 0.6 (SEM) focal regions of enhanced permeability per 1000 cells. To study the effect of culture time on monolayer integrity, electrical conductivities of nine BAEC monolayers were measured daily using a Millipore electrical resistance system. Electrical resistance increased from 4.5 ohm·cm2 at Day 2 to a peak level of 11.4 ohm·cm2 at Day 7 and then decreased daily to 4.0 ohm·cm2 by Day 12. The in vitro BAEC monolayer has many of the transport characteristics of intact vessels, making these techniques useful in physiologic studies of the endothelial transport barrier. These methods provide relatively simple means of assessing the integrity of endothelial cell monolayers grown on porous substrates.