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

Abstract

SUMMARY: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) X 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-cm 2 at Day 2 to a peak level of 11.4 ohm" cm 2 at Day 7 and then decreased daily to 4.0 ohm. cm 2 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.

‘What controls aqueous humour outflow resistance?’

“…Junctions between cells continue to mature long after the cells have become confluent (Albelda et al, 1988;Sill et al, 1992;Yaccino et al, 1997;Underwood et al, 1999;Kaida et al, 2000;Penfold et al, 2000). These studies have shown that the permeability to solutes, electrical resistance, and hydraulic conductivity can continue to change one to 2 weeks post confluence in endothelial monolayers.…”

“…These in vitro cell layers show large (micro-sized) intercellular gaps (Underwood et al, 1999;Alvarado et al, 2004) that are not seen physiologically. The permeability of vascular monolayers are reported to be 10-100 times greater than for intact endothelia, presumably due to such defects (Albelda et al, 1988;Sill et al, 1992). Formation of large blebbing structures in the cells have also been seen when these cellular layers are perfused from the basal to apical direction (Alvarado et al, 2004), but these structures are different in size and morphological characteristics from giant vacuoles that are seen in Schlemm's canal cells from intact tissues (Bill, 1970;Johnstone and Grant, 1973;Ethier, 2002).…”

Rho/Rho-Kinase Pathway Contributes to C-Reactive Protein–Induced Plasminogen Activator Inhibitor-1 Expression in Endothelial Cells

“…21 Cells were grown to confluence in DMEM (Sigma) with 10% fetal bovine serum (Biowest, Ringmer, UK), 100 U/mL penicillin, and 100 g/mL streptomycin in 95% O 2 /5% CO 2 at 37°C and were used between passages 3 and 9.…”

Shear-Induced Increase in Hydraulic Conductivity in Endothelial Cells Is Mediated by a Nitric Oxide–Dependent Mechanism

“…22 Cells were plated at a density of 2.5ϫ10 5 cells/cm 2 on polycarbonate membrane Transwell filters pretreated with gelatin (5 mg/L, type A from porcine skin) and fibronectin (30 g/mL). Cells between passages 6 and 12 were used in experiments.…”