An in vitro model for endothelial permeability: assessment of monolayer integrity.

Abstract

An essential component of any in vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9 +/- 11.4 omega.cm2, 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to 3H-inulin demonstrated a linear relationship between the luminal concentration of 3H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of 3H-inulin, was 2.01 +/- 0.076 x 10(-4) ml/min (r2 = .9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17 +/- 0.427 x 10(-6) cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07 +/- 0.617 x 10(-6) cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to 3H-inulin (1.43 +/- 0.445 x 10(-5) cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.