Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay (original) (raw)

The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer's tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell ® tracer assays and two commercial impedance devices (xCELLigence ® , ECIS ®). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments. The vascular endothelium, a cell monolayer lining the blood vessels, operates as a size-selective and semi-permeable extravasation filter for plasma proteins, solutes, and fluids, thus controlling tissue homeostasis 1. Dysfunction of the endothelial barrier, e.g. by opening intercellular junctions, and the subsequent edema formation is a hallmark of inflammatory processes and associated with a plethora of severe diseases, e.g. atherosclerosis, rheumatoid arthritis, or asthma. Unfortunately, anti-edematous drugs that specifically interact with barrier-regulating processes in endothelial cells have not been described yet. Consequently, there is a great need for basic research and drug discovery programs involving both academic and industrial partners. Both, the in-depth elucidation of endothelial barrier regulation and the comprehensive screening of potential anti-edematous compounds must be pursued. Quantitative, sensitive, and high-throughput approaches are, thus, required to monitor barrier properties of endothelial cell monolayers in vitro mimicking physiological and patho-physiological conditions. Two techniques have been considered as gold standard throughout the last decades: (i) Measurement of macromolecular permeation (e.g. of fluorescence-labeled dextran) across a vascular endothelial cell layer as direct indicator for transendothelial solute flux and (ii) determination of transendothelial electrical resistance (TER) as measure for ionic permeability through intercellular clefts 2-9. Both approaches have their individual advantages and limitations with respect to their sensitivity, time resolution, artifacts, and practicability. For either type of