Barrier integrity assay
A unique, dynamic and real-time barrier integrity assay
The barrier function of endothelium and epithelium can be studied in the OrganoPlate®. Tissue are seeded against an ECM gel to form tubular structures with tight junctions. Within a few days, these perfused tubules become fully polarized and leak-tight. The barrier integrity assay is a multi-purpose assay to study barrier function of these tissues over time e.g. during compound exposure (figure 1). Furthermore, this assay can also be used for the set up and optimizing tissue models. Since disruption of tight junctions or changes in cell morphology affect barrier function, the barrier integrity assay provides great insight and is more informative than standard viability assays.
The lumen of the tubule is continuously perfused with medium (A). For the assay, we replace the blank medium with medium containing a fluorescent dye (B). In a leak-tight tubule, the dye will remain in the lumen compartment (C). If the barrier is disrupted from the start or becomes disrupted following toxicant exposure, the dye can diffuse into the adjacent channel containing an ECM gel (D). Using a standard fluorescent microscope, this process can be monitored by imaging over time.
Barrier function can be analyzed by comparing the fluorescent intensity of the ECM channel to the intensity of the perfusion channel (endothelial/epithelial tubule). If the monolayer is leak-tight, the mean intensity ratio will be nearly zero and depending on the tissue, the monolayer can remain leak-tight for several weeks. In contrast, a (partially) leaky tubule will show an increasing ratio of intensities over time and ultimately will result in a ratio of 1. The timing and slope of the increasing ratio offer valuable information on barrier function and toxic effects of compounds.