Transwell Permeability Assay for Compounds

Commercially-available 12-well Transwells (Corning, Corning, NY, USA), with polyester membranes, were utilized to assess static compound permeability in the absence of flow-induced shear stress. The area of each Transwell membrane was 1.12 cm² with 0.4 µm diameter pores and 0.5% porosity. Epithelial cells were seeded in the Transwell top apical compartment at a density of 2 × 10⁶ cells/mL. Once the cell monolayer had reached confluency, the 500 µL culture media in the top compartment was replaced with a solution of endothelial cell media mixed with various fluorescently-labeled compounds at a pre-determined initial concentration. The bottom basolateral compartment was filled with 1500 µL fresh endothelial cell culture media, and the Transwell was placed in an incubator for 2 h. Then, liquid samples of 100 µL from both compartments were aliquoted into a 96-well plate. Fluorescent intensities of the collected samples were measured, using a BioTek Synergy 2 Plate Reader (BioTek, Winooski, VT, USA), and converted to molecular concentrations as reported elsewhere [21 (link)].