In Vitro Blood-Brain Barrier Permeability Assay

This assay was performed using the model published by Cecchelli and co-workers^{53 (link)}. Endothelial cells and pericytes were defrosted in gelatin-coated Petri dishes (Corning). Pericytes and endothelial cells were cultured in DMEM pH 6.8 or in supplemented endothelial cell growth medium (Sciencells), respectively. After 48 h, pericytes (50,000 cells/well) and endothelial cells (80,000 cells/well) were seeded in gelatin-coated 12-well plates or in Matrigel-coated 12-well Transwell inserts (Corning), respectively. Medium was changed every 2–3 days and assays were performed 7–8 days after seeding. Lucifer Yellow (50 µM) was used as internal control (P_app < 15·10⁻⁶ cm/s). LY fluorescence was measured in a 96-well plate with a Fluoroskan Ascent Microplate Fluorometer (Thermo Fisher Scientific). Compounds were dissolved in Ringer-HEPES at a concentration of 200 µM. Then, 500 µL of the compound and 1,500 µL of Ringer-HEPES alone were introduced in the apical or in the basolateral compartments, respectively. The plates were set on at 37 °C for 2 h. The solutions from both compartments were then recovered and quantified by HPLC and identified by MALDI-TOF.