Transwell Co-culture for Permeability

On Day 0, 12-well Transwell inserts were coated with 10 μg/ml human recombinant laminin 511-E8 in sterile PBS or 10 μg/ml BSA control coating, and left to dry for 1 h at 37 °C. After coating, H69 cholangiocytes were seeded in the apical compartment at 100,000 cells per insert. On Day 4, PBMCs were isolated from healthy volunteers, and T lymphocytes were activated with PMA and ionomycin, after which 200,000 cells were added to the basolateral compartment. The apical compartment was refreshed with H69 culture medium and the basolateral compartment contained supplemented IMDM with or without activated T lymphocytes. Co-culture was left overnight. On Day 5, 4 kDa fluorescein isothiocyanate (FITC)-Dextran permeability assays were performed. For this, medium was refreshed with 800 μl DMEM supplemented with 10% FBS and 37.5 U/ml (1%) penicillin, 37.5 μg/ml (1%) streptomycin on the basolateral side, and 250 μl of supplemented DMEM containing 1 mg/ml 4 kDa FITC–Dextran on the apical side. At timepoint 0, 100 μl of medium was transferred to a black 96-well plate to determine potential background fluorescence. In addition, an empty unseeded Transwell insert was included to determine maximal permeability of the insert itself. At t = 60, 120, 180, and 240 min, 100 μl of basolateral medium was collected per experimental condition and transferred to the black 96-well plate. FITC–Dextran fluorescence (excitation 490 nm, emission 520 nm) was measured using the CLARIOstar apparatus.