Figure 4

Characterisation of RBEC monolayer barrier function induced by co-culture in optimised EBM-2/EGM-2 media. (a) Development and stabilisation of TEER for passaged RBECs grown on cell culture inserts in the optimised EBM-2/EGM-2 conditions over a two week period. TEER was measured following equilibration of the inserts in cell culture medium to room temperature, n = 5 independent cell culture experiments. (b) Relationship between pre-experimental TEER and permeability to Lucifer yellow over 90 minutes at 37°C in the EBM-2/EGM-2 media conditions. Data was fitted to a one-phase exponential decay curve, R² = 0.78, n = 6 independent cell culture experiments, equivalent to 71 inserts in total. Peak TEER at room temperature in this experiment reached as high as 999 Ω × cm²_. (c) Permeability of Lucifer yellow (50 μg/mL) and FITC-labelled dextrans (10 mg/mL) of increasing hydrodynamic radius across RBEC monolayers in the optimised EBM-2/EGM-2 media conditions. Pe was calculated over a 90 minute time-course and was plotted versus the hydrodynamic radius of each molecule. Data was fitted to a one-phase exponential decay curve, R² = 0.86, n = 2 independent cell culture experiments, equivalent to 6 inserts in total for each molecule tested.