To assess monolayer integrity, an inulin-FITC (0.1 mg/ml in PBS) leakage assay was performed, as described in Jochems et al. (2019 (link)). The intraluminal inlet of the chamber was connected to warm inulin-FITC reservoir via a peristaltic pump (Ismatec Reglo, Cole parmer, Wertheim, Germany). The HFM was then perfused with inulin-FITC with a molecular weight of 3–6 kDa at a flow rate of 0.1 mL/min for 10 min. Samples were taken from the apical compartment. Fluorescence of the samples was measured at excitation wavelength of 492 nm and emission wavelength of 518 nm using Tecan infinite M200PRO plate reader (Tecan Austria GmbH).
The values of the apparent permeability Papp (in cm/s), which indicate the leakage of inulin-FITC, were calculated according to:
where dQ/dt indicates the appearance rate of inulin-FITC over time (relative fluorescence unit/s), A is the surface area of the exposure area and C0 is the initial concentration of the warm inulin-FITC reservoir. As a control, coated-fibers without cells were used to determine leakage through the fiber which was then set to 100% leakage for further calculations.
The values of the apparent permeability Papp (in cm/s), which indicate the leakage of inulin-FITC, were calculated according to:
where dQ/dt indicates the appearance rate of inulin-FITC over time (relative fluorescence unit/s), A is the surface area of the exposure area and C0 is the initial concentration of the warm inulin-FITC reservoir. As a control, coated-fibers without cells were used to determine leakage through the fiber which was then set to 100% leakage for further calculations.