Permeability Study on HCE-T Cell Culture Model

Transepithelial electrical resistance (TEER) reflects the tightness of the intercellular junctions closing the paracellular cleft, therefore the overall tightness of cell layers of biological barriers. TEER was measured to check the barrier integrity by an EVOM volt-ohmmeter (World Precision Instruments, Sarasota, FL, USA) combined with STX-2 electrodes, and was expressed relative to the surface area of the monolayers as Ω × cm². TEER of cell-free inserts was subtracted from the measured data.

HCE-T cells were seeded at a density of 10⁵ cells onto Transwell inserts (polycarbonate membrane, 0.4 µm pore size, 1.12 cm² surface area; 3401, Corning Life Sciences, Tewksbury, MA, USA) and cultured for 5–8 days at liquid–liquid and for 5–8 days at air–liquid interface. The culture medium was changed and TEER was checked every second day.

For the permeability experiments the inserts were transferred to 12-well plates containing 1.5 mL Ringer buffer in the acceptor (lower/basal) compartments. In the donor (upper/apical) compartments 0.5 mL buffer was pipetted containing different formulations (F1–F5) of PR for 30 minutes. To avoid unstirred water layer effect, the plates were kept on a horizontal shaker (120 rpm) during the assay. Samples from both compartments were collected and the PR concentration was detected by HPLC.

To determine the tightness of the cornea epithelial culture model two marker molecules were tested.34 In the donor compartments 0.5 mL buffer containing fluorescein (10 μg/mL; M_w: 376 Da) and Evans blue labeled albumin (167.5 μg/mL Evans blue dye and 10 mg/mL bovine serum albumin; M_w: 67.5 kDa) was added. The inserts were kept in the multiwell plates on a horizontal shaker (120 rpm) for 30 minutes, then the concentrations of the marker molecules in the samples from the compartments were determined by a fluorescence multiwell plate reader (Fluostar Optima, BMG Labtechnologies, Germany; fluorescein: excitation wavelength: 485 nm, emission wavelength: 520 nm; Evans-blue labeled albumin: excitation wavelength: 584 nm, emission wavelength: 680 nm).

The apparent permeability coefficients (P_app) were calculated as described previously.34 Briefly, cleared volume was calculated from the concentration difference of the tracer in the acceptor compartment (Δ[C]_A) after 30 minutes and donor compartments at 0 hour ([C]_D), the volume of the acceptor compartment (V_A; 1.5 mL) and the surface area available for permeability (A; 1.1 cm²) using Equation 1: