4.4.1. Human RPMI 2650 Nasal Epithelial Cell Culture

Human RPMI 2650 nasal epithelial cells were purchased from ATCC (cat. no. CCL 30) and used until passage 50 for the experiments. For the cell culturing, Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Pan-Biotech GmbH, Aidenbach, Germany) and 50 µg/mL gentamicin were used. In addition, the cells were kept in a humidified 37 °C incubator with 5% CO₂. All of the plastic surfaces were coated with 0.05% collagen in sterile distilled water before cell seeding in culture dishes, and the medium was changed every 2 days. The cells were trypsinized with 0.05% trypsin 0.02% EDTA solution when they reached about 80–90% confluency in the dishes. To induce tighter epithelial barrier properties, retinoic acid (10 µM) and hydrocortisone (500 nM) were added to the cells 1 day before the experiment [81].

For the permeability measurements, RPMI 2650 epithelial cells were co-cultured with human vascular endothelial cells [82] to create a more physiological barrier [83]. The endothelial cells (≤P8) were grown in an endothelial culture medium (ECM-NG, Sciencell, Carlsbad, CA, USA) supplemented with 5% FBS, 1% endothelial growth supplement (ECGS, Sciencell, Carlsbad, CA, USA), and 0.5% gentamicin on 0.2% gelatin-coated culture dishes.