Primary nasal epithelial cells were obtained by nasal brushing as previously described [
11,
15,
16]. Briefly, the nasal epithelial cells were obtained by brushing the inferior surface of the middle turbinate of both nostrils twice by using cytology brushes (Dent-o-care, London, UK). Next, the freshly brushed tissue was seeded in collagen-coated (Advanced BioMatrix Inc., San Diego, CA, USA) 12.5 cm
2 cell culture flasks (BD Bioscience, USA) in Bronchial Epithelial Growth Medium (BEGM, Lonza, Switzerland) supplemented with Single Quots (Lonza, Switzerland) and Primocin (100 μg/ml, InvivoGen, US) in a humidified incubator at 37 °C. The CF cells were additionally treated with Amphotericin B (250 μg/ml; Sigma Aldrich, US) and Ceftazidime (100 μg/ml, GlaxoSmithKline, Switzerland) during five days after sampling [
16]. Out of 15 CF patients brushed, 5 cultures were lost due to poor cell growth during the expansion phase of the cultures. The 10 CF patients from which successful ALI cultures could be established and used for further investigation are presented in Table
1. We obtained 0.4 to 1.5 million viable cells per CF patient after brushing and the time to reach confluence during the expansion phase was 7 to 15 days. The time to confluence during the expansion phase was directly dependent on the number of cells obtained after brushing. The success rate of culture establishment for healthy donors was higher compare to CF patients (7 healthy donors brushed resulted in 6 cultures successfully established). Of note, when growing on the inserts, we obtained a 100% success in differentiation of the cultures. Once confluent under submerged condition, the cells where seeded at a density of 60,000 cells per insert onto 24-well inserts with a pore size of 0.4 μm at 37 °C, 5% CO
2 (Greiner Bio-One, Austria). Cells were grown on the insert membranes under submerged conditions by adding 200 μl of BEGM apically and 450 μl of BEGM in the basal chamber until they reached confluence (2–3 days post seeding). Cell cultures were then washed with phosphate buffered saline (PBS) 1X w/o Ca
2+ and Mg
2+ and culture medium was then changed to complete PneumaCult™-ALI Medium (Stemcell Technologies, CA) following the manufacturer’s instruction. Briefly, cell cultures were exposed to air on the apical side and provided with 450 μl of complete PneumaCult™-ALI Medium in the basal compartment to promote mucociliary differentiation. The basal medium was changed every second day. Twenty-one to 28 days post exposing the cells to air at 37 °C, 5% CO
2, fully differentiated primary CF airway epithelial cell cultures were obtained and showed evidence of mucus production and ciliary beating. According to the supplier’s specifications, the differentiated CF nasal epithelial cells were therefore used at 21 to 28 days post exposure to air for further experiments. The control bronchial ALI cultures form 2 different donors used for the evaluation of CFTR expression by confocal microscopy were obtained commercially (Epithelix Sàrl, Geneva, Switzerland).