Airway epithelial cell medium

Middle and distal airways from C57BL/6J and Ch25h^−/− mice were isolated and incubated ex vivo as described (Yildirim et al, 2008). Briefly, after sacrifice by a ketamine–xylazine over dose, the trachea was cannulated, the lungs removed from the thorax and infused with 1% low‐melting agarose dissolved in 1:1 Ham‘s F12 nutrient medium (Sigma‐Aldrich) and distilled water (Gibco, Life Technologies). Airways were dissected under a microscope (Zeiss) from the left lung after the agarose had solidified on ice for 30 min. The isolated airways were washed and cultured in airway epithelial cell medium (PromoCell) at 37°C, 5% CO₂.

All cells, including co-cultures, were cultured at 37°C and 5% CO₂ in a standard culture incubator and exposed to hypoxia using an atmosphere-regulated workstation set to 37°C, 5% CO₂ and 1% O₂ (Invivo 400, Baker-Ruskinn Technologies). Calu-3 cells were cultured in Advanced DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/mL penicillin and 10 mg/mL streptomycin (Invitrogen). HEp-2 and BEAS-2B cells were cultured in DMEM (Sigma-Aldrich) with the same supplements as above. Human PBECs were obtained using flexible fibreoptic bronchoscopy under light sedation with fentanyl and midazolam from healthy control volunteers and all participants provided written informed consent. The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361). Airway epithelial cells were cultured in Airway Epithelial Cell medium (PromoCell, Heidelberg, Germany) in submerged culture. PBECs were cultured on PureCol-coated 0.4 μm pore polyester membrane permeable inserts (Corning) in serum-free airway epithelial cell media, brought to air-liquid interface, replacing basal media with ALI media (StemCell Pneumacult). The media was exchanged every 2 days and apical surfaces washed with PBS weekly to disperse accumulated mucus for at least 6 weeks.