Airway epithelial cell growth medium supplement

ALI cells were generated and cultured as described previously (Dalskov et al, 2020 (link); Olagnier et al, 2020 (link)). In brief, primary nasal cells were isolated using a nasal brush (Dent‐O‐Care). Cells were cultured as a monolayer in tissue culture flasks coated with 0.1 mg/ml of Bovine type I collagen solution (Sigma‐Aldrich). At passage two, cells were seeded at 2–3 × 10⁴ cells on 6.5 mm Transwell membranes (Corning) coated with 30 μg/ml of Bovine type I collagen solution and cultured in 2 × P/S (200 U/ml Pen/strep DMEM‐low glycose (Sigma‐Aldrich) mixed 1:1 (v/v) with 2× Monolayer medium (Airway Epithelium Cell Basal Medium, PromoCell, supplemented with 2 packs of Airway Epithelial Cell Growth Medium Supplement, PromoCell, without triiodothyronine +1 ml of 1.5 mg/ml BSA). When cultures reached confluency, Air–liquid interface (Wahl et al) is introduced and medium is changed to ALI medium (Pneumacult ALI medium kit (StemCell) + ALI medium supplement (StemCell) + 100 U/ml Pen/strep) supplemented with 0.48 μg/ml of hydrocortisone (StemCell) and 4 μg/ml of heparin (StemCell). Cells were allowed to differentiate for at least 21 days, as verified by extensive cilia beating and mucus covering, prior to experiment initiation.