All cell culture experiments were performed at the Erasmus MC Cell biology facility. Primary airway cells from newborn CFTR KO and control piglets and human lungs were isolated and cultured in parallel at ALI on permeable membranes to allow differentiation to airway epithelial cells (Methods BEGM) as described in Stolarczyk et al. (2016, 2018). Undifferentiated CF and non-CF primary epithelial cells frozen in liquid nitrogen were obtained from the CF Translational Center, McGill University, Montreal, Canada where they were prepared according to a protocol previously described in detail (Matthes et al., 2018). Non-CF undifferentiated primary epithelial cells from Erasmus MC were obtained stored in liquid nitrogen and cultured using essentially the same method (Amatngalim et al., 2018). In all experiments reported here CF and non-CF cells were obtained as undifferentiated primary bronchial epithelial cells, expanded submerged on collagen-fibronectin coated flasks in KSFM + ROCKinhibitor up to passage 3, and subsequently differentiated in parallel on 12 mm transwell inserts with 0.4 μl pore polyester membrane (Costar) in ALI culture (BEGM, LONZA), as described previously (Stolarczyk et al., 2016, 2018). Human and neonatal CFTR KO and WT Pig BEC-ALI cultured for 21 days, used in all experiments, showed comparable trans-epithelial resistance (range 400–800 Ω•cm2, N = 65, average 550 ± 150 SD Ω•cm2). Differentiated epithelial morphology as shown by lateral cadherin (ECAD) and tight junction (ZO-1) staining, and 20–30% ciliated cells (Tubulin IV) was verified in all experimental groups. Markers of secretory cells (MUC5B, MUC5AC, and SCGB3A1) were also tested under these conditions on randomly selected samples. No obvious differences between CF and non-CF were observed by visual inspection (data not shown), representative images of non-CF patients, three donors, are shown in the Supplement (Supplementary Figure S1).

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