Snapwell filter supports

Tissue was collected by scrape biopsy, and HNE cell isolation was performed as described (53 (link)), followed by conditional reprogramming (54 (link)). HNE carrying rare CFTR mutations were gifts from W. Finkbeiner (UCSF) or were purchased from the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy. For functional measurements, HNE were seeded at a density of 5 × 10⁵ cell/filter on 1.12 cm² Snapwell filter supports (Corning) and differentiated under ALI by culturing in PneumaCult-ALI medium (Stemcell Technologies) for ≥ 3 weeks.

CFBE41o- cells, provided by D. Gruenert (University of California in San Francisco, USA), were maintained in MEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 2 mM L-glutamine and 10 mM HEPES on human fibronectin-coated plastic dishes (Ehrhardt et al., 2006 (link)). CFBE41o- cell lines expressing inducible CFTR variants with a 3HA tag in the fourth extracellular loop were generated using the ClonTech pLVX-Tight-Puro lentivirus technology as previously described (Veit et al., 2012 (link)). For experiments, CFBE41o- cells were seeded at a density of 2 × 10⁴ cells/well into 96-well plates or 1 × 10⁵ cells/filter on 1.12 cm² Snapwell filter supports (Corning). CFTR expression was induced with 50–250 ng/mL of doxycycline for at least 4 days. BHK-21 cells were cultured in DMEM/F12 (50:50) medium (Wisent) supplemented with 5% FBS. The cells were grown to at least 70% confluence before treatment with drugs. 16HBE14o-cells genome-edited to produce the homozygous CFF-16HBEge R1162X and W1282X-CFTR cell lines were obtained from the Cystic Fibrosis Foundation (Valley et al., 2019 (link)).

HNE cell isolation from tissue collected by scrape biopsy was performed as described [36 (link)]. HNE were expanded in presence of irradiated feeder cells in Rock inhibitor containing medium F, a process termed conditional reprogramming [37 (link)]. Functional measurements were performed in filter grown differentiated HNE. For this purpose, HNE were seeded at a density of 5 × 10⁵ cell/filter on 1.12 cm² Snapwell filter supports (Corning) and differentiated under air-liquid interface by culturing in PneumaCult-ALI medium (Stemcell Technologies) for ≥three weeks.