Cell culture of bronchial epithelial cells

The CFBE41o- cells are derived from a cystic fibrosis patient and homozygous for ΔF508 mutation [37, 38]. For standard cultivation, the cells were maintained in MEM supplemented with 10% FCS, 1% NEAA, and 600 mg/l glucose at 5% CO₂ atmosphere and 37 °C, respectively. Cells were fed every 2–3 days and passaged weekly.

For the infection experiments, the CFBE41o- cells were seeded with a density of 50,000 cells/well in 12-well Transwell® plates with a pore size of 0.4 µm.

The human bronchial epithelial cell line Calu-3 HTB-55 (ATCC, passage 31–51) was also used as a model cell line for the in vitro model. The cells were maintained in MEM with 10% FCS, 1% NEAA, and 1% sodium pyruvate at 5% CO₂ atmosphere and 37 °C. Cells were fed every other day and passaged weekly. For infection experiments, cells were seeded with a density of 100,000 cells/well in 12-well Transwell® plates with a pore size of 0.4 µm.

CFBE41o- or Calu-3 cells were lifted to ALI 2–3 days after seeding on Transwell® inserts and fed every other day until forming a confluent monolayer, and barrier properties exceeded 300 Ω*cm² (i.e., 7 days for CFBE41o- or 10–12 days for Calu-3 cells). Both cells were used for up to 20 passages and checked for mycoplasma contamination every 2 months, with the MycoAlert™ mycoplasma detection kit (LONZA), following the manufacturer’s instructions.