Appendix A.2.3. Procedure of Cell Cultivation

The cells, after being removed from the brushes, are resuspended in 1.6 mL PC-Ex-Pl medium (as described above), and 1 mL is then used to be seeded in one coated well of a 12-well plate (called passage 0). The day after the brushing, medium from the previously seeded well is transferred to a 15 mL tube, centrifuged (300 rcf, 5 min, RT; all subsequent centrifugation steps are performed with the same settings unless otherwise stated), resuspended in 1 mL Accutase (Sigma-Aldrich, #A6964-100 mL), and kept for 30 min at 37 °C. Subsequently, the cell suspension is centrifuged and again resuspended in 1 mL PC-Ex-Pl and added to a second coated and rinsed well in the same 12-well plate; One mL of fresh PC-Ex-Pl is added to this first well with the already adhered cells. We usually change media on Monday mornings, and Wednesday and Friday afternoons. Ideally, the media of passage 0 cells is changed every day or at least every second day. However, we do not change media over the weekends. The cells are cultured until a maximum of 60–80% confluence is reached (usually after 3–4 days). If cells do not reach 60–80% confluence after one week, depending on the growth process, they are still passaged, in the hope that the provision of a new stimulus might improve growth. Cells are lifted using Accutase (8–10 min at 37 °C, 1 mL for a well of a 12-well plate, flushed off with the Accutase at least 5 times). The cells in the Accutase are transferred to a 15 mL tube, containing 10 mL RPMI, and centrifuged. After aspiration of the supernatant, the cell pellet is resuspended in 1 mL PC-Ex-Pl medium and seeded in a pre-coated (incubation with 2 mL diluted PureCol for 30 min at 37 °C, see preparation of well plates) T25 tissue culture flask (blue vented cap, Falcon, Corning, NY, USA; #353108) (with 5 mL of media in total) until again confluence of a maximum of 60–80% is reached (usually after 5–7 days; rather use of the cells at 50% confluence than risking reaching >80%, especially over the weekend). Then, the cells are lifted and collected again with Accutase (see above) and seeded in uncoated tissue culture transwell (Corning Transwell polyester membrane inserts, pore size 0.4 μm, diameter 12 mm, CLS3460-48EA, Sigma-Aldrich) at a density of 100,000 cells/transwell and supplemented with 1 mL in the lower, basolateral chamber, where no cells are, and 0.5 mL PC-Ex-Pl in the upper, apical chamber, where the cells were seeded into. We usually seed out into three inserts. The leftover cells are frozen in vials with roughly one million cells (using 1 mL of CyroStor freezing media, Sigma-Aldrich, #C2874) as backup if the cell culture would not successfully differentiate. Medium is again changed three times/week (see above). One day after the cells reach complete confluence (usually 2–5 days after seeding), they are exposed to the air-liquid interface (ALI) and fed with 700 μL/well PC-ALI (Stemcell, cat#05001, preparations see above) media from the basolateral chamber only.

After seven or more days at ALI, cultures undergo a weekly PBS wash (Dulbecco’s Phosphate Buffered Saline with MgCl₂ and CaCl₂ (PBS++), Sigma D8537-500 mL) to remove excess mucus. Before the medium is changed, 500 μL of PBS++ is added to the apical chamber of the insert. The plates are then left at RT for 15 min. After this, the apical PBS and the basal medium is removed and 700 μL of fresh PC-ALI medium is added. Normally after about 7–10 days at the ALI, the cell cultures start to produce mucus, and after about 14–21 days, the first moving cilia were seen. Cell cultures used for the experiments are exposed for ≥28 days at the ALI. Cultures that are at ALI for over 4 weeks are transferred to maintenance and the medium is only changed once per week, along with a PBS wash (see above).