Pneumacult ex plus medium

Nasal brushings were placed in PneumaCult-Ex Plus medium (STEMCELL Technologies, Cambridge, UK) and cells dissociated from the brush by gentle agitation. The cells were seeded into a single well of collagen (PureCol from Sigma-Aldrich) coated plate and once confluent, the cells were passaged and expanded further in a T25 flask. The cells were passaged a second time and seeded onto transwell inserts (6.5 mm diameter, 0.4 μm pore size, Corning) at a density of 24,000 cells per insert. Cells were cultured in PneumaCult-Ex Plus medium (STEMCELL Technologies, Cambridge, UK) until confluent, at which point the media was replaced with PneumaCult-ALI medium in the basal chamber and the apical surface exposed to provide an air-liquid interface (ALI). Ciliation was observed between 4–6 weeks post transition to ALI.

HBECs, from Lonza, were cultured in suspension in PneumaCult-Ex Plus Medium according to manufacturer’s instructions (STEMCELL Technologies, Cambridge, Massachusetts, USA). To generate ALI cultures, HBECs were plated on collagen-coated transwell inserts with a 0.4-micron pore size (Costar, Corning, Tewksbury, Massachusetts, USA) at 5 × 10⁴ cells/ml per filter and inserted into 24-well culture plates. Cells were maintained for the first 3 days in PneumaCult-Ex Plus Medium, then changed to PneumaCult-ALI Medium (STEMCELL Technologies) containing the ROCK inhibitor Y-27632 for 4 days. Fresh medium, 100 μl in the apical chamber and 500 μl in the basal chamber, was provided every day. On day 7, medium at the apical chambers were removed, while basal chambers were maintained with 500 μl of PneumaCult-ALI Medium. HBECs were maintained at an ALI for 28 days, allowing them to differentiate. Medium in the basal chamber was changed every 2 to 3 days (500 μl).

Normal human bronchial epithelial (NHBE, Lonza, cat# CC-2540 S, Germany) are available in our laboratary and routinely cultured in air liquid interface (ALI) as described [9 (link), 10 (link), 14 (link)].
Briefly, cells were cultured in a T75 flask for 2–4 days until they reached 80% confluency. The cells were detached using TrypLE (Thermo Fisher Scientific) and seeded onto GrowDexT (UPM)-coated 0.33 cm² porous (0.4 μm) polyester membrane inserts with a density of 1 × 10⁵ cells per Transwell (Costar, Corning, New York, NY, USA). The cells were grown to near confluency in submerged culture for 2–3 days in specific epithelial cell growth medium according to the manufacturer´s instructions (PneumaCult™-Ex Plus Medium, Stemcell, cat# 05040, Germany). Cultures were maintained in a humidified atmosphere with 5% CO₂ at 37 °C and then transferred to ALI culture in PneumaCult™-ALI medium (Stemcell, cat# 05001, Germany) for another 30 days until fully differentiated.

The immortalized human bronchial epithelial cell line, BEAS-2B (European Collection of Cell Cultures) was cultured in PneumaCult™-Ex Plus Medium (Stemcell Technologies, United Kingdom) supplied with 50x extra supplement; hydrocortisone (Stemcell Technologies, United Kingdom) and penicillin-streptomycin solution (Gibco, Sweden) was added to the complete cell medium. It is important to note that the cell medium is free from FBS and bovine pituitary extract (BPE). Hence, the cell culture medium can be considered “animal-free” (Oredsson et al., 2019 (link)). Furthermore, BEAS-2B cells are often grown on a substrate of fibronectin, collagen, and serum albumin of bovine origin. However, we were able to maintain cells without pre-coating with extracellular matrix proteins (Supporting Information), thus avoiding the use of animal proteins. Hence, the cells were seeded in 75 cm² tissue culture flasks without pre-coating and expanded until 70–80% confluence for further studies under static or dynamic conditions, as described below.

A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C 5% CO₂. pHCECs were maintained in Human Corneal Epithelial Cell Growth Medium (Tebu-bio) at 37°C 5% CO₂.
Regarding pHNECs, patients’ pHNECs were collected on superior turbinates using smear brushes at the Hospital of Toulouse, France. After brushing back cells in collection medium, centrifugation was performed for 5 min 400 g at 4°C. Pellet was resuspended in 4 ml TrypLE express (GIBCO) + 20 µl Sputolysin (200X) and incubated at 37°C for 5 min to disrupt mucus. TrypLE was diluted with 4 ml of Advanced DMEM F12-.
Pellet was recovered after centrifugation and culture was continued in the expansion medium Pneumacult. After a week of proliferation, basal cells were counted and seeded onto collagen-coated (0.03 mg/ml) and maintained in Pneumacult Ex Plus Medium (StemCell) at 37°C 5% CO₂.

The hPAECs were collected via bronchial brushing from three study participants who gave written informed consent and were recruited from the outpatient clinics of the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). The study was approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University. Further details regarding the demographic characteristics are shown in Supplementary Table S1.
The cells were detached from the brush in DMEM containing 1% (v/v) penicillin and streptomycin. After centrifugation at 500×g for 5 min, the cells were re-suspended in PneumaCult™-Ex Plus Medium (05040, STEMCELL, Canada) containing 1% (v/v) penicillin and streptomycin and cultured at 37 °C in a 5% CO₂-gassed humidified atmosphere. The cells were subcultured until 50–60% confluence. The characteristic of the hPAECs is shown in Supplementary Fig. S7a.