Pneumacult ali basal medium

Transwells (6.5 mm) with 0.4-μm-pore polyester membrane inserts (Corning Inc.) were coated with PureCol for 20 minutes before cell seeding. NHBE cells (5×10^4) suspended in 200 μl of complete AEC medium were seeded in the apical part of a Transwell. Subsequently, 500 μl of complete AEC medium was added to the basal part of the Transwell. When the cells formed a confluent layer on the Transwell insert, the AEC medium was removed from the apical part, and PneumaCult-ALI basal medium (Stemcell Technologies) with the required supplements (Stemcell Technologies), 2% penicillin/streptomycin and 1% amphotericin B (complete ALI basal medium) was added to the basal part. The ALI medium in the basal part was changed every other day, and the apical surface was washed with 1x Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific) once per week initially but more frequently when more mucus was observed on later days. However, the thickness of the mucus was not determined. All the cells were differentiated for at least 4 weeks at 37°C in an incubator with 5% CO₂.

Transwells (6.5 mm) with 0.4 µm-pore polyester membrane inserts (Corning Inc.) were coated with PureCol for 20 min before cell seeding. NHBE cells (5 × 10⁴) suspended in 200 µl of complete AEC medium were seeded in the apical part of each Transwell. Then, 500 µl of complete AEC medium was added to the basal part of the Transwell. When the cells formed a confluent layer on the Transwell insert, the AEC medium was removed from the apical insert, and PneumaCult-ALI basal medium (Stemcell Technologies) with the required supplements (Stemcell Technologies), 2% penicillin/streptomycin and 1% amphotericin B was added to the basal chamber. The ALI medium in the basal chamber was changed every other day. The apical surface was washed with 1x Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific) once per week initially but more frequently when more mucus was observed on later days (when it was difficult to see the apical cells, it was determined that the difficulty was probably due to a thick layer of mucus). All cells were differentiated for up to four weeks (at 37 °C with 5% CO₂) until the desired cellular and physiological properties of an epithelial layer were obtained, such as a CBF greater than 6 Hz and a TEER greater than 500 ohm/cm².

We used a previously described protocol (12 (link), 58 (link), 116 (link)). Briefly, 6.5 mm transwells with a 0.4 μm pore polyester membrane insert (Corning, Inc.) were coated with PureCol for 20 minutes before cell seeding and then removed. NHBE cells (5×10⁴) suspended in 200 μl of AEC medium were seeded on the apical part of a transwell. Then, 500 μl of AEC medium was added to the basal part of a transwell. When cells formed a confluent layer on the transwell, the AEC medium was removed from the apical part and replaced with PneumaCult-ALI basal medium (Stemcell Technologies) with the required supplements (Stemcell Technologies), 2% penicillin/streptomycin and 1% amphotericin B in the basal part. The ALI medium was changed from the basal medium every other day. Apical surfaces were washed with 1× Dulbecco’s phosphate buffer saline (DPBS) (Thermo Fisher Scientific) once per week initially but washed more frequently when higher mucus was observed in later days. All cells were differentiated for up to four weeks (37 °C with 5% CO₂) until the cellular and physiological properties of the epithelial layer were obtained.

HAOs were cultured at the air-liquid interface (ALI) for differentiation. Organoids were collected in cold basal medium and dissociated as described above. After spin and medium aspiration, cells were resuspended in warm basal medium with 2% (v/v) BME2 and seeded onto pre-coated 2% BME2 in 6.5-mm insert of a 24-well plate Transwell Permeable Support (Costar). Cells were incubated for 1 hour at 37°C and then 500 μL of warm HAO medium was added to the bottom wells of the 24-well plate. Cells were grown to confluency for 1 week before the ALI was established by removal of apical side medium and substitution of basolateral medium for 1:1 (v/v) HAO medium and with Pneumacult ALI basal medium (Stem Cell Technologies, 05002). Basolateral medium was changed every 3–4 days, and mucus was washed from the apical side Cells were differentiated at the ALI for at least 1 month.