Primary human bronchial epithelial cell culture for air–liquid interface (ALI)

Human bronchial epithelial cell culture kit (Catalog #: CC-2540S) was purchased from Lonza Bioscience (Walkersville, MD). Cells were re-initiated, seeded and maintained according to the company’s protocols. Briefly, cells were initially plated for expansion into in a 75 cm² tissue culture flask at 3500 cells/cm² and maintained in defined serum free Bronchial Epithelial Growth Medium supplemented with corresponding BulletKit™ (Lonza #: CC-3170). After expansion, cells were seeded on rat-tail collagen, 30.0 µg/ml (Corning-Costar, Cambridge MA #354236), coated trans-well plates (Corning-Costar #354236) at 50,000 cells/well and allowed to reach confluence. After confluence was achieved cells were “air lifted” (removal of all media,allowing direct air exposure) and the basal chamber media was changed to B-ALITM Differentiation Media (Lonza Catalog #00193517) supplemented with B-ALITM SingleQuots™ (Lonza Catalog #00193515). Media changes were made to basal chamber daily until treatments.

When confluent, air-exposed ALI-HBECs were washed once with 150 µl PBS on the apical side of the transwell insert to clear mucus. Two hours later, the plates/cells were exposed to freshly generated CSE (see above). Cell exposure was conducted in a modified hypoxic chamber for 5 min to either cigarette smoke at a concentration of 186.7 mg/m³ (TSP) or to filtered ambient (“sham”) air. Immediately after, residual smoke (or sham air) was removed by ventilation with ambient air for 5 min and cells were subsequently placed back in the incubator overnight. At 24 h after cigarette smoke exposure, ALI-HBEC were washed apically with PBS and ready for SARS-CoV-2 infection.