MDCK and NHBE Cell Culture Protocol

Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA, USA) were serially passaged and maintained in culture in modified Eagle's medium (CellGro, Herndon, VA, USA) supplemented with L-glutamine (2 mM), 5% fetal bovine serum and antibiotics at 37 °C with 5% CO₂. Normal human bronchial epithelial (NHBE) cells (Lonza, Walkersville, MD, USA) from two healthy male donors (two and four years old) were expanded, cryopreserved and maintained in culture in an air/liquid interface (ALI) system, as previously described.^{27 (link), 28 (link)} Briefly, cells were plated in 0.33 cm² transwell inserts (Corning, Corning, NY, USA) and allowed to differentiate. An ALI was established when the cells reached 98%–100% confluence. Media from the apical surface of the cells was removed, and the cells were exposed to a humidified 95% air/5% CO₂ environment. The basolateral media or the ALI media, which was supplemented with SingleQuot growth factors (Lonza), was changed every 48 h for a minimum of six weeks. During every media change, the apical surface of the cells was washed with sterile phosphate-buffered saline (PBS) to remove mucus.