Polarized Human Airway Epithelial Cell Cultures

The primary HAE-ALI cultures HAE-ALI^B3-20, HAE-ALI^B4-20, and HAE-ALI^B9-20 were provided by the Cells and Tissue Core of the Center for Gene Therapy, University of Iowa (18 (link), 55 (link)– (link)57 (link)). These polarized HAE-ALI cultures were derived from three independent donors. The freshly isolated human bronchial epithelial cells from the donor tissues were seeded onto collagen-coated, semipermeable polycarbonate membrane inserts (0.33 cm², 0.4-μm pore size, Costar Transwell, catalog no. 3413, Corning, New York), and grown at an ALI as previously described (58 (link)). The cultures were maintained in USG medium containing 2% Ultroser G (USG) serum substitute (Pall BioSepra, France). After 3 to 4 weeks of culture at an ALI, the polarized culture was fully differentiated. The polarity of the HAE was determined for the TEER using an epithelial volt-ohm meter (Millipore). A value of 1,000 Ω·cm² or higher was chosen for SARS-CoV-2 infection as we previously used for HBoV1 infection (50 (link), 59 (link)). HAE-ALI^L209 cultures on 1.1 cm² Millicell-PCF (Millipore, Billerica, MA) were provided by Dr. Matthias Salathe, which were generated following a published method (60 (link)) using primary airway bronchial epithelial cells isolated from the lung of a donor (L209).