Isolation and Culture of Porcine Airway Epithelial Cells

Primary porcine airway epithelial cells were isolated from pigs of a local slaughterhouse. PTEC and PBEC were obtained from swine trachea and bronchi, respectively. Primary cells were harvested as previously described18 (link) and were seeded on type I collagen (Sigma)-coated flasks with bronchial epithelial cell serum-free growth medium (BEGM). The BEGM was modified from previous studies28 29 (link) and contained the BEBM basal medium (Lonza) supplemented with the required additives. PTEC and PBEC were transferred to type IV collagen-coated Transwell^® polycarbonate membrane (24 well, 0.4 μm pore size, Corning Costar) at a density of 2.5 × 10⁵ cells per filter support with the air-liquid interface (ALI) medium consisting of a mixture of DMEM (Gibco) and BEBM basal medium (1:1) with additives described previously28 . After PTEC and PBEC reached confluence, the cells were maintained under ALI conditions for at least 4 weeks at 37 °C in a humidified 5% CO₂ atmosphere. Both cultures were validated for porcine specific respiratory tract pathogens including porcine circovirus-2, porcine reproductive and respiratory syndrome virus, porcine cytomegalovirus, porcine influenza A virus, porcine respiratory coronavirus, Mycoplasma hyorhinis and Mycoplasma hyopneumoniae by multiplex Polymerase Chain Reaction (PCR)30 . All PTEC and PBEC used in this study were free from the above mentioned pathogens.