Cultivation of Alveolar Cell Lines and Primary Endothelial Cells

Human epithelial NCl-H441 cells (which express alveolar type II cell markers [Ren et al., 2016 (link)]) were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in Roswell Park Memorial Institute medium (RPMI) (Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO) and 1% penicillin-streptomycin (Lonza, Basel, Switzerland). NC1-H441 cells were used at passages 3–8. Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from Sciencell (Carlsbad, CA) and cultured in endothelial cell growth medium (Sciencell) in fibronectin coated tissue culture flasks (2 μg/cm², Sigma). HPMEC cells were used at passages 3–7, as at higher passages these cells can demonstrate senescence (Shen et al., 1995 (link)). Madin-Darby canine kidney (MDCK) cells were obtained from ATCC and cultured in Dulbecco modified Eagle medium (Gibco) between passages 20 and 50. All cell lines were maintained in a humidified 37°C incubator with 95% O₂ and 5% CO₂. Each cell line has no mycoplasma contamination to report.