Endothelial and Fibroblast Cell Isolation

The use of embryos for cell culture was approved by the University of Queensland animal ethics committee (SCMB/002/18). Seventeen-day-old chicken and twenty-one-day-old Pekin duck embryonated eggs were purchased from Darling Downs Hatchery (Queensland, Australia). Primary aortic endothelial cells were cultured from the aortic arches of chicken and duck embryos as described previously using EGM-2MV medium (Lonza, Basel, Switzerland) with 10% FCS (Gibco, Waltham, MA, USA) [7 (link),8 (link)]. Primary chicken and duck embryo fibroblasts were cultured as described previously [9 (link),10 (link)]. Madin–Darby Canine Kidney (MDCK) cells were obtained from American Type Culture Collection (ATCC), and cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% FCS (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). NCI-H441 cells (human lung epithelial cells) were obtained from ATCC and cultured in RPMI (Gibco, Waltham, MA, USA) medium supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco). Primary human pulmonary microvascular endothelial cells were obtained from Sciencell (Carlsbad, CA, USA) and cultured in an endothelial cell growth medium (Sciencell, Carlsbad, CA, USA). All cell lines of mammalian origin were incubated at 37 °C 5% CO₂ whilst all avian cells were cultured at 40 °C 5% CO₂ unless otherwise stated.