Bronchial epithelial growth medium (begm)

The Syrian hamster pancreatic ductal adenocarcinoma (PDAC) cell lines HPD1NR (maintained in Roswell Park Memorial Institute (RPMI) 1640 with 10% FCS), Hap-T1, SHPC6, IPAN and the renal tumor cell line HaK were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS. SHPC6 and IPAN were kindly provided by W. Wold (St. Louis University, St. Louis, MO, USA). HPD1NR and Hap-T1 cell lines were purchased from the German Collection of Microorganisms and Cell Cultures. The HaK cell line was purchased from the American Type Culture Collection (ATCC). JH293, the human kidney epithelial cell line transformed with Ad5 DNA, was obtained from the Cancer Research UK Central Cell Services (London, United Kingdom) and maintained in DMEM with 10% FCS. Normal human bronchial epithelial cells (NHBE) were obtained from Cambrex (Cambridge, UK) and maintained in Bronchial Epithelial Growth Medium (BEGM) (Cambrex). Primary culture hamster hepatocytes (maintained in DMEM with 10% FCS) were isolated and cultured in our lab. All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ and confirmed to be mycoplasma-free before being used experimentally.

HBECs were cultured in bronchial epithelial growth medium (BEGM, Clonetics, San Diego, CA, USA or PneumaCult-Ex Medium, STEMCELL Technologies, Cambridge, UK) and subsequently seeded into 12-well plates (Nunc Technologies, Carlsbad, CA, USA). For experiments cells were exposed to H₂O₂ (Sigma-Aldrich, Stockholm, Sweden) for 30 min before stimulation with 10 μg/ml poly(I:C) (InvivoGen, San Diego, CA, USA) for 3 and 24 h. Cell lysates were obtained for gene and protein expression analysis and cell-free supernatants were obtained for analysis of protein release.

HBECs (3x10⁴ cells/mL) were cultured in bronchial epithelial growth medium (BEGM, Clonetics, San Diego, CA, USA) until 80% confluence in type-I bovine collagen-coated (Advanced BioMatrix, San Diego, USA) 12-wells plates (Nunc Technolgies, Carlsbad, CA, USA) at 37°C, 5% CO₂ in air. Cells were treated with 10 µg/mL imiquimod (Tocris Bioscience, Bristol, UK) with or without 10 µg/mL polyinosine-polycytidylic acid (poly(I:C)) (InvivoGen, San Diego, US) or SARS-CoV-2 SP1 for 3, 24 or 48 hours. Cell-free supernatants were collected for protein release analysis and cell lysates were collected for protein or gene expression analysis.

Tracheobronchial tissues were obtained according to standard guidelines from left-over tracheobronchial tissue of donor lungs, of whom no further information was available and treated with pronase, as described previously [13 (link)]. Primary bronchial epithelial cell (PBECs) were cultured on coated flasks with 30 μg/ml collagen, 30 μg/ml fibronectin, and 10 μg/ml BSA in serum-free hormonally supplemented bronchial epithelium growth medium BEGM (Clonetics, LONZA, Breda, The Netherlands) and passaged twice. PBECs were seeded in coated permeable polyester membrane (0.4 μm) (Corning-Costar, Corning, NY). After reaching confluence, cells were exposed to air and cultured under ALI conditions as described previously [12 (link)]. Mucociliary differentiation was confirmed between days 14 and 28 after exposure to ALI by transepithelial electrical resistance (TEER) measurement and by presence of mucus and cilia. The study protocol was consistent with the Research Code of the University Medical Center Groningen (http://www.rug.nl/umcg/onderzoek/researchcode/index) and national ethical and professional guidelines (“Code of conduct; Dutch federation of biomedical scientific societies”; http://www.federa.org).

BEAS-2B cells (transformed human bronchial epithelial cells) were obtained from the American Type Culture Collection through LGC Promochem AB (Borås, Sweden). The BEAS-2B cells were grown in serum-free Bronchial Epithelial Growth Medium (BEGM; Clonetics, Walkerwille, MD, USA) at 37 °C in a humidified atmosphere of 5% CO₂. In total, 60,000 cells were plated on 24-well plates (for cytotoxicity and comet assays; Nunc, Roskilde, Denmark) 24 h before treatment. For the micronucleus assay, 300,000 BEAS-2B cells were seeded 24 h before treatment in six-well plates (Nunc, Roskilde, Denmark).