Culturing Human Lung Cell Lines

As a growth medium, the Minimum Essential Medium Eagle (MEM, Sigma-Aldrich) commercial solution was enriched with 1 mM of penicillin, streptomycin, and 0.25 mM L-glutamine (Sigma-Aldrich). Two MEM-enriched solutions were used: one with 10% Fetal Bovine Serum (FBS-MEM) and one without (MEM, Sigma-Aldrich). Human epithelial lung cancer cell line (A549, CCL-185, passage number: 1) and fibroblast lung cell line (MRC-5, CCL-171, passage number: 30) were purchased from the American Type Culture Collection (ATCC). In FBS-MEM, cells were maintained as a monolayer culture at 37 °C in a humidified 5% CO₂ atmosphere. Cells were sub-cultured according to the protocols recommended to the cell’s phenotype and described previously in Jakubczak et al. [23 (link)]. Cells were detached from the culture flasks using 2 mL of 0.05% trypsin-EDTA solution (trypLE Express, Gibco) for 5 min, suspended in 6 mL of FBS-MEM, and centrifuged for 5 min at 1500 rpm (Universal 32 Tabletop Centrifuge, Hettich, Tuttlingen, Germany). The supernatant was removed, and cell pellets were resuspended in the proper amount of FBS-MEM to obtain the cell density of 1 × 10⁵ cells/mL.