Hsaecs

Human primary small airway epithelial cells (HSAECs, ATCC, Manassas, VA, USA) were cultured and expanded in airway cell basal medium following manufacturer instructions. L-MSCs harvested from the 3D cultures in HC-L-ECM and LC-L-ECM, or control L-MSCs (cultured in 2D) were co-cultured in direct contact with HSAECs at a ratio L-MSCs:HSAECs of 1:4 in 12-wells plates with HSAECs medium. After 16 h of seeding, the cultures were washed twice with PBS 1X and replaced by serum-free media with either 20 µg/mL of lipopolysaccharides (LPS) or serum-free media for control, following a protocol for an in vitro model of acute respiratory distress syndrome (ARDS) adapted from [46 (link)]. After 40 h of co-culture, inflammatory (IL-6 and IL-8) cytokines were measured from the supernatants by enzyme-linked immunosorbent assay (ELISA) following the DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN, USA) instructions.

Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO₂ environment (19 (link), 23 (link)). RSV Long strain was prepared by sucrose cushion ultracentrifugation and titered by methylcellulose plaque assay (19 (link)). hSAECs were infected for 24 h at a multiplicity of infection (MOI) of 1.0 prior to harvest. For pharmacological induction of the UPR, hSAECs were treated for indicated times with various standard concentrations of tunicamycin (TM, 0.5-1 μg/ml) or thapsigargin (Tg, 50-100 nM). The kinase-inhibiting RNase attenuator (KIRA)-8, a selective IRE1α RNase inhibitor, was directly added to the SAGM at a concentration of 10 μM (31 (link)). The reagent was from MedChemExpress (South Brunswick Township, NJ, USA).