COPD Primary Airway Cell Protocol

The primary HBECs from COPD and non-COPD donors were obtained from the commercial suppliers (Lonza Inc. or MatTek Corp). All primary cell lines were grown and treated in bronchial epithelial cell growth media (BEGM from Lonza or UNC MLI Tissue Procurement and Cell Culture Core). Air-liquid interface (ALI) cultures of primary cells were grown in BEGM and differentiated in bronchial ALI (B-ALI) differentiation media (Lonza or UNC MLI Tissue Procurement and Cell Culture Core), as described previously (11 (link)). ALI cultures were seeded at a density of 5 x 10⁵ cells/cm² on collagen IV-coated Costar^® 6.5 mm Transwells with 0.4 µm pore polyester membranes (Corning Costar Corporation). Cells were differentiated for a minimum of 21 days before treatments. Epithelial differentiation was confirmed by live cell imaging of ciliary beatings and mucus glycoprotein expression. For CSE preparation, CS particulate matter collected on the filter membranes from mainstream smoke of 3RF4 research cigarettes (courtesy Philip Kuehl, Lovelace Biomedical) were used and final treatments at 20 µg/ml CSE were used. Apical side epithelial cells were exposed to treatments for 30 minutes at each treatment point. In addition, paraffin-embedded tissue sections human COPD and healthy control lungs were obtained from the LTRC.