Bronchial epithelial growth medium (begm)

Basal cell–derived 3D spheres were obtained following published protocols (40 (link), 42 (link)). Cells in passage 1 were trypsinized and resuspended (3 × 10⁴ cells/mL) in BEGM* (Lonza, see Supplemental Table 4 for details of media composition) containing 5% growth-factor-reduced Matrigel (Corning). A total of 65 μL of the cell suspension was plated in each well of a nonadherent 96-well plate precoated with 30 μL of a 25% solution of Matrigel (Corning) in BEGM* (Lonza). ROCK inhibitor (5 μm, Y-27632) was added at seeding only, and cultures were fed or treated on days 3, 8, and 14 of culture with 70 μL of BEGM*. On day 21, plates containing 3D spheres were placed on ice. The spheres were washed with ice-cold PBS 1X to remove the medium and Matrigel and then collected into Eppendorf tubes. Cells were incubated for 1 hour at room temperature with 4% PFA (Thermo Fisher Scientific), washed with 1X PBS, and embedded in Histogel (Thermo Fisher Scientific) following the manufacturer’s instructions. Histogel-embedded spheres were processed and embedded in paraffin at Morphisto GmbH as detailed above for the tissue specimens.

NHBE culture was performed as previously described (40 (link)). Briefly, primary NHBEs (Lonza, Basel, Switzerland) of four genetically independent donors were grown as monolayers in 100% humidity and 5% CO₂ at 37 °C in serum-free defined growth media (BEGM, Lonza). NHBEs (passage 3) were used at ∼80% confluence in 6-well plates. To avoid gene expression changes or influences by growth factors in the BEGM medium, cells were rested in basal medium (BEBM, Lonza) for 12 h, then stimulated with HDM extract at a final concentration of 40µg/mL (CITEQ), recombinant human IL-4 at 50 ng/ml (R&D Systems, Minneapolis, MN, USA) for 6 h. When indicated, cells were pretreated for two h prior to stimulation with the AhR inhibitor CH-223191 (Sigma-Aldrich) at a concentration of 1µM/mL. For RNA analysis, harvested cells were lysed in RLT buffer (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol (Roth, Karlsruhe, Germany) directly in the cell culture well.

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.

The immortalized human bronchial epithelial cell line BEAS-2B and NSCLC cell lines, including NCI-H1299, NCI-H1975, A549, HCC827 and PC9, were obtained from the American Type Culture Collection (ATCC). All cells were cultured according to the manufacturer’s instructions. BEAS-2B was grown in BEGM (Lonza/Clonetics) with all the additives provided with the BEGM kit except for GA­1000 (gentamycin-amphotericin B mix). NSCLC cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Gibco) and 100 U/ml penicillin/streptomycin (Invitrogen).

Primary MAECs isolated from mouse trachea and immortalized human airway epithelial cells (HAECs) provided by S. Randell (University of North Carolina, Chapel Hill, NC, USA) described previously [36 (link)] were cultured on Transwell membranes (Corning, New York, NY, USA) as described previously [37 (link)]. The primary cells were cultured in bronchial epithelial growth medium (BEGM) and small AEC growth media (SAGM) (Lonza, Basel, Switzerland), respectively, supplemented with growth factors (BEGM and SAGM Singlequots; Lonza) and incubated at 37 °C under an atmosphere containing 5% carbon dioxide as described [38 (link)]. MAECs were isolated from murine tracheas after overnight digestion with pronase, washing in phosphate buffered saline (PBS) and were cultured as previously described [39 (link)].