Transwell clear

Primary normal Human Bronchial Epithelial (NHBE) cells were isolated from left over tissues after lung transplantation following the approved protocol^{51 (link)} and were received from Marsico Lung Institute/Cystic Fibrosis Center at the University of North Carolina, Chapel Hill (Chapel Hill, NC). Then NHBE cells were cultured as previously described^{29 (link),52 (link),53} . Cells at passage 2 were transferred to microporous polyester inserts (0.4 um pore size, Transwell-Clear; Corning Costar, Corning, NY) and fed with a 1:1 mixture of BEBM and Dulbecco’s Modification of Eagle’s Media (Mediatech, Herndon, VA). Media was applied apically and basally until the cells were confluent and then basally after an air–liquid interface (ALI) was established. Cells were cultured at ALI for 14 days to promote relatively stable expression of goblet and ciliated cells before exposure to e-cig smoke solution. Mature, well-differentiated monolayers of cells were then exposed to control (ultrapure water dissolved in culture medium, water/medium: 2% v/v) or e-cig smoke solution (containing 2 ppm diacetyl, water/medium: 2% v/v) on the apical side for 6 or 24 h (n = 3 subjects, each treatment was performed in duplicate). After incubation, LDH release in the culture medium was measured for cytotoxicity assay. Total RNAs were isolated from the cells using miRNeasy kit (Qiagen) for RNA -Seq analysis.

The human mucoepidermoid lung cancer cell line NCI-H292 (herein referred to as H292) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). H292 cells were cultured in RPMI 1640 media (LGC Standards, Middlesex, UK) supplemented with 10% fetal bovine serum (Invitrogen) and antibiotic-antimycotic solution (Invitrogen) in a 5% CO₂ incubator. Cultures were sub-cultured every 7 days by treatment with trypsin-EDTA solution (Sigma). For establishment of ALI, H292 cells were plated (2 x 10⁵ cells per well) on non-coated polyester Transwell membranes (12 mm diameter, 0.4 μm-pore size, 12-well plate, Transwell Clear; Corning Costar) and maintained in RPMI 1640 until confluent. Once confluent, media from the apical chamber of the Transwell was removed and only applied to the basolateral chamber to establish the ALI. Cells were maintained at ALI for 48 h before being used for experiments.

PBECs were plated into fibronectin/collagen-coated filter inserts of 12 mm diameter (Costar-Corning Transwell Clear, pore size: 0.4 µm). After reaching confluency, endothelial monolayers were supplied with 550 nM hydrocortisone, 250 μM CPT-cAMP and 17.5 μM RO-201724 and placed into the CellZscope instrument (nanoAnalytics, Münster, Germany). TEER was followed continuously. Treatments were applied after approx. 24 h, when TEER reached plateau. TEER was further monitored for 48 h.

Porous membrane supports (Transwell-Clear, 12 mm diameter, 0.4 μm pore size, Corning Incorporated, NY, USA) were coated with 10 μg/cm² collagen (Sigma-Aldrich, Munich, Germany). Caco-2 cells were seeded onto collagen-coated porous membrane supports at a density of 2 × 10⁵ cells/cm² and placed in a Transwell chamber. Medium was changed twice a week. Cells were grown for at least 3 weeks to build a confluent monolayer. Experiments were performed between day 21 and 28 after seeding.