Evaluating E-Cig Exposure on NHBE Cells

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.