Isolation and Characterization of Human Lung Fibroblasts

Surgical lung biopsy tissue was obtained from consented patients under protocols approved by the Institutional Review Board of Weill Cornell Medical College and included macroscopically normal surgical waste tissue specimens. Unidentified waste tissue specimens were used for human lung fibroblast cultures. Lung fibroblasts were isolated from human lung tissue specimens. The human lung tissue was digested with type I collagenase solution (Thermo Fisher, Waltham, MA, United States 1%) and then spun down. Cell pellets were resuspended in DMEM/F12 (Corning, Corning, NY, United States) with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, United States) and 1% penicillin/streptomycin/amphotericin (Corning, Corning, NY, United States) and the cells were plated into T75 tissue culture flasks. Before treatments cells were left quiescent for 24 h. Treatments included exposure to MC-EXO (40 μg total protein), TGF-β (Peprotech, Cranbury, NJ, United States 10 ng/ml) with or without the TGF-βR1 inhibitor SB525334 (Selleck Chemicals, Houston, TX, United States 10 μM). Cells were pretreated with the inhibitor or vehicle [phosphate-buffered saline (PBS) or ethanol] 15 min before treatment with TGF-β and MC-EXO.