METHODS Isolation and culture of HTGM and CFTGM www.selleckchem.com/products/Vandetanib.html cells. The Committee on Human Research at the University of California San Francisco approved the use of human tissues for these studies. Nondiseased human bronchial tissues used for immunohistochemistry were obtained from excess donor tissue following lung transplantation surgery. For cell culture, we obtained tracheas and mainstem bronchi from non-CF and CF patients from autopsies performed within 24 h after death. All non-CF postmortem tissues were from individuals without significant pulmonary disease. Strips of epithelium were removed from the trachea and bronchi, and the gland-rich submucosal tissues were then dissected from between the cartilaginous rings. From these pieces of submucosal tissue, we isolated small segments of gland tubules and acinar structures by enzymatic digestion as described previously (60).

These gland fragments were plated into T25 flasks in 1:1 mixture of DMEM and Ham’s F-12 medium (DMEM/F12) supplemented with 20% FBS, penicillin (105 U/l), streptomycin (100 mg/l), gentamicin (100 mg/l) and amphotericin B (2.5 mg/l). During the first hours of culture, the gland fragments attached. After 8�C24 h, cultures were rinsed with PBS and plating medium was replaced with bronchial epithelial growth medium (BEGM; Lonza, Basel, Switzerland). Subsequently, medium was changed every 24 h for 3 days and every 2 days thereafter. Cultures derived from CF patients received additional PBS rinses prior to media changes to prevent microbial contamination. In BEGM, there was robust growth of cells from the attached gland fragments.

After the cultures became ~80% confluent, cells were removed by trypsinization (0.05% trypsin, 0.02% EDTA) and plated (3 �� 105 cells) onto 12-mm cell culture inserts (Transwell polycarbonate membranes, 0.4-��m pore diameter; Corning, Corning, NY) coated with human placental collagen (11). Plating Anacetrapib medium used for the isolated cells was the same as for acini but lacked penicillin, streptomycin, and amphotericin B and contained less gentamicin (50 mg/l). After 12�C18 h, cultures were rinsed with PBS and ��HTGM medium�� was added to the outside of the insert (basolateral side of the cells). HTGM medium was composed of DMEM/F12 supplemented with insulin (10 ��g/ml), transferrin (5 ��g/ml), retinoic acid (10?6 M), hydrocortisone (0.5 ��g/ml) triidothyronine (20 ng/ml), epidermal growth factor (25 ng/ml), bovine serum albumin (2 mg/ml), 0.1% Ultroser G serum substitute (Pall, Port Washington, NY). All cultures were maintained in a humidified incubator (5% CO2, 37��C). Confluency of cells on the inserts was revealed when medium ceased to leak through to the inside of the insert, and the mucosal surface of the cells appeared dry.