Materials and Methods

Isolation and Culture of Airway Epithelial Explants Inferior nasal tissues were obtained from six patients undergoing elective excision of turbinates for nasal obstruction. Human tracheobronchial airways (distal trachea and first to third generation bronchi) were dissected from tissues removed from five patients (three with CF, one with bronchiolitis, and one with primary pulmonary hypertension) undergoing lung transplantation at the University of North Carolina and at 1 h postmortem from one patient with no apparent lung disease. Although we are interested in investigating the effects of several 5'-nucleotides on goblet cell function in CF, the availability of such tissues is limiting. Consequently, we chose to emphasize studies with UTP over ATP on CF tissues because of its potentially greater therapeutic value (see reference 27). All procedures were reviewed and approved by the University of North Carolina Committee for the Protection of Rights of Human Subjects. Human airway epithelial explants were isolated and cultured using minor modifications to the technique previously described for canine tracheal epithelium (11). Briefly, tissues were pinned out with the mucosal surface uppermost. A solution containing collagenase (100 Mandl U/ml; Boehringer-Mannheim, Indianapolis, IN) and dispase (1 U/ml, grade IT; Boehringer-Mannheim) in phosphate-buffered saline (PBS) was injected under the epithelium, and the tissue was bathed in Joklik's MEM containing: I roM CaClz, 1 roM dithiothreitol (Sigma Chemical Co., St. Louis, MO), 0.01% DNAse 1 (Sigma), and hexokinase (1 U/ml; Boehringer-Mannheim). After a 30-min incubation at 37 C in a 5% COz incubator, the epithelium was scraped from the tissue with a glass coverslip and suspended in bathing medium. The suspended sheet of epithelium was cut into 3-to 5-mm pieces, which were explanted, mucosal surface uppermost, onto a synthetic Vitrogen (Celtrix Pharmaceuticals, Santa Clara, CA)-coated nitrocellulose substrate and allowed to attach by incubation overnight at air: liquid interface in 5% COz at 37 C. The culture medium in contact with the basolateral surface of the substrata was hormone-supplemented Ham's F12 containing 3T3-conditioned medium, as described previously (II), with the addition of hexokinase (I U/ml; Boehringer-Mannheim). After overnight incubation, the explants were washed by immersion in culture medium and returned to airliquid interface culture. The explants were maintained in this configuration with daily changing of the medium until used, usually within 2 to 3 days.