Propagation of differentiating normal human tracheobronchial epithelial cells in serum-free medium

Serial-passage cultures of normal human tracheobronchial (TB) epithelial cells that exhibit functional differentiation have been established in serum-free medium supplemented with bovine pituitary extract (25 micrograms/ml), insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), EGF (5 ng/ml), 10(-6)M each of ethanolamine and phosphoethanolamine, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 80 hours. Further, the passaged cultures retained differentiated morphology as evidenced by secretion of glycoproteins, binding of concanavalin A lectin, and presence of alcian blue and periodic acid Schiff-positive material in their cytoplasm. Ultrastructural observations further supported the functional epithelial nature of the cultures. Most cells exhibited characteristic microvilli on cell surfaces and showed junctional complexes between them. The cytoplasm contained a large number of perinuclear secretory vesicles, a characteristic feature of the differentiated cells. These cultures provide an excellent model to study factors that regulate synthesis and secretion of glycoproteins in normal human TB cells.