Normal Human Tracheobronchial Epithelial Cells Research Articles

Resistance to serum-induced terminal differentiation in normal human tracheobronchial epithelial cells after in vivo exposure to 7,12-dimethylbenz[a]anthracene.

Normal human tracheobronchial epithelial cells (NHTBECs) from nine donors were used to repopulate de‐epithelialized rat tracheas. After transplantation into nude mice and treatment with 7,12‐dimethylbenz[a]anthracene (DMBA), the transplants were removed at 3, 4, 5 and 6 months. Epithelial cells from DMBA‐treated tracheas were subculturable. Epithelial cells from most untreated tracheas were not subculturable. After treatment with 0.5,1, 2, 4, 6 and 8% serum, cells exhibited increased subculturability after in vivo treatment with DMBA, did not terminally differentiate and were still proliferating even in medium containing 8% serum. Karyotypes from these cells showed considerable aneuploidy. Although these cells did not survive for more than 10 subcultures (42 weeks), this was considerably longer than the survival of control cells. Because of their longer survival, resistance to serum‐induced terminal differentiation and chromosome alterations, they were considered to be phenotypically altered or partially transformed cells produced by in vivo treatment of human cells with a chemical carcinogen.

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.