Simple technique for culture of highly differentiated cells from dog tracheal epithelium

Cultures of dog tracheal epithelium have proved very useful in studies of ion transport. Their short-circuit current (Isc), however, is usually much less than the original tissue. We have tested a variety of conditions in an attempt to produce large numbers of cells with electrical properties comparable with the original tissue. Of several growth supports, human placental collagen (HPC) gave the best results. When plated at 2.5 x 10(5) cells/cm2 onto HPC, cells grown in serum-free, growth factor-supplemented medium (GF medium) showed increases in cells per unit area, thickness of cell sheet, numbers of domes, numbers of apical microvilli, and degree of basolateral membrane interdigitation compared with cells grown in medium containing 5% fetal calf serum (FCS medium). Transepithelial resistance (Rte) and the increases in Isc and intracellular Ca in response to isoproterenol were also increased. However, baseline Isc and adenosine 3',5'-cyclic monophosphate levels were not changed. The improved electrical properties were maintained for up to 4 mo. GF medium combined with an air interface produced further increases in Rte, Isc, and changes in Isc in response to amiloride and isoproterenol. Ultrastructural features such as the presence of cilia, greater thickness of the cell sheet, and increased amplification of apical and basolateral membranes also indicated improved differentiation. Our results show that GF medium and an air interface can be combined with a simple growth support and a relatively low-plating density to allow the easy production of greater than 500 cm2 of cultured cells from a single trachea, with a level of differentiation similar to that of the original tissue.