CILIOGENESIS OF RAT TRACHEAL EPITHELIAL-CELLS IN-VITRO

To facilitate the studies of the factors and processes regulating the differentiation and function of ciliated cells of the airways, methods for culturing dissociated airway epithelial cells that promote de novo ciliogenesis in vitro have been developed for hamster, rat, dog, guinea pig, and human respiratory cells. Studies have demonstrated the importance of a preformed extracellular matrix and/or culture on permeable supports at an air-liquid interface for the development of ciliated cells. This chapter describes the procedures used in developing an in vitro system that supports proliferation and then mucociliary differentiation of dissociated primary rat tracheal epithelial (RTE) cells. Under isolation and culture of cells, the chapter discusses solutions and materials and procedures. Cannulas, used for the isolation of RTE cells, are made from 5-cm pieces of polyethylene tubing. One end of the tubing is pressed against a heated scalpel blade to form a slight flange. These are stored in 70% ethanol. There are details on the basic and complete mediums, bovine pituitary extract and tissue culture insert. The procedure describes the isolation of rat tracheal epithelial cells, plating and growth of cells, differentiation of rat tracheal epithelial cells, and quantitation of ciliated cell differentiation. Ciliated cells are visible in the viable cultures by phase microscopy. To estimate the extent of ciliated cell differentiation in large numbers of cultures, an immunostaining procedure was developed using a monoclonal antibody, RTE-3, which reacts specifically with ciliated cells. RTE-3 can also be used to identify ciliated cells in cytospins prepared from dissociated cultures. If some of the growth-stimulating compounds are removed from the medium after the cells have reached confluence (day 7), the number of ciliated cells present at day14 can be two- to fourfold higher than that obtained when the cells are cultured with complete medium. © 1995, Academic Press Inc.