Growth and differentiation of hamster tracheal epithelial cells in culture

Abstract

The purpose of these studies was to define culture conditions that support growth and differentiation of normal epithelial cells obtained from hamster tracheas. Epithelial cells from tracheas of adult hamsters were collected using enzymatic procedures and cultured under various conditions. The medium used consisted of a 1:1 mixture of medium 199 and Dulbecco's modified Eagle's medium with 2% fetal bovine serum, which was conditioned by mouse 3T3 cells before use. Insulin, transferrin, hydrocortisone, epidermal growth factor, and an extract from bovine hypothalamus were used as supplements. When seeded on uncoated or collagen-coated tissue culture dishes, the hamster cells grew only poorly. When the cells were seeded on collagen gels, however, rapid and prolonged growth ensued. The cultures had a population doubling time of 20 hr and a colony-forming efficiency of 7-10%, and they could be grown for up to three passages. Growth was dependent on the presence of transferrin, insulin, epidermal growth factor, and 3T3 conditioning factors in the medium. The latter could be omitted if the concentration of serum was increased. Less important for growth was the presence of hydrocortisone and bovine hypothalamus extract. In contrast to results with tracheal epithelial cells from adult rabbits, rats, and mice, differentiation into ciliated cells regularly occurred in cultures of cells derived from hamster tracheas. The appearance of ciliated cells in the cultures was dependent on the presence of collagen gel as a substratum and of 3T3 conditioning factors in the medium. In addition, there were numerous cells that contained electron-dense cytoplasmic granules. The granules were not stained by dialyzed iron, which stains acidic glycoproteins, but were stained positively by periodic acid-Schiff reagents and the periodic acid-thiocarbohydrazide-silver proteinate method, suggesting the presence of secretory granules containing neutral glycoproteins. A similar staining pattern was observed for the secretory granules of intact hamster tracheas. The culture system described supports growth and cellular differentiation of normal tracheal epithelial cells of hamsters. We believe therefore that it will be a useful model for studying the regulation of tracheal cell function on the cellular and biochemical level.