Clonal proliferation of rat tracheal epithelial cells in serum-free medium and their responses to hormones, growth factors and carcinogens

Abstract

A serum- and feeder cell-free medium has been developed for the proliferation of rat tracheal epithelial (RTE) cells at clonal density. In this medium, RTE cells continue to proliferate for several weeks after cells in serum containing medium on feeder cells have begun to differentiate. The responsiveness of RTE cells to selected hormones and growth factors was determined using a clonal growth assay. The colony-forming efficiency (CFE) of RTE cells was reduced greater than 85% when bovine pituitary extract or bovine serum albumin were deleted from the medium and 45-70% reductions in CFE were observed when insulin, hydrocortisone, epidermal growth factor or cholera toxin were deleted. RTE cells also require high concentrations of Ca2+ (0.8 mM) for maximal clonal proliferation in this medium. The induction by carcinogens of preneoplastic RTE cell variants resistant to serum-mediated squamous differentiation was compared in serum-free medium and in serum-containing medium on feeder cells. N-methyl-N'-nitro-N-nitrosoguanidine was considerably more cytotoxic and effective as a transforming agent on an equivalent dose basis for RTE cells in serum-free medium. In contrast, (+/-)-7B,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene was equally cytotoxic and transforming under both culture conditions. This serum-free culture system for primary RTE cells will be useful in studies on the control of normal epithelial cell proliferation and differentiation by defined growth factors and in studies on the cellular changes involved in carcinogenesis.