Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured "on top" and "inside" different extracellular matrix substitutes

Abstract

An essentially pure population of bile ductular epithelial cells isolated from bile duct-ligated rats was placed in primary culture by plating the cells either "on top" of or "inside" different extracellular matrix substitutes, including basement membrane Matrigel, type I collagen gel, and agarose gel. Plating efficiencies of greater than 60% were obtained when the cells were seeded in the presence of 1.0% fetal calf serum on top of Matrigel and collagen gel, but there was very little if any cell attachment to the agarose surface. In contrast, the cells could be maintained equally well and at very similar densities when they were cultured inside the various gel substances, including agarose. Regardless of substratum condition, bile ductular cells at 10 days in primary culture expressed specific activities of the marker enzymes gamma-glutamyl transpeptidase and leucine aminopeptidase which were significantly higher than those shown by freshly isolated cells. On the other hand, alkaline phosphatase activity of the cells became undetectable by day 3 of culture when they were cultured on top of either Matrigel or collagen gel but was retained at approximately 50% of its original level in cells cultured for 10 days within Matrigel or agarose gel. Treatments with dexamethasone or hydrocortisone (i.e., 10(-6) M) inhibited the increase in gamma-glutamyl transpeptidase activity of the cultured cells but did not affect the other enzyme changes. Subcultures of the bile ductular epithelial cells were developed by passing the cells in the presence of 10% fetal calf serum on surfaces coated with either type I collagen or Matrigel. In either case, cells subjected to at least 4-6 passages (up to 100 days of culture) were still characterized by a high gamma-glutamyl transpeptidase activity. Preliminary results obtained with cells plated at very low density within Matrigel also indicated the development of cell growths that appeared to be organized in the form of distinct acinar-like structures.