Isolation and maintenance of monolayer hepatocytes from the livers of toxin-treated rats

Hepatocytes were isolated from male Sprague-Dawley rats 30 min after oral challenge with carbon tetrachloride (CCl4), or 1 hr after ip injection of D(+)-galactosamine (GAL). Cell preparations of comparable yield and initial viability were obtained from toxin-treated and respective control animals with equivalent 1-hr attachment efficiencies to tissue culture plastic. Cells from toxin-treated rats exhibited significant degeneration over 48 hr in culture. This degeneration included loss of viable cell density on the monolayer and increased lactate dehydrogenase activity in the culture media compared to controls. Toxicity expressed in culture was dose dependent for both CCl4 (0.1-2.5 ml/kg) and GAL (100-400 mg/kg). Loss of cell viability in vitro and ultrastructural degeneration followed a time course consistent with hepatocellular necrosis produced by these agents in vivo. As a model for studying late occurring events in the progression from initiation of toxic cell injury to cell death, this methodology offers several potential advantages over strict in vivo or in vitro models. The analytical advantages of an in vitro cell model are incorporated with initiation of injury in vivo by physiologically relevant doses of hepatotoxins. Limited bioactivating potential of monolayer hepatocytes, and time course limitations of suspension hepatocytes for toxicity studies, are also circumvented in this model.