Purification and transplantation of hepatocytes from livers of carcinogen-treated rats

Young adult male F344 rats were given 90 ppm diethylnitrosamine in drinking water for 5 weeks. Cells were obtained in suspension from the livers of these animals by in situ perfusion of collagenase. Cells were separated in a 2.7-16% (wt/wt) continuous gradient of Ficoll in tissue culture medium in the Sorvall TZ-28 reorienting zonal rotor for 10 minutes at 4 degrees C with a centrifugal force of 12 X g at the sample-gradient interface (26 X g at the gradient-cushion interface). Up to half a billion liver cells were separated without exceeding the band capacity. In all experiments the purest fractions contained more than 90% hepatocytes. After centrifugation, 72.4 +/- 6.6% of the purified hepatocytes had histochemically demonstrable gamma-glutamyl transpeptidase (GGT). When purified hepatocytes were injected into the mesenteric veins of rats given a diet containing 0.02% (wt/wt) N-2-fluorenylacetamide for 7 days and subjected to partial hepatectomy, all rats that received these cells developed foci that exhibited histochemically demonstrable GGT. Hepatocytes with histochemically demonstrable GGT make up all or part of what previously has been referred to as "liver colony-forming units." With the TZ-28 reorienting zonal rotor, these cells can be purified in biochemically preparative quantities.