Purification of Cells from Livers of Carcinogen-treated Rats by Free-Flow Electrophoresis

The tumorigenic roles of specific types of cells that emerge during chemical hepatocarcinogenesis in rats could potentially be determined if the specific types of cells could be purified adequately. Type II cells, defined (J. M. Jacobs, T. P. Pretlow, N. Fausto, A. M. Pitts, and T. G. Pretlow, J. Natl. Cancer Inst., 66: 967–973, 1981) as small, slowly sedimenting cells with histochemically demonstrable γ-glutamyl transpeptidase, have oval nuclei similar in size to those of lymphocytes. Adult male F344 rats were fed a choline-deficient diet containing 0.05% ethionine for 4 weeks. Liver cells were obtained in suspension by in situ perfusion of collagenase. A two-step process, i.e. , sedimentation in an isokinetic gradient followed by free-flow electrophoresis, was used to purify type II cells. We obtained preparations of cells with 32.3 ± 5.0% (S.D.) type II cells, 13.4 ± 2.0% erythrocytes, 52.9 ± 3.3% small nucleated cells, and only 1.4 ± 0.3% hepatocytes. The small nucleated cells were Kupffer cells and cells smaller than Kupffer cells that included many lymphocytes and unidentified cells similar in size to lymphocytes. Because both the electrophoretic mobilities and the rates of sedimentation of type II cells and hepatocytes are different, there is some advantage to be obtained from the sequential use of these techniques that exploit independent differences in the physical properties of these cells.