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Liver parenchymal cells were isolated from adult rats by digesting liver slices or perfusing liver with collagenase. The cell yields were 1.5 X 10(7) and 1.0 X 10(8) cells/g liver from slices and perfused liver, respectively, and in both cases the cell viabilities and attachment efficiencies were over 90% and 60%, respectively. The cells were viable for more than one week when cultured in Williams medium E with 10% fetal bovine serum, and addition of insulin and dexamethasone enhanced the maintenance of cell viability. Various biochemical functions or freshly isolated cells and cultured cells were compared in this medium. In freshly isolated cells, induction of tyrosine transaminase [EC 2.6.1.5] by dexamethasone was low and none of the hormones examined stimulated protein synthesis; but when the cells had been cultured for a few days, induction of tyrosine transaminase became prominent, and insulin and dexamethasone stimulated protein synthesis and glucagon inhibited their effect. About half the synthesized proteins were secreted into the medium and among these proteins, albumin, transferrin, fibrinogen, and lipoproteins were identified immunochemically and electrophoretically. It was also shown that the polysomes in freshly isolated cells were almost completely disaggregated, but that in cells after a few days culture they were reaggregated. These results showed that freshly isolated cells have impaired functions, but that after culture for a few days the cells recover various liver functions and thus become more suitable for use in biochemical studies on liver functions.

In primary monolayer cultures of rat mature hepatocytes, many metabolic functions as well as cell growth are regulated by cell density. There are two types of regulatory response of these functions to change of cell density. Growth-related functions, such as DNA synthesis, induction of glucose-6-phosphate dehydrogenase, 2-aminoisebutyric acid transport, synthesis of cellular protein, and cholesterogenesis, are stimulated by low cell density. In contrast, functions related to hepatocyte-specific characters, such as the inductions of tyrosine aminotransferase, serine dehydratase, and malic enzyme and synthesis of triglycerides, are stimulated by high cell density. The reciprocal responses of these cellular activities to cell density were mimicked by addition of plasma membranes purified from adult rat liver to hepatocytes cultured at low cell density. The modulator activity was heat labile and trypsin sensitive. The activity was also found in plasma membranes from kidney, brain, and erythrocytes, although the specific activities of these preparations seemed to be different. These results suggest that the reciprocal regulations of cell growth and hepatocyte-specific functions are mediated by some surface components via cell-cell contact.Adult rat hepatocytes in primary culture retain in vivo levels of various liver functions and respond to various hormones (1)(2)(3)(4) Cell Isolation and Monolayer Culture. Parenchymal hepatocytes were isolated from male Wistar rats weighing 150-200 g by in-situ perfusion on the liver with collagenase, essentially as described by Seglen (11). The isolated cells were suspended at 5 X 10' cells per ml in Williams medium E containing 2% calf serum and 2 nM insulin. The cell density at inoculation was varied from 5 X 105 to 2.5 x 106 cells per 5-cm (diameter)Corning plastic culture dish. Over 80% of the cells became attached in 1 hr at 37C under 5% C02/30% 02 in air. After culture for 2 hr. the medium -was changed to hormone-free Williams medium E containing 5% calf serum. In experiments on the effect of addition of plasma membranes, rat hepatocytes were inoculated at a low density (7 x 105 cells per 5-cm dish). After 2 hr. the medium was changed to hormone-free medium containing 5% calf serum and then various amounts of plasma membranes were added to the cultures. Hormones were added to the medium after 22 hr of culture. Incubation was continued for 24 or 45 hr further and then various activities were assayed.Assay of DNA Synthesis. DNA synthesis was assayed by measuring incorporation of [3H]thymidine into DNA with or without hydroxyurea in 2 hr. Radioactivity in the hot trichloroacetic acid-soluble fraction was measured as described (6). Assays of Enzyme Activities. The cells were harvested with a rubber policeman in 0.2-1.0 ml of 20 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM dithiothreitol and 5 mM EDTA for the glucose-6-phosphate dehydrogenase (Glc-6-P-DHase) assay, in 0.1-0.5 ml of 5 mM Tris-HCl buffer (pH