Primary cultures of liver cells isolated from adult rats by trypsin and collagenase perfusion techniques were carried out to compare cytologic and biochemical properties between the differently prepared cells. Trypsin-dispersed cells consisted of comparatively smaller cells, whereas collagenase-dispersed cells consisted of larger cells. The cell attachment efficiency on culture day 1 was about twice as high in the liver cells prepared with collagenase than those prepared with trypsin. Mature hepatocytes isolated by collagenase perfusion could be maintained in the primary culture for a longer period than those isolated by trypsin perfusion. Epithelial-like clear cells started to grow much earlier in the primary culture of the trypsin-dispersed liver cells than in that of the collagenase-dispersed liver cells. Earlier proliferation of epithelial-like clear cells could not be induced by in vitro trypsinization of the collagenase-dispersed liver cells. Both kinds of enzymatically prepared liver cells showed albumin production and exhibited glucose 6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9, G6Pase) and tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate amino-transferase, EC 2.6.1.5, TAT) activities for 1 week in the primary culture. Albumin production was higher in the liver cells prepared with collagenase than those prepared with trypsin, whereas G6Pase activity was almost the same between them. TAT activity up to culture day 2 was about 3-fold higher in the liver cells prepared with collagenase than in those prepared with trypsin. Combined supplementation of dexamethasone (1 X 10(-5)M) and insulin (10 micrograms/ml) consistently improved the cell attachment efficiency and was very effective in the maintenance of mature hepatocytes in both types. Furthermore, these hormones enhanced the albumin production and TAT activity in both types.
The expression of gamma-glutamyltranspeptidase (gamma-GTP) and the relationship between this enzyme activity and tumorigenicity were studied in 38 tumorigenic and non-tumorigenic epithelial cell lines derived from livers of normal and aminoazo-dye-fed rats. Nine of 22 non-tumorigenic epithelial cell lines exhibited gamma-GTP-positive cells, whereas 9 of 16 tumorigenic epithelial cell lines did not exhibit gamma-GTP-positive cells. This finding revealed that no correlation existed between the acquisition of gamma-GTP activity and tumorigenicity. None of 10 spontaneously transformed epithelial cell lines, which consisted of 8 tumorigenic and 2 non-tumorigenic cell lines, exhibited gamma-GTP-positive cells. On the other hand, 16 of 28 transformed epithelial cell lines, which were derived from aminoazo-dye-fed rat livers and aminoazo-dye-treated cultures of normal rat liver cells, included gamma-GTP-positive cells in various percentages. As described above, in contrast to in vitro spontaneous transformation of rat liver cells, it is obvious that the carcinogen-induced transformation of rat liver cells in vitro as well as in vivo was frequently accompanied by the acquisition of gamma-GTP-activity. Therefore, it is concluded that the expression of gamma-GTP in liver cells may be mainly associated with exposure to chemical carcinogens.
Primary mass cultures of isolated liver cells, which prepared from normal adult rat by a collagenase-liver-perfusion technique, were treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) to study the production of transformed liver cells. Enzymatically isolated liver cells had high sensitivity to 3'-Me-DAB in primary culture. By 2- or 6-day treatment, mature hepatocytes remarkably decreased in their numbers due to cytotoxic effect of 3-Me-DAB, but thereafter active proliferation of epithelial-like clear cells was observed. Six-day treatment induced epithelial-like clear cells with gross chromosomal abnormalities, although 2-day treatment failed to induce transformed cells. However, the transformed epithelial-like clear cells with chromosomal abnormalities were negative for gamma-glutamyl-transpeptidase (GGT).
Summary. Isolated liver cells, which were prepared from adult rats by a trypsin-liver-perfusion technique, were treated with dexamethasone or hydrocortisone at a concentration of 7.7 × 10 -6 M for 8 days in primary culture. The treated cultures displayed homogeneous population consisting of epithelial-like clear cells, while the untreated cultures displayed mixed population consisting of epithelial-like clear cells and fibroblast-like cells. The epithelial-like clear cells, which proliferated in the cultures treated with glucocorticoids for 8 days in primary culture, did not show any morphological changes following cultivation in glucocorticoid-free medium. After continuous glucocorticoid-treatment for more than 1 month, the treated cultures showed relatively low cell densities at confluence. The surface area of individual epithelial-like clear cells in the cultures treated with glucocorticoids for long periods of time was evidently greater than that in the cultures treated for only 8 days. The epithelial-like clear cells had glucose 6-phosphatase and tyrosine aminotransferase activities even though the levels of these enzymeactivities were very low compared with those in rat liver homogenates.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.