Comparison of cytologic and biochemical properties between liver cells isolated from adult rats by trypsin perfusion and those isolated by collagenase perfusion

Abstract

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.

Enhancing effect of S-(1,2-dicarboxyethyl)glutathione on epidermal growth factor-stimulated DNA synthesis in primary cultures of adult rat hepatocytes

“…The attached cells were polygonal cells with granular cytoplasm (mature hepatocytes), which arranged themselves to form cords and clusters, as reported previously [9]. Even after change to the serum-free basal medium following the 1-h attachment period, the attached hepatocytes survived well.…”

Promotion of 3′‐methyl‐4‐dimethylaminoazobenzene‐initiated adult rat liver cells to malignant state by phenobarbital in culture

“…In the PB-treated cultures, ppm (Kitagawa et al, 1984). On the other hand, we have also observed that PB causes a dose-dependent increase in the number of marker-chromosome-positive and GGT-positive cultures in the 3'-Me-DABinitiated primary rat liver cell cultures within the dose range of 0.75 to 3 m~, and that the optimal concentration of PB is 1.5 m~ for such promotion (Miyazaki et al, 1985). In this study, therefore, the putative preneoplastic epithelial-type cell lines, which were derived from the 3 '-Me-DAB-initiated cultures of normal adult rat liver cells, were treated with PB at the optimal concentration of 1.5 m~.…”

Effect of various barbituric acid derivatives on survival of functional hepatocytes from adult rats in primary culture

“…Four-milliliter and 10-ml aliquots of the cell suspensions were inoculated into 60-and 100-ram Falcon plastic dishes, respectively, and cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C. To enhance the attachment efficiency of inoculated hepatocytes, dexamethasone and insulin were added to the cultures at 10 ~tM and 10 ~tg/ml, respectively, during only the first 24 h [21]. Following the 1-day attachment period, the culture medium was replaced by the drug-supplemented basal media, or the basal medium supplemented with or without 0.5% DMSO for control cultures.…”

“…However, after a few days in culture, the hepatocytes enter a period of degeneration and most of the measured liver functions begin to decline rapidly [1,2,21]. Such a rapid and consistent loss in cell function and viability makes it difficult to perform experiments in which cells are to be incubated for long periods of time.…”

“…Mainly two different approaches, namely selective supplementation of medium with various factors and use of appropriate substrata have been attempted. Supplementation of the media with hormones, such as dexamethasone and insulin, has greatly enhanced the attachment efficiency of hepatocytes and prolonged the hepatocyte survival to a certain extent [10,11,21,22]. On the other hand, several studies have focused on substratum requirements for hepatocyte attachment and survival in primary culture.…”