Summary
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 aminotransferase, 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 × 10−5 M) and insulin (10 µg/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.
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References
Berry MN, Friend DS (1969) High-yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. J Cell Biol 43:506–520
Bissell DM, Hammaker LE, Meyer UA (1973) Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J Cell Biol 59:722–734
Bonney RJ, Becker JE, Walker PR, Potter VR (1974) Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis. In Vitro 9:399–413
Chapman GS, Jones AL, Meyer UA, Bissell DM (1973) Parenchymal cells from adult rat liver in nonproliferating monolayer culture. II. Ultrastructural studies. J Cell Biol 59:735–747
Chen PS Jr, Toribara TY, Warner H (1956) Microdetermination of phosphorus. Anal Chem 28:1756–1758
Granner DK, Tomkins GM (1970) Tyrosine aminotransferase (rat liver). Methods Enzymol 17A:633–637
Grisham JW, Thal SB, Nagel A (1975) Cellular derivation of continuously cultured epithelial cells from normal rat liver. In: Gerschenson LE, Thompson EB (eds) Gene expression and carcinogenesis in cultured liver. Academic Press, New York San Francisco London, pp 1–23
Howard RB, Christensen AK, Gibbs FA, Pesch LA (1967) The enzymatic preparation of isolated intact parenchymal cells from rat liver. J Cell Biol 35:675–684
Jacob ST, Bhargava PM (1962) A new method for the preparation of liver cell suspensions. Exp Cell Res 27:453–467
Koide H, Oda T (1959) Pathological occurrence of glucose-6-phosphatase in serum in liver diseases. Clin Chim Acta 4:554–561
Laishes BA, Williams GM (1976) Conditions affecting primary cell cultures of functional adult rat hepatocytes. I. The effect of insulin. In Vitro 12:521–532
Laishes BA, Williams GM (1976) Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12:821–832
Lowry OH, Rosebrough NJ, Farr AL (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193:265–275
Masuji H, Nakabayashi H, Sato J (1974) Long-term cultivation of two diploid epithelial cell lines derived from normal rat liver cells. Acta Med Okayama 28:281–293
Miyazaki M (1977) Primary culture of adult rat liver cells. I. Preparation of isolated cells from trypsin-perfused liver of adult rat. Acta Med Okayama 31:351–360
Miyazaki M (1978) Primary culture of adult rat liver cells. II. Cytological and biochemical properties of primary cultured cells. Acta Med Okayama 32:11–22
Miyazaki M, Wahid S, Matsuura K, Tsunashima M, Nakabayashi H, Miyano K, Sato J (1982) Effect of 3′-methyl-4-dimethylaminoazobenzene on liver cells from adult rat in primary culture. Res Exp Med (Berl) 181:27–38
Miyazaki M, Wahid S, Miyano K, Sato J (1982) Selective growth of epithelial-like clear cells from adult rat liver by short-term exposure to glucocorticoids in primary culture. Res Exp Med (Berl) 181:189–196
Miyazaki M, Watanabe A, Wahid S, Tsunashima M, Sato J (1979) Preparation of anti-rat albumin antiserum and its application to radioimmunoassay. Acta Med Okayama 33:409–413
Miyazaki M, Watanabe A, Shiraishi M, Hoshika T, Miyano K, Sato J (1978) Primary culture of adult rat liver cells. III. Hormonal effects on cytological and biochemical properties of primary cultured cells. Acta Med Okayama 32:85–96
Nakabayashi H (1981) Spontaneous neoplastic transformation in vitro of a diploid clone of rat liver cells under conditions of large inoculum size of cells at the transfer. Gann 72:377–387
Ontko JA (1967) Chylomicron, free fatty acid and ketone body metabolism of isolated liver cells and liver homogenates. Biochim Biophys Acta 137:13–22
Plageman PGW, Swim HE (1966) Propagation of lactic dehydrogenase-elevating virus in cell culture. Proc Soc Exp Biol Med 121:1147–1152
Rappaport C, Howze GB (1966) Dissociation of adult mouse liver by sodium tetraphenylboron, a potassium complexing agent. Proc Soc Exp Biol Med 121:1010–1016
Schneider WC, Potter VR (1943) Assay of animal tissues for respiratory enzymes; succinic dehydrogenase and cytochrome oxidase. J Biol Chem 149:217–227
Tanaka K, Sato M, Tomita Y, Ichihara A (1978) Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis. J Biochem (Tokyo) 84:937–946
Tokiwa T, Nakabayashi H, Miyazaki M, Sato J (1979) Isolation and characterization of diploid clones from adult and newborn rat liver cell lines. In Vitro 15:393–400
Tokiwa T, Sato J (1982) Effect of 3′-methyl-4-dimethylaminoazobenzene in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations in a diploid clone derived from normal rat liver cells in culture. In Vitro 18:501–509
Tsutamune T (1975) Establishment of five cell lines from normal adult rat livers in vitro. Acta Med Okayama 29:137–145
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Miyazaki, M., Tsunashima, M., Wahid, S. et al. Comparison of cytologic and biochemical properties between liver cells isolated from adult rats by trypsin perfusion and those isolated by collagenase perfusion. Res. Exp. Med. 184, 191–204 (1984). https://doi.org/10.1007/BF01852393
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DOI: https://doi.org/10.1007/BF01852393