Advertisement

In Vitro

, Volume 12, Issue 7, pp 521–532 | Cite as

Conditions affecting primary cell cultures of functional adult rat hepatocytes

1. The effect of insulin
  • Brian A. Laishes
  • Gary M. Williams
Article

Summary

The conditions for obtaining representative, primary adult rat hepatocyte cultures were explored. The methods applied included enzymatic liver perfusion which was nondestructive to hepatocytes, the prevention of aggregation of dissociated cells and the selective attachment of viable cells. These procedures yielded a recovery of 50% of the liver cells which gave rise to cultures representing 14% of the total liver cells. The cultures were composed of homogeneous epithelial-like cells cytologically similar to hepatocytes and possessed a number of liver-specific enzymes. There was virtually no cell division initially and most cells died between 24 and 48 hr. Insulin enhanced the attachment of the liver cells, altered their morphology, but did not prolong cell survival.

Key words

hepatocyte insulin tissue culture perfusion liver 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Katsuta, H., and T. Takaoka. 1963. Carcinogenesis in tissue culture. II. Cultivation of normal rat liver cells. Japan J. Exp. Med. 33: 265–275.Google Scholar
  2. 2.
    Biberfeld, P., T. L. E. Ericsson, P. Perlman, and M. Z. Raftell. 1966. Ultrastructural features of in vitro propagated rat liver cells. Z. Zellforsch. 71: 153–168.PubMedCrossRefGoogle Scholar
  3. 3.
    Gerschenson, L. E., and D. Casanello. 1968. Metabolism of rat liver cells cultured in suspension: insulin and glucagon effects on glycogen level. Biochem. Biophys Res. Comm. 33: 584–589.PubMedCrossRefGoogle Scholar
  4. 4.
    Coon, H. 1969. Clonal culture of differentiated cells from mammals: rat liver cell culture. Carnegie Inst. Washington Year Book 67: 419–421.Google Scholar
  5. 5.
    Williams, G. M., E. K. Weisberger, and J. H. Weisburger. 1971. Isolation and long-term cell culture of epithelial-like cells from rat liver. Exp. Cell Res. 69: 106–112.PubMedCrossRefGoogle Scholar
  6. 6.
    Rutzky, L. P., W. G. Taylor, and R. W. Pumper. 1971. In vitro stimulation of rat liver cells by homologous partial hepatectomy serum. In Vitro 7: 1–5.PubMedCrossRefGoogle Scholar
  7. 7.
    Leffert, H. L., and D. Paul. 1972. Studies of primary cultures of differentiated fetal liver cell. J. Cell Biol. 52: 559–568.PubMedCrossRefGoogle Scholar
  8. 8.
    Chessebeuf, M., A. Olsson, P. Bournot, J. Desgres, M. Guiguet, G. Maume, B. F. Maume, B. Perissel, and P. Padieu. 1974. Long term cell culture of rat liver epithelial cells retaining some hepatic functions. Biochimie 56: 1365–1379.PubMedCrossRefGoogle Scholar
  9. 9.
    Bausher, J., and W. I. Schaeffer. 1974. A diploid rat liver cell culture. 1. Characterization and sensitivity to aflatoxin B. In Vitro 9: 286–293.CrossRefGoogle Scholar
  10. 10.
    Borek, C., S. Higashino, and W. R. Lowenstein. 1969. Intercellular communication and tissue growth. IV. Conduction of membrane junctions of normal and cancerous cells in culture. J. Membr. Biol. 1: 274–293.CrossRefGoogle Scholar
  11. 11.
    Gerschenson, L. E., M. Andersson, J. Molson, and T. Okigaki. 1970. Tyrosine transaminase induction by dexamethasone in a new rat liver cell line. Science 170: 859–861.PubMedCrossRefGoogle Scholar
  12. 12.
    Iype, P. T. 1971. Cultures from adult rat liver cells. I. Establishment of monolayer cell-cultures from normal liver. J. Cell. Physiol. 78: 281–288.PubMedCrossRefGoogle Scholar
  13. 13.
    Diamond, L., R. McFall, Y. Tashiro, and D. Sabatini. 1973. The WIRL-3 rat liver cell lines and their transformed derivatives. Cancer Res. 33: 2627–2636.PubMedGoogle Scholar
  14. 14.
    Williams, G. M., and J. M. Gunn. 1974. Long-term cell culture of adult rat liver epithelial cells. Exp. Cell Res. 89: 139–142.PubMedCrossRefGoogle Scholar
  15. 15.
    Waymouth, C., H. W. Chen, and B. G. Wood. 1971. Characteristics of mouse liver pharenchymal cells in chemically defined media. In Vitro 6: 371.CrossRefGoogle Scholar
  16. 16.
    Kaighn, M. E., and A. M. Prince. 1971. Production of albumin and other serum proteins by clonal cultures of normal human liver. Proc. Natl. Acad. Sci. U.S.A. 68: 2396–2400.PubMedCrossRefGoogle Scholar
  17. 17.
    Harkness, R. D. 1957. Regeneration of liver. Br. Med. Bull. 13: 87–93.PubMedGoogle Scholar
  18. 18.
    Daoust, R. 1958. The cell population of liver tissue and the cytological references bases. In: R. W. Brauer (Ed.),Liver Function. Publ. No. 4. Am. Inst. Biol. Sci., Washington, D.C., pp. 3–10.Google Scholar
  19. 19.
    Alwen, J., and J. J. Gallhai-Atchard. 1972. A method of maintaining parenchymal cells from adult rat liver in vitro. J. Cell Sci. 11: 249–256.PubMedGoogle Scholar
  20. 20.
    Bissell, D. M., L. Hammaker, and U. A. Meyer. 1973. Parenchymal cells from adult rat liver in nonproliferating monolayer culture. J. Cell Biol. 59: 722–734.PubMedCrossRefGoogle Scholar
  21. 21.
    Bonney, R. J., J. E. Becker, P. R. Walker, and V. R. Potter. 1974. Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis. In Vitro 9: 399–413.CrossRefGoogle Scholar
  22. 22.
    Bonney, R. J. 1974. Adult liver parenchymal cells in primary culture: characteristics and cell recognition standards. In Vitro 10: 130–142.PubMedCrossRefGoogle Scholar
  23. 23.
    Puvion, E., J. Garrido, and A. Viron. 1974. Biologie cellulaire. Technique rapide d’isolement et de survie en monocouches, d’hepatocytes de rat adulte. C. R. Acad. Sci. [D] (Paris) 279: 509–512.Google Scholar
  24. 24.
    Phillips, M. J., M. Oda, V. D. Edwards, G. R. Greenberg, and K. N. Jeejeebhoy. 1974. Ultrastructural and functional studies of cultured hepatocytes. Lab. Invest. 31: 533–542.PubMedGoogle Scholar
  25. 25.
    Seglen, P. O. 1972. Preparation of rat liver cells. I. Effect of CA2+ on enzymatic dispersion of isolated perfused liver. Exp. Cell Res. 74: 450–454.PubMedCrossRefGoogle Scholar
  26. 26.
    Seglen, P. O. 1973. Preparation of rat liver cells. II. Effects of ions and chelators on tissue dispersion. Exp. Cell Res. 76: 25–30.PubMedCrossRefGoogle Scholar
  27. 27.
    Seglen, P. O. 1973. Preparation of rat liver cells. III. Enzymatic requirements for tissue dispersion. Exp. Cell Res. 82: 391–398.PubMedCrossRefGoogle Scholar
  28. 28.
    Ballard, F. J., and R. W. Hanson. 1969. Purification of phosphoenolpyruvate carboxykinase from the cytosol fraction of rat liver and the immunochemical demonstration of differences between this enzyme and the mitochondrial phosphoenolpyruvate carboxykinase. J. Biol. Chem. 244: 5625–5630.PubMedGoogle Scholar
  29. 29.
    Sapico, V., L. Shear, and G. Litwack. 1974. Translocation of inducible tyrosine aminotransferase to the mitochondrial fraction. Facilitation by acute uremia and other conditions. J. Biol. Chem. 249: 2122–2129.PubMedGoogle Scholar
  30. 30.
    Gunn, J. M., H. Shinozuka, and G. M. Williams. 1976. Enhancement of phenotypic expression in cultured malignant liver epithelial cells by a complex medium. J. Cell. Physiol., 87: 79–88.PubMedCrossRefGoogle Scholar
  31. 31.
    Adelman, R. C., P. D. Spolter, and S. Weinhouse. 1966. Dietary and hormonal regulation of enzymes of fructose metabolism in rat liver. J. Biol. Chem. 241: 5467–5472.PubMedGoogle Scholar
  32. 32.
    Farina, F. A., J. B. Shatton, H. P. Morris, and S. Weinhouse. 1974. Isozymes of pyruvate kinase in liver and hepatomas of the rat. Cancer Res. 34: 1439–1446.PubMedGoogle Scholar
  33. 33.
    Stich, H. F., and R. H. C. San. 1970. DNA repair and chromatid anomalies in mammalian cells exposed to 4-nitroquinoline-1-oxide. Mutat. Res. 10: 389–404.PubMedGoogle Scholar
  34. 34.
    Alwen, J., and A. M. Lawn. 1974. The reaggregation of adult rat liver cells maintained in vitro. Exp. Cell Res. 89: 197–205.PubMedCrossRefGoogle Scholar
  35. 35.
    Williams, G. M., J. M. Elliott, and J. H. Weisburger. 1973. Carcinoma after malignant conversion in vitro of epithelial-like cells from rat liver following exposure to chemical carcinogens. Cancer Res. 33: 606–612.PubMedGoogle Scholar
  36. 36.
    Williams, G. M. 1975. The study of chemical carcinogenesis using cultured rat liver cells. In: L. E. Gerschenson, and E. B. Thompson (Eds.),Gene Expression and Carcinogenesis in Cultured Liver. Academic Press, New York, pp. 480–487.Google Scholar
  37. 37.
    Walker, P. R., R. J. Bonney, J. E. Becker, and V. R. Potter. 1972. Pyruvate kinase, hexokinase, and adolase isozymes in rat liver cells in culture. In Vitro 8: 107–114.PubMedCrossRefGoogle Scholar
  38. 38.
    Williams, G. M., K. Stromberg, and R. Kroes. 1973. Cytochemical and ultrastructural alterations associated with confluent growth in cell cultures of epithelial-like cells from rat liver. Lab. Invest. 29: 293–303.PubMedGoogle Scholar
  39. 39.
    Berry, M. N., and D. S. Friend. 1969. High-yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43: 506–520.PubMedCrossRefGoogle Scholar
  40. 40.
    Crisp, D. M., and C. I. Pogson. 1972. Glycolytic and gluconeogenic enzyme activities in parenchymal and non-parenchymal cells from mouse liver. Biochem. J. 126: 1009–1023.PubMedGoogle Scholar
  41. 41.
    Webster, S. H., E. J. Liljegren, and D. J. Zimmer. 1947. Organ: body weight ratios for liver, kidneys and spleen of laboratory animals. Am. J. Anat. 81: 477–513.CrossRefGoogle Scholar
  42. 42.
    Ingebretsen, W. R. Jr., and S. R. Wagle. 1972. A rapid method for the isolation of large quantities of rat liver parenchymal cells with high anabolic rates. Biochem. Biophys. Res. Comm. 47: 403–410.PubMedCrossRefGoogle Scholar
  43. 43.
    Howard, R. B., and L. A. Pesch. 1968. Respiratory activity of intact, isolated parenchymal cells from rat liver. J. Biol. Chem. 243: 3105–3109.PubMedGoogle Scholar
  44. 44.
    Allard, C., R. Mathieu, G. DeLamirande, and A. Cantero. 1952. Mitochondrial population in mammalian cells. I. Description of a counting technic and preliminary results on rat liver in different physiological and pathological conditions. Cancer Res. 12: 407–412.PubMedGoogle Scholar
  45. 45.
    Laird, A. K., and A. D. Barton. 1961. Cell proliferation in precancerous liver: relation to presence and dose of carcinogen. J. Natl. Cancer Inst. 27: 827–839.PubMedGoogle Scholar
  46. 46.
    Berry, M. N. 1974. High-yielded preparation of morphologically intact isolated parenchymal cells from rat liver. In: S. Fleischer, and L. Packer (Eds.),Methods in Enzymology Volume XXXII. Academic Press, New York, pp. 625–632.Google Scholar
  47. 47.
    MacDonald, R. A. 1961. Lifespan of liver cells. Arch. Intern. Med. 107: 335–343.PubMedGoogle Scholar
  48. 48.
    Wagle, S. R., W. R. Ingebretsen, Jr., and L. Sampson. 1973. Studies on the in Vitro effects of insulin on glycogen synthesis and ultrastructure in isolated rat liver hepatocytes. Biochem. Biophys. Res. Comm. 53: 937–943.PubMedCrossRefGoogle Scholar

Copyright information

© Society for In vitro Biology 1976

Authors and Affiliations

  • Brian A. Laishes
    • 1
  • Gary M. Williams
    • 1
  1. 1.Fels Research Institute and Department of PathologyTemple University School of MedicinePhiladelphia

Personalised recommendations