The restoration of the functions of serially passaged calf hepatocytes by spheroid formation

  • Fumiko Karikusa
  • Yoshio Sawasaki
Growth, Differentiation and Senescence


A serial cultivation system of hepatocytes was established for the first time using calf liver as a cell source and, repeating passage of more than 30 cumulative population doublings (PDs), was obtained in the presence of long-acting ascorbic acid derivative (L-ascorbic acid 2-phosphate) and epidermal growth factor. The complete purification of hepatocytes was achieved by repeating ethylenediaminetetraacetic acid (EDTA) treatment, by which hepatocytes were easily detached from the culture dish, leaving most of the nonparenchymal cells on the dish. As the population cumulatively doubled, the cell density and albumin-synthesizing ability decreased gradually, and doubling time has exceeded 120 h at about 30 cumulative PDs. In serially passaged cells, the hepatocyte-specific histochemical and biochemical markers—including glucose-6-phosphatase, ornithine carbamoyltransferase, glutamate hydrogenase, and ammonia-metabolizing activities—have been lost after 20 cumulative PDs. However, when these passaged cells were allowed to form spheroids, the morphologic and biochemical characteristics of hepatocytes have rapidly been restored to levels comparable to those in younger generations. Because no extrinsic factor was needed for this restoration, three-dimensional cell-cell interaction would be indispensable for the differentiation of the hepatocytes. The routine serial cultivation of hepatocytes and their redifferentiation by spheroid formation will be useful for studying metabolism, gene regulation, and transplantation of hepatocytes.

Key words

hepatocytes serial cultivation spheroid redifferentiation cell-cell interaction 


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  1. 1.
    Alpini, G.; Lenzi, R.; Zhai, W. R., et al. Isolation of a nonparenchymal liver cell fraction enriched in cells with biliary epithelial phenotypes. Gastroenterology 97:1248–1260;1989.PubMedGoogle Scholar
  2. 2.
    Berry, M. N.; Halls, H. J.; Grivell, M. B. Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions. Life Sci. 51:1–16;1992.PubMedCrossRefGoogle Scholar
  3. 3.
    Braun, L.; Mead, J. E.; Panzica, M., et al. Transforming growth mechanism of growth regulation. Proc. Natl. Acad. Sci. USA 85:1539–1543;1988.PubMedCrossRefGoogle Scholar
  4. 4.
    Fausto, N.; Mead, J. E. Regulation of liver growth: protooncogenes and transforming growth factors. Lab. Invest. 60:4–13;1989.PubMedGoogle Scholar
  5. 5.
    Folkman, J.; Haudenschild, C. C.; Zetter, B. R. Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. USA 76:5217–5221;1979.PubMedCrossRefGoogle Scholar
  6. 6.
    Gonchoroff, N. J.; Greipp, P. R.; Kyle, R. A., et al. Monoclonal antibody reactive with 5-bromo-2′-deoxyuridine that does not require DNA denaturation. Cytometry 6:506–512;1985.PubMedCrossRefGoogle Scholar
  7. 7.
    Gospodarowicz, D.; III, C. R. Do plasma and serum have different abilities to promote cell growth? Proc. Natl. Acad. Sci. USA 77:2726–2730;1989.CrossRefGoogle Scholar
  8. 8.
    Grisham, J. W. Cell types in long-germ propagable cultures of rat liver. Ann. NY Acad. Sci. 349:128–137;1989.CrossRefGoogle Scholar
  9. 9.
    Hata, R.; Ninomiya, Y.; Sano, J., et al. Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes). J. Cell. Physiol. 122:333–342;1985.PubMedCrossRefGoogle Scholar
  10. 10.
    Hata, R.; Senoo, H. L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of three-dimensional tissuelike substance by skin fibroblasts. Methods Cell Biol. 138:8–16;1989.Google Scholar
  11. 11.
    Hernandez, J.; Zarnegar, R.; Michalopoulos, G. K. Characterization of the effects of human placental HGF on rat hepatocytes. J. Cell. Physiol. 150:116–121;1992.PubMedCrossRefGoogle Scholar
  12. 12.
    Hoffman, B.; Piasecki, A.; Paul, D. Proliferation of fetal rat hepatocytes in response to growth factors and hormones in primary culture. J. Cell. Physiol. 139:654–662;1989.CrossRefGoogle Scholar
  13. 13.
    Hoshi, H.; Kan, M.; McKeehan, W. Direct analysis of growth factor requirements for isolated human fetal hepatocytes. In Vitro Cell. Dev. Biol. 23:723–732; 1987.PubMedCrossRefGoogle Scholar
  14. 14.
    Houck, K. A.; Zaneger, R.; Muga, S. J. Acidic fibroblast growth factor (HBGF-1) stimulates DNA synthesis in primary rat hepatocyte cultures. J. Cell. Physiol. 143:129–132; 1990.PubMedCrossRefGoogle Scholar
  15. 15.
    Koide, N.; Sakaguchi, K.; Koide, Y., et al. Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments. Exp. Cell Res. 186:227–235; 1990.PubMedCrossRefGoogle Scholar
  16. 16.
    Landry, J. D.; Bernier, C.; Ouellet, R., et al. spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J. Cell Biol. 101:914–923; 1985.PubMedCrossRefGoogle Scholar
  17. 17.
    Li, A. P.; Colburn, S. M.; Beck, D. J. A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids. In Vitro Cell. Dev. Biol. 28:673–677; 1992.CrossRefGoogle Scholar
  18. 18.
    Lyons, B. L.; Schwarz, R. I. Ascorbate stimulation of PAT cells causes an increase in transcription rates and decrease in degradation rates of procollagen mRNA. Nucleic Acids Res. 12:2569–2579; 1984.PubMedCrossRefGoogle Scholar
  19. 19.
    Michalopoulos, G.; Cianciulli, H. D.; Novotny, A. R., et al. Liver regeneration studies with rat hepatocytes in primary culture. Cancer Res. 42:4673–4682; 1982.PubMedGoogle Scholar
  20. 20.
    Mizutani, A. Cytochemical demonstration of ornithine carbamoyltransferase activity in liver mitochondria of rat and mouse. J. Histochem. Cytochem. 16:172–180; 1968.PubMedGoogle Scholar
  21. 21.
    Nakamura, T.; Nishizawa, T.; Hagiya, M., et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 342:440–443; 1989.PubMedCrossRefGoogle Scholar
  22. 22.
    Nomura, H.; Ishiguro, T.; Morimoto, S. Studies on L-ascorbic acid derivatives. III. Bis (L-ascorbic acid-3, 3′) phosphate and L-ascorbic acid 2-phosphate. Chem. & Pharm. Bull. (Tokyo) 17:387–393; 1969.Google Scholar
  23. 23.
    Oberhammer, F. A.; Pavelka, M.; Sharma, S., et al. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor β-1. Proc. Natl. Acad. Sci. USA 89:5408–5412; 1992.PubMedCrossRefGoogle Scholar
  24. 24.
    Okuda, H.; Fujii, S.; Kawashima, Y. A direct colorimetric determination of blood ammonia. Tokushima J. Exp. Med. 12:11–23; 1976.Google Scholar
  25. 25.
    Oshita, M.; Takeda, H.; Kamiyama, Y., et al. A direct method for the estimation of ornithine carbamoyltransferase activity in serum. Clin. Chim. Acta 67:145–152; 1976.CrossRefGoogle Scholar
  26. 26.
    Podah, H. Cellular functions of ascorbic acid. Biochem. Cell Biol. 68:1166–1173; 1990.CrossRefGoogle Scholar
  27. 27.
    Prockop, D. J.; Berg, R. A.; Kivirikko, K. I., et al. Intracellular steps in biosynthesis of collagen. In: Ramachandran, G. N.; Reddi, A. H., eds. Biochemistry of collagen. New York: Plenum Press; 1976:163–273.Google Scholar
  28. 28.
    Schmidt, E. W.; Schmidt, F. W. Glutamate dehydrogenase. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis. 3rd ed. Basel, Switzerland: Verlag Chemie; 1983:216–227.Google Scholar
  29. 29.
    Seglen, P. O. Preparation of isolated rat liver cells. In: Prescott, D. M., ed. Methods in cell biology. Vol. 13. New York: Academic Press; 1976:29–83.Google Scholar
  30. 30.
    Tajima, S.; Pinnel, S. R. Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA. Biochem. Biophys. Res. Commun. 106:632–637; 1982.PubMedCrossRefGoogle Scholar
  31. 31.
    Takahashi, K.; Hata, J.; Mukai, K., et al. Close similarity between cultured human omental mesoterial cells and endothelial cells in cytochemical markers and plasminogen activator production. In Vitro Cell. Dev. Biol. 27A:542–548; 1991.PubMedCrossRefGoogle Scholar
  32. 32.
    Takahashi, K.; Kiguchi, T.; Sawasaki, Y., et al. Lung capillary endothelial cells produce and secrete urokinase-type plaminogen activator. Am. J. Respir. Cell Mol. Biol. 7:90–94; 1992.PubMedGoogle Scholar
  33. 33.
    Takezawa, T.; Yamazaki, M.; Mori, Y., et al. Morphological and immunocytochemical characterization of a heterospheroid composed of fibroblasts and hepatocytes. J. Cell Sci. 101:495–501; 1992.PubMedGoogle Scholar
  34. 34.
    Thornton, S. C.; Mueller, S. N.; Levine, E. M. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science 222:623–625; 1983.PubMedCrossRefGoogle Scholar
  35. 35.
    Tong, J. Z.; De Lagausie, P.; Furlan, V., et al. Long-term culture of adult rat hepatocyte spheroids. Exp. Cell Res. 200:326–332; 1992.PubMedCrossRefGoogle Scholar
  36. 36.
    Tsao, M.; Smith, J. D.; Nelson, K. G., et al. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of “oval” cells. Exp. Cell Res. 154:38–52; 1984.PubMedCrossRefGoogle Scholar
  37. 37.
    Wachstein, M.; Meisel, M. E. Histochemistry of hepatic phosphatases at a physiologic pH. Am. J. Clin. Pathol. 27:13–23; 1957.PubMedGoogle Scholar
  38. 38.
    Yamada, N.; Okano, T.; Sakai, H., et al. Thermo-responsive polymeric surface; control of attachment and detachment of culture cells. Makromol. Chem. Rapid Commun. 11:571–576; 1990.CrossRefGoogle Scholar
  39. 39.
    Yaswen, P.; Hayner, N. T.; Fausto, N. Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and preneoplastic livers. Cancer Res. 44:324–331; 1984.PubMedGoogle Scholar
  40. 40.
    Yokota, S.; Mori, M. Immunoelectron microscopical localization of ornithine transcarbamylase in hepatic parenchymal cells of the rat. Histochem. J. 18:451–457; 1986.PubMedCrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 1996

Authors and Affiliations

  • Fumiko Karikusa
    • 1
  • Yoshio Sawasaki
    • 1
  1. 1.Department of BiochemistryNational Defense Medical CollegeSaitamaJapan

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