In Vitro Cellular & Developmental Biology

, Volume 21, Issue 4, pp 221–228 | Cite as

Trout hepatocyte culture: Isolation and primary culture

  • James E. Klaunig
  • Randall J. Ruch
  • Peter J. Goldblatt
Article

Summary

Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×106 liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep450 activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d of culture. Culture temperature also influenced thep450 activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and the appearance of bundles of intermediate filaments also were observed with increased time in culture.

Key words

trout hepatocyte culture teleost liver 

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References

  1. 1.
    Bailey, G. S.; Taylor, M. J.; Selivonchichy, D. P. Alfatoxin B1 metabolism and DNA binding in isolated hepatocytes from rainbow trout (Salmo gairdneri). Carcinogenesis 3: 511–518; 1982.PubMedCrossRefGoogle Scholar
  2. 2.
    Burton, K. A. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 315–323; 1956.PubMedGoogle Scholar
  3. 3.
    Fry, J.R.; Bridges, J.W. Use of primary hepatocyte cultures in biochemical toxicology. In: Hodges, E.; Bend, J. R.; Philpot, R.M., eds. Review in biochemical toxicology. New York: Elsevier/North Holland; 1979: 201–247.Google Scholar
  4. 4.
    haschemeyer, A.E.V.; Matthews, R.W. Temperature dependency of protein synthesis in isolated hepatocytes of antarctic fish. Physiol. Zool. 56: 78–87; 1983.Google Scholar
  5. 5.
    Hayashi, S.; Osshiro, Z. Gluconeogenesis and glycolysis in isolated perfused liver of the eel. Bull. Jpn. Soc. Sci. Fish. 41: 201–208; 1975.Google Scholar
  6. 6.
    Hazel, J.R.; Prosser, C.L. Incorporation of 1-14C-Acetate into fatty acids and sterols by isolated hepatocytes of thermally acclimated rainbow trout (Salmo gairdneri). J. Comp. Physiol. 134: 321–329; 1979.Google Scholar
  7. 7.
    Ishikawa, T.; Shimamiwe, T.; Takayama, S. Histologic and electron microscopy observation on diethylnitrosamine-induced hepatomas in small aquarium fish (Oryzias latipes). J. Natl. Cancer Inst. 55: 909–916; 1975.PubMedGoogle Scholar
  8. 8.
    Klaunig, J.E.; Lipsky, M.M.; Trump, B.F.; Hinton, D.E. Biochemical and ultrastructural changes in teleost liver following subacute exposure to PCB. J. Environ. Pathol. Toxicol. 2: 953–963; 1979.PubMedGoogle Scholar
  9. 9.
    Klaunig, J.E.; Goldblatt, P.J.; Hinton, D.E.; Lipsky, M.M.; Chacko, J.; Trump, B.F. Mouse liver cell culture. I. Hepatocyte isolation. In Vitro 17: 913–925; 1981.PubMedCrossRefGoogle Scholar
  10. 10.
    Klaunig, J.E.; Goldblatt, P.J.; Hinton, D.E.; Lipsky, M.M.; Knipe, S.M.; Trump, B.F. Morphologic and functional studies of mouse hepatocytes in primary culture. Anat. Rec. 204: 231–243; 1982.PubMedCrossRefGoogle Scholar
  11. 11.
    Klaunig, J.E. Primary culture of mouse hepatocytes: Effects of insulin and dexamethasone. In: Harris, R.A.; Cornell, N.W., eds. Isolation, characterization and use of hepatocytes. Elsevier Science Publishing Co., New York; 1983: 93–98.Google Scholar
  12. 12.
    Klaunig, J.E. Establishment of fish hepatocyte cultures for use in in vitro carcinogenicity studies. Natl. Cancer Inst. Mongr. 65: 163–173; 1984.Google Scholar
  13. 13.
    Maslansky, C.; Williams, G.M. Primary cultures and levels of cytochrome P450 in hepatocytes from mouse, rat, hamster, and rabbit liver. In Vitro 18: 683–693; 1981.CrossRefGoogle Scholar
  14. 14.
    Porthe-Nibelle, J.; Lahlow, B. Mechanisms of glucocorticoid uptake by isolated hepatocytes of the trout. Comp. Biochem. Physiol. 69B: 425–433; 1981.Google Scholar
  15. 15.
    Saez, L.; Golcoechea, O.; Anthauer, R.; Krauskopf, M. Behavior of RNA and protein synthesis during the acclimatization of the carp. Studies with isolated hepatocytes. Comp. Biochem. Physiol. 72B: 31–38; 1982.Google Scholar
  16. 16.
    Sato, S; Matsushima, T.; Tanaka, N.; Sugimura, T.; Takashima, F. Hepatic tumors in the guppy induced by aflatoxin B1, dimethylnitrosamine, and 2-acetylaminofluorene. J. Natl. Cancer Inst. 50: 767–778; 1973.PubMedGoogle Scholar
  17. 17.
    Sinnhuber, R.O.; Hendricks, J.D.; Wales, J.H.; Putnam, G.B. Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. NY Acad. Sci. 298: 389–408; 1977.CrossRefGoogle Scholar
  18. 18.
    Sirica, A.E.; Pitot, H.C. Drug metabolism and effects of carcinogens in cultured hepatic cells. Pharmacol. Rev. 31: 205–227; 1980.Google Scholar
  19. 19.
    Tomatis, L.; Partensky, C.; Montesano, R. The predictive value of mouse liver tumor induction in carcinogenicity testing—A literature survey. Int. J. Cancer 12: 1–20; 1973.PubMedCrossRefGoogle Scholar
  20. 20.
    Walton, M.J.; Cowey, C.B. Gluconeogenesis by isolated hepatocytes from rainbow troutSalmo gairdneri. Comp. Biochem. Physiol. 62B: 75–79; 1979.Google Scholar
  21. 21.
    Williams, G.M.; Bermudez, E.; Scaramuzzino, D. Rat hepatocyte primary cell cultures. Improved dissociation and attachment technologies and the enhancement of survival by culture medium. In Vitro 13: 809–817; 1978.CrossRefGoogle Scholar
  22. 22.
    Wolf, K.; Mann, J.A. Poikilotherm vertebrate cell lines and viruses. A current listing for fishes. In Vitro 16: 168–179; 1980.PubMedGoogle Scholar

Copyright information

© Tissue Culture Association, Inc 1985

Authors and Affiliations

  • James E. Klaunig
    • 1
  • Randall J. Ruch
    • 1
  • Peter J. Goldblatt
    • 1
  1. 1.Department of PathologyMedical College of OhioToledo

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