Skip to main content
SpringerLink
Log in

Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions

  • Regular Papers
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Summary

Composition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120×g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ashley, W. H. H. Liver. Studies in biology no. 105. Baltimore: University Park Press, 1979:1.

    Google Scholar 

  • Bailey, G. S.; Taylor, M. J.; Selivonchick, D. P. Aflatoxin B1 metabolism and DNA binding in isolated hepatocytes from rainbow trout (Salmo gairdneri). Carcinogenesis 3:511–518; 1982.

    Article  PubMed  CAS  Google Scholar 

  • Bergmeyer, H. U.; Bernt, E. Lactate dehydrogenase: UV-assay with pyruvate and NADH. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis. Vol. 2. New York: Academic Press, Inc.; 1974:574–579.

    Google Scholar 

  • Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254; 1976.

    Article  PubMed  CAS  Google Scholar 

  • Greenwald, P., ed. Consensus Committee, summary and recommendations: a consensus report. In: Greenwald, P., ed. Use of Small Fish Species in Carcinogenicity Testing. Bethesda, MD: National Cancer Institute. Monograph 65; 1984:397–404.

    Google Scholar 

  • Couch, J. A.; Courtney, L. A. N-Nitrosodiethylamine-induced hepatocarcinogenesis in estuarine sheepshead minnow (Cyprinodon variegatus): neoplasms and related lesions compared with mammalian lesions. JNCI 79:297–321; 1987.

    PubMed  CAS  Google Scholar 

  • Couch, J. A.; Courtney, L. A. Attempts to abbreviate time to endpoint in fish hepatocarcinogenesis assays. In: Jolley, R. L.; Bull, R. J.; Davis, W. P., et al., eds. Water chlorination: chemistry, environmental impact and health effects. Vol. 5. Chelsea, MI: Lewis Publishers; 1985:377–398.

    Google Scholar 

  • Droy, B. F.; Miller, M. R.; Freeland, T. M., et al. Immuno-histochemical detection of CCl4-induced, mitosis-related DNA synthesis in livers of trout and rat. Aquat. Toxicol. 13:155–166; 1988.

    Article  CAS  Google Scholar 

  • Dunn, J. C. Y.; Yarmush, M. L.; Koebe, H. G., et al. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J. 3:174–177; 1989.

    PubMed  CAS  Google Scholar 

  • French, S. W. Editorial: Role of canalicular contraction in bile flow. Lab. Invest. 53:245–249; 1985.

    PubMed  CAS  Google Scholar 

  • Frasca, J. M.; Parks, V. R. A routine technique for double-staining ultrathin sections using uranyl and lead salts. J. Cell Biol. 25:157–161; 1965.

    Article  PubMed  CAS  Google Scholar 

  • Gebhardt, R. Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. Gastroenterology 84:1462–1470; 1983.

    PubMed  CAS  Google Scholar 

  • Giles, K. W.; Mayers, A. An improved diphenylamine method for the estimation of deoxyribonucleic acids. Nature 206:93; 1965.

    Article  CAS  Google Scholar 

  • Hampton, J. A.; Lantz, R. C.; Goldblatt, P. J., et al. Functional units in rainbow trout (Salmo gairdneri; Richardson) liver. II. The biliary system. Anat. Rec. 221:619–634; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Hampton, J. A.; Lantz, R. C.; Hinton, D. E. Functional units in rainbow trout (Salmo gairdneri; Richardson) liver. III. Morphometric analysis of parenchyma, stroma and component cell types. Am. J. Anat. 1989 (in press).

  • Hampton, J. A.; McCuskey, P. A.; McCuskey, R. S., et al. Functional units in rainbow trout (Salmo gairdneri) liver. I. Arrangement and histochemical properties of hepatocytes. Anat. Rec. 213:166–175; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Haschemeyer, A.; Mathews, R. Temperature dependency of protein synthesis in isolated hepatocytes of Antarctic fish. Physiol. Zool. 56:78–87; 1983.

    Google Scholar 

  • 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.

    CAS  Google Scholar 

  • Hendricks, J. D. Chemical carcinogenesis in fish. In: Weber, L. J., ed. Aquatic toxicology. New York: Raven Press; 1982: 149–221.

    Google Scholar 

  • Hendricks, J. D.; Meyers, T. R.; Shelton, D. W., et al. Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection. JNCI 74:839–847; 1985.

    PubMed  CAS  Google Scholar 

  • Hightower, L. E.; Renfro, J. L. Recent applications of fish cell culture to biomedical research. J. Exp. Zool. 248:290–302; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Hinton, D. E.; Couch, J. A.; Teh, S. J., et al. Cytological changes during progression of neoplasia in selected fish species. Aquat. Toxicol. 11:77–112; 1988.

    Article  Google Scholar 

  • Hinton, D. E.; Walker, E. R.; Pinkstaff, C. A., et al. A morphological survey of teleost organs important in carcinogenesis with attention to fixation. NCI Monogr. 65:291–320; 1984a.

    CAS  Google Scholar 

  • Hinton, D. E.; Lantz, R. C.; Hampton, J. A. Effect of age and exposure to a carcinogen on the structure of the medaka liver: a morphometric study. NCI Monogr. 65:239–249; 1984b.

    CAS  Google Scholar 

  • Hinton, D. E.; Lantz, R. C.; Hampton, J. A., et al. Normal versus abnormal structure: considerations in morphologic responses of teleost to pollutants. Environ. Health Perspect. 71:139–146; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Ito, S.; Karnovsky, M. J. Formaldehyde-glutaralde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol. 39:168A-169A; 1968.

    Google Scholar 

  • Klaunig, J. E. Establishment of fish hepatocyte cultures for use in vitro carcinogenicity studies. NCI Monogr. 65:163–173; 1984.

    CAS  Google Scholar 

  • Klaunig, J. E.; Ruch, R. J.; Goldblatt, P. J. Trout hepatocyte culture: isolation and primary culture. In Vitro Cell. Dev. Biol. 21:221–227; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Kocal, T.; Quinn, B. A.; Smith, I. R., et al. Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout (Salmo gairdneri). In Vitro Cell. Dev. Biol. 24:304–308; 1988a.

    Article  Google Scholar 

  • Kocal, T.; Crane, T. L.; Quinn, B. A., et al. Degradation of extracellular thymidine by cultured hepatocytes from rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 91B:557–561; 1988b.

    CAS  Google Scholar 

  • Koide, N.; Shinji, T.; Tanabe, T., et al. Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans. Biochem. Biophys. Res. Commun. 16:385–391; 1989.

    Article  Google Scholar 

  • Lipsky, M. M.; Sheridan, T. R.; Bennett, R. O., et al. Comparison of trout hepatocyte culture on different substrates. In Vitro Cell. Dev. Biol. 22:360–362; 1986.

    Article  Google Scholar 

  • Maitre, J.-L.; Valotaire, Y.; Guguen-Guillouzo, C. Estradiol-17β stimulation of vitellogenin synthesis in primary culture of male rainbow trout hepatocytes. In Vitro. Cell. Dev. Biol. 22:337–343; 1986.

    Article  CAS  Google Scholar 

  • Masahito, P.; Ishikawa, T.; Sugano, H. Fish tumors and their importance in cancer research. Jpn. J. Cancer Res. (Gann) 79:545–555; 1988.

    CAS  Google Scholar 

  • McCuskey, P. A.; McCuskey, R. S.; Hinton, D. E. Electron microscopy of cells of the hepatic sinusoids in rainbow trout (Salmo gairdneri). In: Kirn, A.; Knook, D. L.; Wisse, E., eds. Cells of the hepatic sinusoid. Vol. I. Leiden: Netherlands: Kupfer Cell Foundation; 1986:489–494.

    Google Scholar 

  • Moon, T. W.; Walsh, P. J.; Mommsen, T. P. Fish hepatocytes: a model metabolic system. Can. J. Fish Aquat. Sci. 42:1772–1782; 1985.

    Article  CAS  Google Scholar 

  • Parker, R. S.; Morrissey, M. T.; Moldeus, P., et al. The use of isolated hepatocytes from rainbow trout (Salmo gairdneri) in the metabolism of acetaminophen. Comp. Biochem. Physiol. 70B:631–633; 1981.

    CAS  Google Scholar 

  • Schoenhard, G. L.; Hendricks, J. D.; Nixon, J. E., et al. Aflatoxicol-induced hepatocellular carcinoma in rainbow trout (Salmo gairdneri) and the synergistic effects of cyclopropenoid fatty acids. Cancer Res. 41:1011–1014; 1981.

    PubMed  CAS  Google Scholar 

  • Shore, T. W.; Jones, H. L. On the structure of the vertebrate liver. J. Physiol. (Lond.) 10:408–428; 1889.

    CAS  Google Scholar 

  • Simon, R. C.; Dollar, A. M.; Smuckler, E. A. Descriptive classification on normal and altered histology of trout livers. In: Halver, J. E.; Mitchell, I. A.; eds Trout hepatoma research conference papers. Res. Rep. 70, Washington, DC: U. S. Fish and Wildlife Service; 1967:18–28.

    Google Scholar 

  • Sinnhuber, R. O.; Hendricks, J. D.; Wales, J. H., et al. Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. NY Acad. Sci. 298:389–408; 1977.

    Article  CAS  Google Scholar 

  • Smith, C. R.; Oshio, C.; Miyairi, M., et al. Coordination of the contractile activity of bile canaliculi. Evidence from spontaneous contractionsin vitro. Lab. Invest. 53:270–274; 1985.

    PubMed  CAS  Google Scholar 

  • Smith, G. R.; Stearley, R. F. The classification and scientific names of rainbow and cutthroat trouts. Fisheries 14:4–10; 1989.

    Article  Google Scholar 

  • Tanuma, Y.; Ito, T. Electron microscopic study on the sinusoidal wall of the liver of the crucian,Carassius carassius, with special remarks on the fat-storing cell (FSC). Arch. Histol. Jpn. 43:241–263; 1980.

    PubMed  CAS  Google Scholar 

  • Trump, B. F.; Smuckler, E. A.; Benditt, E. P. A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5:343–348; 1961.

    Article  PubMed  CAS  Google Scholar 

  • Vaillant, C.; Le Guellec, C.; Pakdel, F., et al. Vitellogenin gene expression in primary culture of male rainbow trout hepatocytes. Gen. Comp. Endocrinol. 70:284–290; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Venable, J. H.; Coggeshall, R. A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25:407–408; 1965.

    Article  PubMed  CAS  Google Scholar 

  • Wales, J. H.; Sinnhuber, R. O.; Hendricks, J. D., et al. Aflatoxin B1 induction of hepatocellular carcinoma in the embryos of rainbow trout (Salmo gairdneri). JNCI 60:1133–1139; 1978.

    PubMed  CAS  Google Scholar 

  • Walton, M. J.; Cowey, C. B. Gluconeogenesis by isolated hepatocytes from rainbow troutSalmo gairdneri. Comp. Biochem. Physiol. 62B:75–79; 1979.

    CAS  Google Scholar 

  • Watanabe, S.; Smith, C. R.; Phillips, M. J. Coordination of the contractile activity of bile calaniculi. Evidence from calcium microinjection of triplet hepatocytes. Lab. Invest. 53:275–279; 1985.

    PubMed  CAS  Google Scholar 

  • Whitham, M.; Nixon, J. E.; Sinnhuber, R. O. Liver DNA bound in vivo with aflatoxin B1 as a measure of hepatocarcinoma initiation in rainbow trout. JNCI 68:623–628; 1982.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, West Virginia University Health Sciences Center, 26506, Morgantown, West Virginia

    James B. Blair, Michael R. Miller, Donna Pack & Rebecca Barnes

  2. Department of Medicine, School of Veterinary Medicine, University of California, 95616, Davis, California

    Swee J. Teh & David E. Hinton

Authors
  1. James B. Blair
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael R. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Donna Pack
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rebecca Barnes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Swee J. Teh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David E. Hinton
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Supported by grant CA45131 from the National Cancer Institute, Bethesda, MD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blair, J.B., Miller, M.R., Pack, D. et al. Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions. In Vitro Cell Dev Biol 26, 237–249 (1990). https://doi.org/10.1007/BF02624453

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02624453

Key words

Search

Navigation