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Mouse liver cell culture

I. Hepatocyte isolation

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Summary

A method for isolation of mouse liver cells by a two-step perfusion with calcium and magnesium-free Hanks' salt solution followed by a medium containing collagenase is described. Several variations of the commonly used procedure for rat liver cell isolation were quantitatively compared with respect to cell yield and viability. The optimal isolation technique involved perfusion through the hepatic portal vein and routinely produced an average of 2.3×106 viable liver cells/g body weight. Optimal perfusate collagenase concentration was found to be 100 U of enzyme activity per milliliter of perfusate.

Light and electron microscopic evaluation of liver morphology after several steps of the isolation showed distinct morphologic changes in hepatocytes and other liver cells during perfusion. After perfusion with Hanks' calcium-and magnesium-free solution, many hepatocytes exhibited early reversible cell injury. These changes included vesiculation and slight swelling of the endoplasmic reticulum as well as mitochondrial matrix condensation. Subsequent to perfusion with collagenase, the majority of hepatocytes appeared connected to one another only by tight junctional complexes at the bile canaliculi. Multiple evaginations were see on the outer membrane resembling microvilli and probably represented the remains of cell-to-cell interdigitations between hepatocytes and sinusoidal lining cells from the space of Disse. The cytoplasmic injury seen after Hanks' perfusion was reversed after collagenase perfusion. After mechanical dispersion, isolated mouse hepatocytes were spherical in shape and existed as individual cells; many (80 to 85%) were binucleated under phase contrast light microscopy. By electron microscopy, cells appeared morphologically similar in cytoplasmic constitution to that seen in intact nonaltered liver cells.

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References

  1. Berry, M. N.; Friend, D. S. High-yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43: 506–520; 1969.

    Article  PubMed  CAS  Google Scholar 

  2. Seglen, P. O. Preparation of rat liver cells. II. Effects of ions and chelators on tissue dispersion. Exp. Cell Res. 76: 25–30; 1973.

    Article  PubMed  CAS  Google Scholar 

  3. Seglen, P. O. Preparation of rat liver cells. III. Enzymatic requirements for tissue dispersion. Exp. Cell Res. 82: 391–398; 1973.

    Article  PubMed  CAS  Google Scholar 

  4. Williams, G. M.; Bermudez, E.; Scaramuzzino, D. Rat hepatocyte primary cell cultures. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13: 809–817; 1978.

    Article  Google Scholar 

  5. Laishes, B. A.; Williams, G. M. Conditions affecting primary cell cultures of functional adult rat hepatocytes. I. The effect of insulin. In Vitro 12: 521–532; 1976.

    PubMed  CAS  Google Scholar 

  6. Laishes, B. A.; Williams, G. M. Conditions affecting primary cell cultures of function adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12: 821–832; 1976.

    PubMed  CAS  Google Scholar 

  7. Berry, M. N. Metabolic properties of isolated liver cells. J. Cell Biol. 15: 1–8; 1962.

    Article  CAS  Google Scholar 

  8. Berry, M. N.; Simpson, F. O. Fine structure of cells isolated from adult mouse liver. J. Cell Biol. 15: 9–17; 1962.

    Article  Google Scholar 

  9. Harris, C. C.; Leone, C. A. Some effects of EDTA and tetraphenyl-boron on the ultrastructure of mitochondria in mouse liver cells. J. Cell Biol. 28: 405–408; 1966.

    Article  PubMed  CAS  Google Scholar 

  10. Rappaport, C.; Howze, G. B. Dissociation of adult mouse liver by sodium tetraphenylboron: A potassium complexing agent. Proc. Soc. Exp. Biol. Med. 121: 1010–1016; 1966.

    PubMed  CAS  Google Scholar 

  11. Rappaport, C.; Howze, G. B. Further studies on the dissociation of adult mouse tissue. Proc. Soc. Exp. Biol. Med. 121: 1016–1021; 1966.

    PubMed  CAS  Google Scholar 

  12. Renton, K. W.; Deloria, L. B.; Mannering, G. J. Effects of polyribonoinosinic acid polyribocytidylic acid and a mouse interferon preparation on cytochrome P-450-dependen monooxygenase systems in cultures of primary mouse hepatocytes. Mol. Pharmacol. 14: 672–681; 1978.

    PubMed  CAS  Google Scholar 

  13. Williams, G. M. Classification of genotoxic and epigenetic hepatocarcinogens using liver culture assays. Borek, C.; Williams, G. M. eds. Differentiation and carcinogenesis in liver cell cultures. Ann. N.Y. Acad. Sci. 1980; 349: 273–282.

  14. Leibovitz, A. The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. Am. J. Hyg. 78: 173–180; 1963.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  16. Trump, B. F.; Goldblatt, P. J.; Griffin, C. C.; Waraudekar, U. S.; Stovall, R. E. Effects of freezing and thawing on the ultrastructure of mouse parenchymal cells. Lab. Invest. 13: 967–1002; 1964.

    PubMed  CAS  Google Scholar 

  17. Ogawa, K.; Medline, A.; Farber, E. Sequential analysis of hepatic carcinogenesis. A comparative study of the ultrastructure of preneoplastic, malignant, prenatal, postnatal and regenerating liver. Lab. Invest. 41: 22–35; 1979.

    PubMed  CAS  Google Scholar 

  18. Weibel, E. R.; Bolender, R. P. Stereological techniques for electron microscopic morphometry. Hayat, M. A. ed. Principals and techniques of electron microscopy. Vol. 3. New York: Academic Press; 1973: 237–296.

    Google Scholar 

  19. Budd, G. D.; Barnard, E. A.; Porter, C.; Mattimore, S. A. Fluorophosphate-sensitive esterases in mammalian liver. J. Histochem. and Cytochem. 28: 533–542; 1979.

    Google Scholar 

  20. Weibel, E. R. Stereological methods: practical methods for biological morphometry. Vol. 1. New York: Academic Press; 1979.

    Google Scholar 

  21. Gravela, E.; Poli, G.; Albano, E.; Dianzani, M. U. Studies on fatty liver with isolated hepatocytes. Exp. Mol. Pathol. 27: 339–352; 1977.

    Article  PubMed  CAS  Google Scholar 

  22. Lee Jeebhoy, K. N.; Phillips, M. J. Isolated mammalian hepatocytes in culture. Gastroenterology 71: 1086–1096; 1977.

    Google Scholar 

  23. Phillips, M. J.; Oda, M.; Edwards, V. D.; Greenberg, G. R.; Jee Jebhoy, K. N. Utrastructural and functional studies of cultured hepatocytes. Lab. Invest. 31: 533–541; 1974.

    PubMed  CAS  Google Scholar 

  24. Schreiber, G.; Schreiber, M. The preparation of single cell suspension from liver and their use for the study of protein synthesis. Subcell. Biochem. 2: 321–383; 1973.

    Google Scholar 

  25. Grisham, J. W.; Charlton, R. K.; Kaufman, D. G.In vitro assay of cytotoxicity with cultured liver: Accomplishments and possibilities. Environ. Health Perspect. 25: 161–171; 1978.

    Article  PubMed  CAS  Google Scholar 

  26. Bresnick, E.; Vaught, J. B.; Chuang, A. H. L.; Stoming, T. A.; Bockman, D.; Mukhtar, H. Nuclear aryl hydrocarbon hydroxylase and interaction of polycyclic hydrocarbons with nuclear components. Arch. Biochem. Biophys. 181: 257–269; 1977.

    Article  PubMed  CAS  Google Scholar 

  27. Burke, M. D.; Vadi, H.; Jernstrom, B.; Orrenius, S. Metabolism of benzo(a)pyrence with isolated hepatocytes and the formation and degradation of DNA-binding derivatives. J. Biol. Chem. 18: 6424–6431; 1977.

    Google Scholar 

  28. Jones, C. A.; Moore, B. P.; Fry, J. R.; Cohen, G. M.; Bridges, J. W. Studies on the metabolism and excretion of benzo(a)pyrene in isolated adult rat hepatocytes. Biochem. Pharmacol. 27: 693–699; 1978.

    Article  PubMed  CAS  Google Scholar 

  29. Fry, J. R.; Bridges, J. W. Use of primary hepatocyte cultures in biochemical toxicology. Hodgson, E.; Bend, J. R.; Philpot, R. M. eds. Reviews in biochemical toxicology. New York: Elsevier North Holland, Inc.; 1979: 201–247.

    Google Scholar 

  30. Williams, G. M. The use of liver epithelial cell cultures for the study of chemical carcinogenesis. Am. J. Pathol. 85: 739–751; 1976.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pathology, Medical College of Ohio, 43699, Toledo, Ohio

    James E. Klaunig & Peter J. Goldblatt

  2. Department of Pathology, University of Maryland School of Medicine, 21201, Baltimore, Maryland

    Michael M. Lipsky, Jacob Chacko & Benjamin F. Trump

  3. Department of Anatomy, West Virginia University School of Medicine, 26506, Morgantown, West Virginia

    David E. Hinton

Authors
  1. James E. Klaunig
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  2. Peter J. Goldblatt
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  3. David E. Hinton
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  4. Michael M. Lipsky
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  5. Jacob Chacko
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  6. Benjamin F. Trump
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Klaunig, J.E., Goldblatt, P.J., Hinton, D.E. et al. Mouse liver cell culture. In Vitro 17, 913–925 (1981). https://doi.org/10.1007/BF02618288

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  • DOI: https://doi.org/10.1007/BF02618288

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