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Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

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Summary

Leakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2±1.5×10−6 IU assayed at 37°C for 25 preparations of isolated hepatocytes.

The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells.

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References

  1. Seglen, P. O. Preparation of isolated rat liver cells. Prescott, D. M. ed. Methods in cell biology. Vol. 13. New York: Academic Press; 1976: 29–83.

    Google Scholar 

  2. Gebhart, R.; Bellemann, P.; Mecke, D. Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in non-proliferating primary monolayer cultures. Exp. Cell Res. 112: 431–441; 1978.

    Article  Google Scholar 

  3. Fry, J. C.; Jones, C. A.; Weibkin, P.; Bellemann, P.; Bridges, J. W. The enzymic isolation of adult rat hepatocytes in a functional and viable state. Anal. Biochem. 71: 341–350; 1976.

    Article  PubMed  CAS  Google Scholar 

  4. Bissell, D.; Guzelian, P. S. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Borek, C.; Williams, G. M. eds., Differentiation and carcinogenesis in liver cell cultures. Vol. 349, New York: New York Academy of Sciences; 1980: 85–98.

    Google Scholar 

  5. Krebs, H. A.; Lund, P.; Edwards, M. Criteria of metabolic competence of isolated hepatocytes. Reid, E. ed. Cell populations. England: Ellis Horwood Limited; 1979: 1–6.

    Google Scholar 

  6. Baur, H.; Kasperek, S.; Pfaff, E. Criteria of viability of isolated liver cells. Hoppe-Seyler's Z. Physiol. Chem. 356: 827–838; 1975.

    PubMed  CAS  Google Scholar 

  7. Crisp, D. M.; Pogson, C. I. Glycolytic and gluconeogenic enzyme activities in parenchymal and non-parenchymal cells from mouse liver. Biochem. J. 126: 1009–1023; 1972.

    PubMed  CAS  Google Scholar 

  8. Jeejeebhoy, K.; Phillips, J. M. Isolated mammalian hepatocytes in culture. Gastroenterology 71: 1086–1096; 1976.

    PubMed  CAS  Google Scholar 

  9. Grisham, J. W., Use of hepatic cell cultures to detect and evaluate the mechanisms of action of toxic chemicals. Richter, G. W.; Epstein, M. A. eds. International review of experimental pathology. Vol. 20. New York: Academic Press; 1979: 124–210.

    Google Scholar 

  10. Bissell, D. M.; Hammaker, L. E.; Meyer, U. A. Parenchymal cells from adult rat liver in nonproliferating monolayer culture. J. Cell Biol. 59: 722–734; 1973.

    Article  PubMed  CAS  Google Scholar 

  11. Pariza, M. W.; Yager, J. D.; Goldfarb, S.; et al. Biochemical, autoradiographic and electron microscopic studies of adult rat liver parenchymal cells in primary culture. Gerchenson, L. E.; Thompson, E. B. eds. Gene expression and carcinogenesis in cultured liver. New York: Academic Press; 1975: 137–167.

    Google Scholar 

  12. Savage, C. R.; Bonney, R. J. Extended expression of differentiated function in primary cultures of adult liver parenchymal cells maintained on nitrocellulose filters. Exp. Cell Res. 114: 307–315; 1978.

    Article  PubMed  CAS  Google Scholar 

  13. Williams, G. M.; Gunn, J. M. Long term culture of adult rat liver epithelial cells. Exp. Cell Res. 89: 139–142; 1974.

    Article  PubMed  CAS  Google Scholar 

  14. Phillips, H. J. Dye exclusion tests for cell viability. Kruse, P. F., Jr.; Patterson, M. K., Jr. eds. Tissue culture methods and applications. New York: Academic Press; 1973: 406–408.

    Google Scholar 

  15. Tolnai, S. A method for viable cell count. TCA Manual 1: 37–38; 1975.

    Article  Google Scholar 

  16. Lehninger, A. L. Biochemistry. New York: Worth; 1972.

    Google Scholar 

  17. Tolman, K. G.; Peterson, P.; Gray, P.; Hammar, S. P. Hepatotoxicity of salicylates in monolayer cell cultures. Gastroenterology 74: 205–208; 1978.

    PubMed  CAS  Google Scholar 

  18. Anuforo, D. C.; Acosta, D.; Smith, R. V. Hepatotoxicity studies with primary cultures of rat liver cells. In Vitro 14: 981–987; 1978.

    Article  PubMed  CAS  Google Scholar 

  19. Dujovne, C. A.; Shoeman, D.; Bianchine, J.; Lasagna, L. Experimental bases for the different hepatotoxicity of erythromycin preparations in man. J. Lab. Clin. Med. 79: 832–844; 1972.

    PubMed  CAS  Google Scholar 

  20. Jauregui, H. O.; Hayner, N.; Laliberte, R.; Lipsky, M.; McMillan, P.; Galletti, P. M. Procurement of hepatocytes for a hybrid artificial liver. Trans. Am. Soc. Artif. Intern. Organs 25: 487–492; 1979.

    PubMed  CAS  Google Scholar 

  21. 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 

  22. Williams, G. M.; Bermudez, E.; Scaramuzzino, D. Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13: 809–817; 1977.

    Article  PubMed  CAS  Google Scholar 

  23. Scandinavian Society for Clinical Chemistry and Clinical Physiology: Recommended methods for the determination of four enzymes in blood. Scand. J. Clin. Lab Invest. 33: 291; 1974.

    Google Scholar 

  24. Bergmeyer, H. U.; Scheibe, P.; Wahlefeld, A. W. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin. Chem. 24: 58–73; 1978.

    PubMed  CAS  Google Scholar 

  25. Trinder, P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem. 6: 24–27; 1969.

    CAS  Google Scholar 

  26. Henry, R. J. Clinical chemistry principles and technics. New York: Harper and Row; 1968: 664–666.

    Google Scholar 

  27. Pesce, M. A.; Strande, C. S. A new micromethod for determination of protein in cerebrospinal fluid and urine. Clin. Chem. 19: 1265–1267; 1973.

    PubMed  CAS  Google Scholar 

  28. Elevitch, F. R. Fluorometric techniques in clinical chemistry. Boston: Little and Brown; 1973: 238.

    Google Scholar 

  29. 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 

  30. Regoeczi, E.; Taylor, P. The net weight of the rat liver. Growth 42: 451–456; 1978.

    PubMed  CAS  Google Scholar 

  31. Berg, T.; Boman, D.; Seglen, P. O. Induction of tryptophan oxygenase in primary rat liver cell suspensions by glucocorticoid hormone. Exp. Cell Res. 72: 571–574; 1972.

    Article  PubMed  CAS  Google Scholar 

  32. Ontko, J. A. Metabolism of free fatty acids in isolated liver cells. J. Biol. Chem. 247: 1788–1800; 1972.

    PubMed  CAS  Google Scholar 

  33. Wilkinson, J. H. Isoenzymes. 2nd ed. London: Chapman and Hall; 1970.

    Google Scholar 

  34. Hook, M.; Rubin, K.; Oldberg, A.; Obrink, B.; Vaheri, A. Cold-insoluble globulin mediates the adhesion of rat liver cells to plastic petri dishes. Biochem. Biophys. Res. Commun. 79: 726–733; 1977.

    Article  PubMed  CAS  Google Scholar 

  35. Deschenes, J.; Valet, J. P.; Marceau, N. Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin mediated adhesion, spreading and functional activities. In Vitro 16: 722–730; 1980.

    PubMed  CAS  Google Scholar 

  36. Merchant, D. J.; Kahn, R. H.; Murphy, W. H. Handbook of cell and organ culture. Minneapolis: Burgess Publishing; 1964: 195–197.

    Google Scholar 

  37. Searpelli, D. G.; Trump, B. F. Cell injury. Kalamazoo: Upjohn Co.; 1971: 7–64.

    Google Scholar 

  38. Acosta, D.; Puchett, M.; McMillan, R. Ischemic myocardial injury in cultured heart cells: leakage of cytoplasmic enzymes from injured cells. In Vitro 14: 728–732; 1978.

    Article  PubMed  CAS  Google Scholar 

  39. Bissell, D.; Montgomery, M.; Levine, G. A.; Bissell, M. J. Glucose metabolism by adult hepatocytes in primary culture and by cell lines from rat liver. Am. J. Physiol. 234: 122–130; 1978.

    Google Scholar 

  40. Ichihara, A.; Nakamura, T.; Tanaka, K.; Tomita, Y.; Aoyama, K.; Kato, S.; Shinno, H. Biochemical functions of adult rat hepatocytes in primary culture. Borek, C.; Williams, G. M. eds. Differentiation and carcinogenesis in liver cell cultures. Vol. 349. New York: New York Academy of Sciences; 1980: 77–84.

    Google Scholar 

  41. Sinclair, R. Glucose metabolism and dehydrogenase activities in the cytosol and mitochondria of mouse LS cells in chemostat culture. In Vitro 16: 1076–1084; 1980.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pathology, Rhode Island Hospital, 02902, Providence, Rhode Island

    Hugo O. Jauregui M.D., Ph.D., James L. Driscoll, Rhonda Williams-Holland & Milton H. Lipsky

  2. Artificial Organ Laboratory, Division of Biology and Medicine, Brown University, 02902, Providence, Rhode Island

    Nancy T. Hayner & Pierre M. Galletti

Authors
  1. Hugo O. Jauregui M.D., Ph.D.
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  2. Nancy T. Hayner
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  3. James L. Driscoll
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  4. Rhonda Williams-Holland
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  5. Milton H. Lipsky
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  6. Pierre M. Galletti
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Additional information

This study was supported in part by Grants HL-11945-11 and 1-RO1-AM 26520-01A1 from the National Institutes of Health, Bethesda, MD.

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Jauregui, H.O., Hayner, N.T., Driscoll, J.L. et al. Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures. In Vitro 17, 1100–1110 (1981). https://doi.org/10.1007/BF02618612

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  • DOI: https://doi.org/10.1007/BF02618612

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