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Protocol no. 20

ISOLATION OF RAT HEPATOCYTES

Collagenase perfusion of rat liver yields a hepatocyte suspension which may be exposed to test compounds in order to assess their effects on cell viability and enzyme leakage.

CONTACT

Prof. Dr. Claus Jurgen Estler Inst. Pharmacology & Toxicology Universitatsstr. 22 D-W-8520 Erlangen Germany Tel: Germany - 9131 852236

BASIC PROCEDURE

The liver of a rat is cannulated and perfused in situ with buffer and medium, following which it is excised and perfused in a closed system with a collagenase medium. The consistency of the liver becomes soft, due to digestion of the connective tissue. At this point the liver is transferred into culture medium and the Glisson's capsule is cut in order to obtain a crude cell suspension which is then filtered, washed and suspended to the required concentration for culture and testing.

CRITICAL ASSESSMENT

A relatively fast, reproducible, procedure to obtain liver cells compared to certain other techniques, e.g. slicing followed by enzymatic/mechanical dispersion. The avoidance of mechanical force minimizes the risk of cell damage. The pressure of the perfusion media is critical to this isolation procedure. It may be standardised to some degree by keeping the level of the liquid column in the bubble trap as constant and as low as possible. The advantage of using isolated cells is that a number of experiments can be performed on just one rat liver, thus reducing the number of animals required. The estimation of hepatotoxicity in vitro by measurement of enzyme leakage correlates well with in vivo hepatotoxicity. Isolated hepatocytes retain their metabolic activity for several hours in suspension culture. In monolayer cultures, metabolic activity can be induced by substances such as phenobarbitone (refer to e.g. Engelmann et al., 1985; Muakkassah-Kelly et al., 1988; Newman & Guzelian, 1982; Rogiers et al., 1986; Schmetz et al., 1986; Seibert et al., 1989). The basic test system can be used for many types of in vitro assay: to determine cytotoxicity, metabolism, transport of xenobiotics, studies on mutagenic potential, etc. It can be used for initial screening of substances for potential hepatotoxicity, or for research into molecular mechanisms of drug action. However, this method alone cannot completely replace in vivo studies.

REFERENCES

  1. Berry, M.N. & Friend, D.S. (1969) High-yield preparation of isolated rat liver parenchymal cells. Journal of Cell Biology, 43, 506-520. Engelmann, G.L., Richardson, A.G. & Fierer, J.A. (1985) Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes. Arch. Biochem. Biophys., 238, 359-367.
  2. Muakkassah-Kelly, S.F., Biere, F., Waechter, F., Bentley, P. & St„ubli, P. (1988) The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: Effect of nafenopin and electroporation. Experientia, 44, 823-827.
  3. Newman, S., & Guzelian, P.S. (1982) Stimulation of de novo synthesis of cytochrome P-450 by phenobarbital in primary cultures of adult hepatocytes. PNAS, USA, 79, 2922-2926.
  4. Rogiers, V., Adriaenssens, L., Vandenberghe, J., Gepts, E., Callaerts, A., & Vercruysse, A. (1986) Critical evaluation of 7-ethoxycoumarin O-deethylase activity measurement in intact isolated hepatocytes. Xenobiotica, 16, 817-826.
  5. Schmetz, E.G., Hazelton, G.A., Hall, J., Watkins, P.B., Klaassen, C.D., & Guzelian, P.S. (1986) Induction of digitoxigenin monodigitoxoside UDP-glucuronyltransferase activity by glucocorticoids and other inducers of cytochrome P-450p in primary monolayer cultures of adult rat hepatocytes and in human liver. J. Biol. Chem., 261, 8270-8275.
  6. Seglen, P.O. (1976) Preparation of isolated rat liver cells. Methods Cell Biol., 13, 29-83.
  7. Seibert, B., Oesch, F. & Steinberg, P. (1989) Distribution and induction of cytochrome P-450-dependent monooxygenase activities in rat liver parenchymal cell subpopulations separated by centrifugal elutration. Arch. Toxicol., 63, 18-22.
  8. Sippel, H. & Estler, C.-J. (1990) Comparative evaluation of hepatotoxic side effects of various new trypanocidal diamidines in rat hepatocytes and mice. Arzneimittel-Forschung/Drug Research, 40, 290-293.

IP-20 c November 1991