Preparation of Hepatocytes

Daniel R. Mudra1, Andrew Parkinson1

1 XenoTech, LLC, Kansas City, Kansas
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.2
DOI:  10.1002/0471140856.tx1402s08
Online Posting Date:  August, 2001
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This unit describes a process for isolation of intact, viable hepatocytes for use in integrated drug metabolism studies. Isolated hepatocytes are increasingly being used as a biological system for studying the in vitro metabolism of xenobiotics. The isolation of hepatocytes from laboratory animals and nontransplantable human livers donated for research activities involves a two‐step enzymatic digestion of the liver tissue. Two methods for the perfusion of liver tissue are included: in situ perfusion and isolation of hepatocytes and perfusion of excised tissue. The basic protocol also includes suggestions for designing drug metabolism experiments using hepatocytes. The final protocol describes cryopreservation and long‐term storage of isolated hepatocytes.

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Table of Contents

  • Basic Protocol 1: In Situ Perfusion of Laboratory Animal Liver to Isolate Intact Hepatocytes
  • Alternate Protocol 1: Perfusion of a Previously Excised Liver
  • Support Protocol 1: Cryopreservation of Hepatocytes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: In Situ Perfusion of Laboratory Animal Liver to Isolate Intact Hepatocytes

  • Laboratory animal
  • 50 mg/ml sodium pentobarbital dissolved in 5:4:1 (v/v/v) water/propylene glycol/alcohol (e.g., Nembutal, Abbott Laboratories)
  • Iodine surgical scrub (e.g., Betadine)
  • 70% (v/v) ethanol
  • PBS ( appendix 2A), sterile
  • PB‐1 (see recipe), sterile and oxygenated
  • Collagenase
  • PB‐2 (see recipe), sterile and oxygenated
  • DMEM+ (see recipe)
  • 0.04% (w/v) trypan blue
  • 90% (v/v) isotonic Percoll (Sigma‐Aldrich) in PBS ( appendix 2A), store at 2° to 8°C
  • Culture medium (application specific)
  • Surgical tray
  • Gauze pads
  • Scalpel
  • Surgical sutures
  • Perfusion apparatus, including perfusion line, peristaltic pump, and oxygen source (see Fig. )
  • 16‐G surgical catheter
  • Surgical forceps
  • Surgical scissors, sterile
  • 100‐mesh nylon net or cheesecloth, sterile
  • Vacuum aspirator
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3B).

Alternate Protocol 1: Perfusion of a Previously Excised Liver

  • Previously excised liver
  • Plastic cannulas from surgical catheters (or equivalent)
  • Medical adhesive (e.g., Loctite 4013; Loctite)

Support Protocol 1: Cryopreservation of Hepatocytes

  • Isolated hepatocytes (see protocol 1; see protocol 2)
  • Fetal bovine serum (FBS), heat‐inactivated ( appendix 3B)
  • Dimethyl sulfoxide (DMSO)
  • Hepatocyte storage vials (must withstand −196°C)
  • Microprocessor‐controlled programmable freezing chamber (e.g., model 1010; Forma Scientific) with thermometer
  • Liquid nitrogen storage unit
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Literature Cited

Literature Cited
   Bayliss, M.K., Bell, J.A., Wilson, K., and Park, G.R. 1994. 7‐Ethoxycoumarin O‐deethylase kinetics in isolated rat, dog and human hepatocytes. Xenobiotica. 24:231‐241.
   Berry, M.N. and Friend, D.S. 1969. High yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43:506‐520.
   Ekins, S., Murray, G.I., Burke, M.D., Williams, J.A., Marchant, N.C., and Hawksworth, G.M. 1995. Quantitative differences in phase I and II metabolism between rat precision‐cut liver slices and isolated hepatocytes. Drug Metab. Dispos. 23:1274‐1279.
   Guillouzo, A., Rialland, L., Fautrel, A., and Guyomard, C. 1999. Survival and function of isolated hepatocytes after cryopreservation. Chem. Biol. Interact. 121:7‐16.
   Jamieson, N.V., Lindell, S., Sundberg, R., Southard, J.H., and Belzer, F.D. 1988. Preservation of the canine liver for 24‐48 hours using simple cold storage with UW solution. Transplantation. 46:517.
   LeCluyse, E.L., Bullock, P.L., Parkinson, A., and Hochman, J.H. 1996a. Cultured rat hepatocytes. In Models for Assessing Drug Absorption and Metabolism (R.T. Borchardt, P.L. Smith, and G. Wilson, eds.) pp.121‐159. Plenum, New York.
   LeCluyse, E.L., Bullock, P.L., and Parkinson, A. 1996b. Strategies for restoration and maintenance of normal hepatic structure and function in long term cultures of rat hepatocytes. Adv. Drug Deliv. Rev. 22:133‐186.
   Li, A., Gorycki, P., Hengstler, J., Kedderis, G., Koebe, H., Rahmani, R., de Sousas, G., Silva, J., and Skett, P. 1999. Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: Consensus of an international expert panel. Chem. Biol. Interact. 121:117‐123.
   Madan, A., DeHaan, R., Mudra, D., Carroll, K., LeCluyse, E., and Parkinson, A. 1999. Effect of cryopreservation on cytochrome P450 enzyme induction in cultured rat hepatocytes. Drug Metab. Dispos. 27:327‐335.
   Parkinson, A. 1996a. Biotransformation of xenobiotics. In Casarett and Doull's Toxicology: The Basic Science of Poisons, V (C.D. Klaassen, ed.) pp. 113‐186. McGraw Hill, New York (edition VI in press).
   Parkinson, A. 1996b. Overview of current cytochrome P450 technology for assessing the safety and efficacy of new materials. Tox. Pathol. 24:45‐57.
   Parkinson, A., Pearce, R., Madan, A., and Forster, J. 1997. Availability and preservation of human tissues, and the use of human liver microsomes in drug metabolism research. In The Use of Human In Vitro Systems to Support Preclinical Safety Assessment (A. Sundwall, G. Alvan, E. Lindgren, P. Moldeus, T. Salmonsson, and P. Sjoberg, eds.) pp.17‐28. Tryckgrappen. Stockholm, Sweden.
   Quistorff, B., Dich, J., and Grunnet, N. 1989. Preparation of isolated rat liver hepatocytes. In Methods in Molecular Biology, Vol. 5: Animal Cell Culture, (J.W. Pollard and J.M. Walker, eds.) pp.151‐160. Humana Press, Totowa, N.J.
   Seddon, T., Michelle, I., and Chnery, R.J. 1989. Comparitive drug metabolism of diazepam in hepatocytes isolated from man, rat, monkey and dog. Biochem. Pharmacol. 38:1657‐1665.
   Seglen, P.O. 1976. Preparation of isolated rat liver cells. In Methods in Cell Biology, Vol. 19 (D.M. Prescott, ed.) pp.29‐83. Academic Press, New York.
   Zomorodi, K., Carlile, D., and Houston, B. 1995. Kinetics of diazepam metabolism in rat hepatic microsomes and hepatocytes and their use in predicting in vivo hepatic clearance. Xenobiotica. 25:907‐916.
Key References
   LeCluyse et al., 1996a, 1996b. See above. See above
  Describe the steps involved in in situ perfusion and enzymatic digestion of rat liver. The and are based on these methods.
   Madan et al., 1999. See above.
  Describes the steps involved in cell isolation, cryopreservation, and thawing. The and are based on these methods.
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