A Rat Primary Hepatocyte Culture Model for Aging Studies

Swapna V. Shenvi1, Brian M. Dixon1, Kate Petersen Shay1, Tory M. Hagen2

1 Linus Pauling Institute, Oregon State University, Corvallis, Oregon, 2 Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.7
DOI:  10.1002/0471140856.tx1407s37
Online Posting Date:  August, 2008
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Abstract

The purpose of this protocol is to establish a primary hepatocyte culture system as a suitable model to examine age‐related changes in Phase II detoxication gene expression. Hepatocytes are isolated using a two‐step collagenase perfusion technique from young (3 to 6 months) and old (24 to 28 months) rats and placed in primary culture using collagen (Type I)‐coated plates as the extracellular matrix. A supplemented William's E Medium is used as the medium. This culture system maintains hepatocyte viability from both young and old rats for ∼60 hr, as measured by lactate dehydrogenase activity, while also maintaining their respective phenotypes relative to Phase II detoxification. We thus conclude that a collagen‐based cell culture system is suitable to study age‐associated deficits in Nrf2/ARE‐mediated Phase II gene regulation provided that experiments can be conducted within 60 hr after cell isolation. Curr. Protoc. Toxicol. 37:14.7.1‐14.7.10. © 2008 by John Wiley & Sons, Inc.

Keywords: collagen Type 1; William's Medium E; primary hepatocyte cell culture; aging; Phase II detoxification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolating Hepatocytes from Young and Old Rats
  • Basic Protocol 2: Culturing Primary Rat Hepatocytes on Collagen‐Coated Plates
  • Support Protocol 1: Determination of Hepatocyte Viability by Lactate Dehydrogenase (LDH) Release
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Isolating Hepatocytes from Young and Old Rats

  Materials
  • Pre‐Hanks' 1× solution (see recipe), prewarmed to 37°C and bubbled for 30 min with carbogen gas
  • Hanks' I solution with BSA and EGTA (see recipe)
  • Hanks' II solution with CaCl 2 (see recipe)
  • Heparin (0.2% in saline; sterile filtered)
  • Young (4 to 6 months old) and/or old (24 to 28 months old) Fischer 344 rats
  • Ethyl ether
  • Collagenase D (Boeheringer‐Manheim)
  • Krebs' 1× solution, pH 7.4 (see recipe), bubbled for 30 min with carbogen gas
  • DNase I (Sigma)
  • 0.4% (w/v) trypan blue
  • 37°C water bath
  • Perfusion apparatus including:
    • Peristaltic pump and tubing
    • Glass vial for bubble trap
  • Inverted microscope
  • Cotton swabs
  • Vacuum aspirator
  • Carbogen tank and tubing
  • 1‐ml syringe
  • Anesthetizing chamber
  • Nose cone
  • Dissecting steel pan (sacrifice tray)
  • Tape
  • Dissection tools including:
    • Surgeon's scissors
    • Cross‐action forceps
    • Iris scissors
    • Blunt forceps
    • Hemostats
    • 18‐G and 21‐G surgical needles for designing cannulae for young and old rats, respectively
    • 23‐G needle for heparin injection
  • Kimwipes
  • 0.4‐mm surgical sutures
  • Glass rod
  • Gauze pads
  • 500‐ml Erlenmeyer flask
  • 50‐ml round‐bottom flask
  • Rotavapor (Büchi Laborteknik AG)
  • 0.5‐ml microcentrifuge tube
  • Hemacytometer and coverslip

Basic Protocol 2: Culturing Primary Rat Hepatocytes on Collagen‐Coated Plates

  Materials
  • Collagen I (rat tail; Sigma)
  • 50 mM HCl
  • Sterile water
  • Hanks' Balanced Salt Solution (HBSS; Sigma)
  • Complete William's Medium E (Sigma; see recipe)
  • Hepatocyte cell suspension (see protocol 1)
  • 37°C water bath
  • 6‐well plastic cell culture dishes (such as Corning or Nunc brands)
  • Laminar flow hood
  • Pipet tips, sterile

Support Protocol 1: Determination of Hepatocyte Viability by Lactate Dehydrogenase (LDH) Release

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10% Triton X‐100
  • Potassium phosphate buffer (0.1 M, pH 7.4)
  • NADH, 20 mM
  • 20 mM sodium pyruvate
  • 1‐ml microcentrifuge tube
  • UV spectrophotometer
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Figures

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Literature Cited

Literature Cited
   Balaban, R.S., Nemoto, S., and Finkel, T. 2005. Mitochondria, oxidants, and aging. Cell 120:483‐495.
   Beckman, K.B. and Ames, B.N. 1998. The free radical theory of aging matures. Physiol. Rev. 78:547‐581.
   Harman, D. 1996. Aging and disease: Extending functional life span. Ann. N. Y. Acad. Sci. 786:321‐336.
   Humphries, K.M., Szweda, P.A., and Szweda, L.I. 2006. Aging: A shift from redox regulation to oxidative damage. Free Radic. Res. 40:1239‐1243.
   Kitano, S., Fawcett, T.W., Yo, Y., and Roth, G.S. 1998. Molecular mechanisms of impaired stimulation of DNA synthesis in cultured hepatocytes of aged rats. Am. J. Physiol. 275:C146‐C154.
   Lambert, A.J. and Merry, B.J. 2000. Use of primary cultures of rat hepatocytes for the study of ageing and caloric restriction. Exp. Gerontol. 35:583‐594.
   Li, J. and Holbrook, N.J. 2004. Elevated gadd153/chop expression and enhanced c‐Jun N‐terminal protein kinase activation sensitizes aged cells to ER stress. Exp. Gerontol. 39:735‐744.
   Liu, Y., Guyton, K.Z., Gorospe, M., Xu, Q., Kokkonen, G.C., Mock, Y.D., Roth, G.S., and Holbrook, N.J. 1996. Age‐related decline in mitogen‐activated protein kinase activity in epidermal growth factor‐stimulated rat hepatocytes. J. Biol. Chem. 271:3604‐3607.
   Moldeus, P., Hogberg, J., and Orrenius, S. 1978. Isolation and use of liver cells. Meth. Enzymol. 52:60‐71.
   Stevenson, D.J., Morgan, C., McLellan, L.I., and Helen Grant, M. 2007. Reduced glutathione levels and expression of the enzymes of glutathione synthesis in cryopreserved hepatocyte monolayer cultures. Toxicol. In Vitro 21:527‐532.
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