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Isolation of Liver Kupffer Cells

Matthias Froh1,  Akira Konno2,  Ronald G. Thurman2

1University of Regensburg, Regensburg, Germany, Germany
2University of North Carolina, Chapel Hill, North Carolina


Publication Name: 
Current Protocols in Toxicology
Unit Number: 
UNIT 14.4
DOI: 
10.1002/0471140856.tx1404s14
Print Publication Date: 
November, 2002
Online Posting Date: 
February, 2003
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Abstract

Isolation of Liver Kupffer Cells (Matthias Froh, Akira Konno, Ronald G. Thurman, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of several diseases. This unit contains an easy-to-follow procedure for effective isolation of liver Kupffer cells from rats and mice. The protocol provides viable Kupffer cells in large amounts that can be used for further investigations.

     
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Table of Contents

  • Basic Protocol
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
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Materials

 Basic Protocol
 Materials
  • Rat
  • 50 mg/ml Nembutal sodium solution (Abbott Laboratories)
  • 100% (v/v) ethanol
  • CMF-HBSS (see recipe), 37°C
  • 0.02% (w/v) collagenase type IV in HBSS (see recipe), made fresh
  • PBS (see recipe)
  • HBSS (see recipe)
  • 50%/25% two-step Percoll gradient (see recipe) in 50-ml conical centrifuge tubes
  • 0.4% (w/v) trypan blue solution (Sigma)
  • RPMI medium 1640 (Life Technologies) containing 1× antibiotic and antimycotic solution (Sigma) and 10% (v/v) fetal bovine serum (FBS; Life Technologies)
  • 1/2-cc insulin syringe
  • Shaver
  • Surgical tools (sterile), including:
  •     Scissors
  •     Forceps
  • Pins or dots, sterile
  • Sterile threads (~15 to 20 cm long)
  • 20-G catheter
  • Perfusion system that allows regulation of the flow rate
  • Petri dishes, sterile
  • Nylon gauze (50-µm mesh width), sterile
  • 50-ml conical centrifuge tubes
  • Large-capacity centrifuge with adjustable brake, swing-out rotor, and speed up to 2000 × g, 4°C
  • Glass or plastic coverslips or tissue culture dishes, sterile
  • Additional reagents and equipment for counting cells with a hemacytometer and determining viability using trypan blue exclusion (appendix 3B)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 14.4.1
    Animal preparation and operation procedure. (A) Orientation and securing of the animal to the workstation. Recommended incision lines and intraperitoneal injection points are indicated. (B) Opened abdomen after median and horizontal incisions. (C) Loosely tied threads around the inferior vena cava (IVC) and portal vein. (D) Perfused liver after cutting the IVC and the abdominal artery and opening the thorax. The catheter is placed in the portal vein, fixed with a thread, and the IVC is ligated.

    View Image
  • Figure 14.4.2
    Isopycnic (gradient) centrifugation with Percoll. (A) The cell suspension, including mostly nonparenchymal cells (NPCs), is placed on a 50%/25% two-step Percoll gradient before centrifugation. (B) After centrifugation, the cells are separated into four fractions; fraction 1 has the highest concentration of Kupffer cells. PC, parenchymal cell.

    View Image
  • Figure 14.4.3
    Assessment of Kupffer cell (KC) purity and viability. (A) Cell count on a hemacytometer by trypan blue exclusion (viability check). Arrows in left and right panels indicate excluded cells (blue cells). (B) Purity check by the uptake of latex beads in KCs. Left panel is with and right panel is without a green filter, which shows the uptake of latex beads by their fluorescence.

    View Image

Literature Cited

 Literature Cited
    Alpini, G., Lenzi, R., Zhai, W.R., Liu, M.H., Slott, P.A., Paronetto, F., and Tavoloni, N. 1989. Isolation of a nonparenchymal liver cell fraction enriched in cells with biliary epithelial phenotypes. Gastroenterology 97:1248-1260.
    Alpini, G., Phillips, J.O., Vroman, B., and LaRusso, N.F. 1994. Recent advances in the isolation of liver cells. Hepatology 20:494-514.
    Blomhoff, R., Smedsrød, B., Eskild, W., Granum, P.E., and Berg, T. 1984. Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin. Exp. Cell Res., 150:194-204.
    Branster, M.V. and Morton, R.K. 1957. Isolation of intact liver cells. Nature 180:1283-1284.
    Brouwer, A., Barelds, R.J., and Knook, D.L. 1984. Centrifugal separations of mammalian cells. In Centrifugation: A Practical Approach. (D. Rickwood, ed.) pp. 183-218. IRL Press, Oxford.
    Eberth, C.J. 1867. Untersuchungen die ber die Leber der Wirbeltiere. Arch. Mikr. Anat. 3:423-440.
    Friedman, S.L. and Roll, F.J. 1987. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal. Biochem. 161:1233-1247.
    Jones, E.A. and Summerfield, J.A. 1988. The Liver: Biology and Pathobiology, 2nd ed. (I.M. Arias, W.B. Jakoby, H. Popper, D. Schachter, D.A. Shafritz, eds.) pp. 683- 704. Raven Press, New York.
    Knook, D.L. and Sleyster, E.C. 1976. Separation of Kupffer and endothelial cells of the rat liver by centrifugal elutriation. Exp. Cell Res. 99:444-449.
    Kupffer, C. 1876. Über die Sternzellen der Leber. Arch. Mikr. Anat. 12:353-358.
    Miller, S.B., Saccomani, G., Pretlow, T.P., Kimball, P.M., Scott, J.A., Sachs, G., and Pretlow, T.G. 1983. Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis. Cancer Res. 43:4176-4179.
    Morin, O., Patry, P., and Lafleur, L. 1984. Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry. J. Cell Physiol. 119:327-334.
    Munthe-Kaas, A.C., Berg, T., Seglen, P.O., and Seljelid, R. 1975. Mass isolation and culture of rat kupffer cells. J. Exp. Med. 141:1-10.
    Praaning-Van Dalen, D.P., De Leeuw, A.M., Brouwer, A., De Ruiter, C.F., and Knook, D.L. 1982. Ultrastructural and biochemical characterization of endocytic mechanisms in rat liver Kupffer and endothelial cells. In Sinusoidal Liver Cells. (D.L. Knook and E. Wisse, eds.) pp. 271-278. Elsevier Biomedical Press, Amsterdam.
    Rous, P. and Beard, J.W. 1934. Selection with the magnet and cultivation of reticulo-endothelial cells (Kupffer cells). J. Exp. Med. 59:577-591.
    Shaw, R.G., Johnson, A.R., Schulz, W.W., Zahlten, R.N., and Combes, B. 1984. Sinusoidal endothelial cells from normal guinea pig liver: Isolation, culture and characterization. Hepatology 4:591-602.
    Smedsrød, B. and Pertoft, H. 1985. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence. J. Leukoc. Biol. 38:213-230.
    Smedsrød, B., Pertoft, H., Eggertsen, G., and Sundström, C. 1985. Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll, and selective substrate adherence. Cell Tiss. Res. 241:639-649.
    Wake, K., Decker, K., Kirn, A., Knook, D.L., McCuskey, R.S., Bouwens, L., and Wisse, E. 1989. Cell biology and kinetics of Kupffer cells in the liver. Int. Rev. Cytol. 118:173-229.
    Wedl, K. 1854. Grundzüge der pathologischen Histologie. Gustav Fischer, Jena, Germany.
    Zahlten, R.N., Hagler, H.K., Nejtek, M.E., and Day, C.J. 1978. Morphological characterization of Kupffer and endothelial cells of rat liver isolated by counterflow elutriation. Gastroenterology 75:80-87.
 Key References
    Alpini et al., 1994. See above.

Describes different methods for and recent advances in the isolation of NPCs and PCs and provides background information on the characterization of individual liver cells.

    Smedsrød and Pertoft, 1984. See above.

Describes in detail the preparation of hepatocytes and reticuloendothelial cells using collagenase perfusion of the liver, isopycnic sedimentation in Percoll, and selective adherence of the cells—the method on which the Basic Protocol is based.

 Internet Resources
    http://www.apbiotech.com/na/

Web page of Amersham Pharmacia Biotech. A search using “Percoll” will lead to background information about the density-gradient medium.

    minf.vub.ac.be/~kupffer/

Home page of The Kupffer Cell Foundation. Good resource for background information about cells of the hepatic sinusoid.

For more than 30 years, Ron Thurman was an outstanding investigator in the fields of hepatic metabolism, alcoholic liver injury, and toxicology. With his passing, his large family of colleagues and friends lost a most productive and creative researcher and teacher

     
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