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Purification of cultured primary rat hepatocytes using selection with ricin a subunit

Authors

  • David E. Johnston M.D.,

    Corresponding author
    1. Gastroenterology Division, Department of Medicine, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111
    • Division of Gastroenterology, University of New Mexico, ACC-5th Floor, 2211 Lomas Blvd. NE, Albuquerque, NM 87131–5271
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  • Rahul Jasuja

    1. Gastroenterology Division, Department of Medicine, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111
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  • Part of this work was presented at the meeting of the American Association for the Study of Liver Diseases, Chicago, Illinois, November 1–3, 1992, and published in abstract form (Hepatology 1992;16:142A)

Abstract

We had found several methods of hepatocyte isolation to be inadequate for removing Kupffer and endo thelial cells and so developed a method of selectively killing these nonparenchymal cells in hepatocyte cultures using the known selective uptake and toxicity of the protein synthesis inhibitor ricin and its A chain by nonparenchymal cells. Kupffer cells were quantitated with the Ku-1 monoclonal antibody. Endothelial and Kupffer cells were identified by means of fluorescence microscopy after uptake of fluorescent-labeled, acetylated low-density lipoprotein. Freshly isolated hepatocyte suspensions contained 21% ± 1.2% (mean ± S.E.M.) as many nonparenchymal cells as hepatocytes (n=12), including 7.6% ± 1.0% as many Kupffer cells as hepatocytes (n=10). In monolayers of hepatocytes maintained up to 48 hr in serum-free medium, 3% to 7% of cells were Ku-1 positive, and 2% to 11% took up fluorescence-labeled, acetylated low-density lipoprotein. Purification of hepatocytes using Percoll densitygradient centrifugation, elutriation rotor or other means was only partially effective. Hepatocyte monolayers in serum-free medium were incubated with various concentrations of ricin A chain for 1 hr, then washed and studied up to 48 hr later. Optimal treatment with 1.0 ng/ml ricin A chain resulted in decreased nonparenchymal cells 24 hr later and nearly complete loss of Kupffer and endothelial cells at 48 hr. Hepatocyte morphology and protein synthesis were unchanged. Ricin A chain can be used to eliminate Kupffer and endothelial cells from hepatocyte cultures. (Hepatology 1994;20:436–444.)

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