Isolated hepatic lipocytes and kupffer cells from normal human liver: Morphological and functional characteristics in primary culture

Authors

  • Scott L. Friedman M.D.,

    Corresponding author
    1. University of California, San Francisco, Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, San Francisco
    • UCSF Liver Center Laboratory, Building 40, Room 4102, San Francisco General Hospital, San Francisco, CA 94110
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  • Don C. Rockey,

    1. University of California, San Francisco, Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, San Francisco
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  • Richard F. McGuire,

    1. University of California, San Francisco, Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, San Francisco
    Current affiliation:
    1. 400 West Central Avenue, Suite 206, Brea, CA 92621
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  • Jacquelyn J. Maher,

    1. University of California, San Francisco, Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, San Francisco
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  • Janet K. Boyles,

    1. Gladstone Foundation Laboratories and Department of Pathology, University of California, San Francisco, San Francisco, CA 94110
    Current affiliation:
    1. The Vanderbilt University School of Medicine, 203 Light Hall, Garland Ave., Nashville, TN 37232–0685
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  • Glenn Yamasaki

    1. University of California, San Francisco, Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, San Francisco
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Abstract

The development of techniques for isolating hepatic lipocytes (Ito, stellate or fat-storing cells) from rodents has been instrumental in defining their role in hepatic vitamin A storage and fibrogenesis. In this study, we developed a method for the purification of lipocytes and Kupffer cell from wedge sections of normal human liver and examined their properties in primary culture. Sections of donor liver (400 to 600 gm) harvested but not used for transplantation were perfused in situ with University of Wisconsin solution and used for lipocyte isolation within 48 hr. Cells were isolated by catheter perfusion of the wedge through several large vessels with L-15 salts, Pronase and collagenase, followed by Larex density gradient centrifugation. Lipocytes were plated on either uncoated plastic or a basement membrane-like gel. Lipocyte and Kupffer cell yields were 2.3 ± 0.6 × 105 and 8.6 ± 1.4 × 105 cells, respectively, per gram of liver (n = 5). Lipocyte purity was 91% as assessed by vitamin A autofluorescence, and Kupffer cell purity was 83% as determined by uptake of fluorescinated staphylococci. Lipocytes cultured on the plastic spread within 48 to 72 hr, displaying slightly more heterogeneous retinoid droplet size than comparable rat cells; on a basement-membrane gel, the cells remained aggregated and spherical with occasional spindlelike extensions. Lipocytes on plastic expressed procollagens I and III, collagen IV and laminin by immunocytochemistry, and types I, III and IV procollagen messenger RNAs by RNAse protection, Northern blot and polymerase chain reaction, respectively. Transmission electron microscopy of lipocytes at 7 days demonstrated a prominent rough endoplasmic reticulum and contractile filaments. Scanning electron microscopy revealed a smooth cell surface with perinuclear droplets beneath the cell membrane. With continued primary culture on plastic (more than 7 days), cells appeared “activated” (i.e., increased spreading and diminished retinoid droplets) and began proliferating as assessed by nuclear autoradiography and [3H]thymidine incorporation. Kupffer cells observed by scanning electron microscopy in early primary culture displayed prominent membrane ruffling and lamellipodia. In summary, we have established a reproducible method for the isolation and primary culture of human lipocytes and Kupffer cells. (HEPATOLOGY 1992;15:234–243).

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