Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

Robert E. Lanford, Kenneth D. Carey, Larry E. Estlack, G. Con Smith, Rick V. Hay

    Research output: Contribution to journalArticlepeer-review

    87 Scopus citations

    Abstract

    The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [35S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [35S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B h, E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes.

    Original languageEnglish (US)
    Pages (from-to)174-182
    Number of pages9
    JournalIn Vitro Cellular & Developmental Biology
    Volume25
    Issue number2
    DOIs
    StatePublished - Feb 1989

    Keywords

    • hepatocytes
    • lipoprotein synthesis
    • serum-free medium

    ASJC Scopus subject areas

    • Developmental Biology
    • Clinical Biochemistry
    • Cell Biology

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