Long-term preservation of high endocytic activity in primary cultures of pig liver sinusoidal endothelial cells

Abstract

Together with Kupffer cells, liver sinusoidal endothelial cells (LSECs) constitute the most powerful scavenger system in the body. However, studies on LSEC function are hampered by the fact that the cells lose their scavenger ability and start deteriorating after a few days in culture. The purpose of the present study was to improve the conditions of cultivation to prolong the survival of pig LSECs in vitro. We used the high capacity receptor-mediated endocytosis of soluble waste molecules as a marker for functionally intact cells in the cultures. Compared with two commercially-, and two other media specifically designed for use with either SECs or hepatocytes from rat, our newly developed serum-free medium, DM 110/SS, devoid of any components of animal origin, was superior in maintaining the endocytic activity. Of six growth factors studied for their effect on endocytosis, basic fibroblast, and recombinant epidermal, but not vascular endothelial growth factor, were found to be most beneficial. After 8 days in DM 110/SS, LSECs maintained endocytosis via the scavenger receptor, mannose receptor, collagen alpha-chain receptor and the Fc-gamma receptor. All endocytosed ligands, except for aggregated IgG were degraded in 8-day-old cultures. Using the new medium, the cells endocytosed ligands for up to 20 days, and survived for at least an additional 10 days, albeit without the high endocytic activity typical of intact LSECs. Importantly, DNA synthesis in prolonged cultures of LSECs was observed only when maintained in DM 110/SS medium. In conclusion, we describe a protocol for the maintenance of LSECs in culture for the longest period yet reported.