Isolation and primary culture of rat Kupffer cells

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48-110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 microm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80-110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80-120 x 10(6) Kupffer cells per liver.