In vitro Model for Ischemia-Reperfusion Injury of Cultured Kupffer Cells

Hong, Jeong Sook; Sung, Hoon Ki; Song, In Hwan; Kim, Joo Young; Jee, Dae Lim; Sung, Eon Gi

Abstract

The aims of this study were to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells, and were to develop in vitro system which could provide a tool for the study of ischemia-reperfusion injury. Kupffer cells were isolated following sequential collagenase digestion of the liver by perfusion and enrichment of a nonparenchymal cell fraction by a double-densities gradient centrifugation step using Percoll and were selected by allowing them to adhere to culture vessel for 2 h at 37 degrees C under 5% CO2. The purity of obtained Kupffer cell was about 90% assessed by the phagocytosis of 3 micrometer latex beads. This method for Kupffer cell isolation resulted in yields of 1~5 x10(7) Kupffer cells per liver and Kupffer cells were preserved in maintenance cultures for 10 days. The phagocytic capacity of cultured Kupffer cells was measured according to the amount of latex beads incorporated into the cytoplasm. Larger round Kupffer cells in the culture had higher phagocytic capacity compared with smaller round or irregular shaped Kupffer cells. The different phagocytic capacity of Kupffer cells which was dependent on size and shape in vivo was well preserved during culture. The experimental group of Kupffer cells in culture were sequentially treated with ischemia and reperfusion at 1h and 30 min. The ratio of Kupffer cells having latex beads in their cytoplasm was significantly increased compared with control (p<0.01). This result was able to explain the Kupffer cells' activation after ischemia-reperfusion injury in vivo. In conclusion, Kupffer cells in this culture well resembled the cells in vivo and this in vitro model could provide a valuable tool for the study of Kupffer cells with a key role in pathophysiology of ischemia-reperfusion injury.