Isolation And Culture Of Kupffer Cells And Its Role And Mechanism In Allo-geneic Liver Transplantation

Part one: Isolation purification and culture of kupffer cells from the rat and human liverObjective:To establish a reliable and simple technique for isolation and purification of rat kupffer cells, then to explore an efficient and convenient way to isolate and purify human kupffer cells from the liver biopsies specimen of patients undergoing hepatic surgery, so as to further investigate the biological function of the kupffer cells in liver transplantation.Methods:Livers got from twenty-four SD rats and six cases of human liver biopsies specimen were used in this experiment. The rat liver was separated and perfused through hepatic portal vein with collagenase to obtain liver cell suspension, while human liver wedge biopsies were perfused via hepatic vein and cut into small tissue fragments to be further digested with collagenase at 37℃ for 30 minutes to get liver cell suspension. The filtrated cell suspension was centrifuged at 50g to remove hepatocytes, Then the non-parenchymal cells was collected and resuspended in 25%(v/v) percoll solution. Lastly kupffer cells were separated from other non-parenchymal cells by inconsequential density gradient centrifugation with percoll, and the kupffer cells were further enriched by selective attachment according to attaching time and culture conditions. Kupffer cells were identificated by endocytosis, and the viability was determined by Typan blue exclusion staining.Results:...