Primary Human Hepatocytes: Isolation, Culture And Reversible Immortalization

Part One:Primary Porcine Hepatocytes Isolation, Culture and Reversible Immortalization[Aims] To establish a method of porcine hepatocyte isolation, and subsequently to develop reversible immortalized porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T)[Methods] Porcine hepatocytes were isolated with a revised four-step retrograde perfusion method. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination.[Results] Porcine hepatocytes were isolated from six pig tissue samples (Experimental Animal Center, Tongji Medical University) using the four-step retrograde perfusion method. The method resulted in a hepatocyte yield of 9.4±7.5×106 cells/g liver. The viability of the cells was 94.6±4.2%. The resultant hepatocytes with high viability were successfully immortalized with retroviral vector SSR#69. The immortalized clones showed the typical morphological appearance and were selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes.[Conclusions] In conclusion, we herein describe a modified method of hepatocyte islolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination. Part Two:Isolation and Culture of Primary Human Hepatocytes[Background and Aims] There are few studies investigating resected specimens with hepatic benign disease on hepatocyte isolation and therefore, available data about the effect of the donor liver on isolated hepatocyte yield are very sparse. To improve cell availability, we investigate the influence of warm and cold ischemia time and liver donor characteristics on the outcome of freshly isolated hepatocytes from surgically removed liver tissue.[Methods] Hepatocytes were isolated from the livers of patients with calculus of intrahepatic duct and hemangiomas with a revised four-step retrograde perfusion method. We investigate the influence of warm and cold ischemia time and liver donor characteristics on the outcome of freshly isolated hepatocytes from surgically removed liver tissues.[Results] Samples were collected from a total of 13 patients with calculus of intrahepatic duct (n=3) and hemangiomas of liver (n=10) undergoing partial hepatectomy in Tongji Hospital (Wuhan, China). Thirteen liver specimens, weighing 15g to 42 g, were immediately taken from the margin of the removed samples and placed into ice-cold ringer's lactate solution to transfer to the laboratory. The warm ischemia time (WIT) was 10-35 min, and the cold ischemia time was 15-35 min. The method resulted in a total cell yield of 4.8±2.1×106 cells/g liver. The viability of the cells was 78.1±10.4%(62%-93%).[Conclusions] Data from our work revealed that patient sex, age and blood group have no correlation with cell yield and viability. A four-step retrograde perfusion method could isolate high quality hepatocytes, but the procedure should be modified for small specimens without a visible vessel orifice on the cut surface. Warm ischemia time can affect the outcome of hepatocytes isolation. we considered that mild cirrhotic livers shouldn't be arbitrarily excluded from cell isolation. Part Three:Reversible Immortalization of Primary Human Hepatocytes[Background and Aims] The utility of cultured hepatocytes is hampered by difficulties in timely obtaining populations of primary cells which have a limited life span in vitro. An attractive alternative source of hepatocytes would be immortalized cells which could make unlimited supplies of cells feasible and exhibit the characteristics of differentiated hepatocytes. Cre/LoxP-mediated reversible immortalized hepatocytes with SV40T could provide an unlimited supply of cells for research and clinical use.[Methods] Primary human hepatocytes were immortalized with retro viral vector SSR#69 expressing S V40T and hygromycin-resistance genes flanked by paired loxP recombination targets. The immortalized clones showed the typical morphological appearance were selected by clone rings and expanded in culture. Then,SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination.[Results] The freshly isolated primary human hepatocytes with high viability were successfully immortalized with retro viral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. The immortalized clones showed the typical morphological appearance and were selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes.[Conclusions] In conclusion, we herein successfully established reversible immortalization of primary human hepatocytes mediated by retro viral transfer and site-specific recombination. Furthermore, the established method of reversible immortalization would be used for transfering SV40T/hTERT.