Establishment Of A Hepatocyte Line From Human

The bioartificial liver support system (BALs) is a promising therapy for the treatment of acute hepatic failure(AHF) and fulminant hepatic failure(FHF) with the developments in isolation of hepatocyte, culture of high density and bioreactor. The promising effects have been confirmed in animal experiments and clinical tests. As a main biomaterial of BALs, the hepatocytes perform an important role. Although primary human hepatoyctes are most suitable to BALs and the extracorporeal model of drug metabolism, they have many disadvantages such as limited lifespan, functional activities decline rapidly after several days in culture and the organ shortage all over the world. So it is too difficult to isolate normal human hepatocyte applicated in BALs. As another ideal hepatocyte resource for BALs and hepatocyte transplantation, the fetal heaptocytes not only maintain the functions of proliferation and differentiation in extracorporeal culture, but also have a lower immunogenicity. Because of ethnical problems, the fetal hepatocyte can't applicated in clinical therapy.Many researcher around the world want to use immortal hepatocyte line such as C3A, HepG2 and CL-1 to overcome above difficulties. Extracorporeal bioartificial liver support system(EBLSS) has been established with immortal C3A and HepG2 cell line for treatment of FHF animal models and the II clinical test of EBLSS with C3A cell has been approved in USA. In order to Wang's report, the immortalized human hepatoma cell lineC3A has lower viability, metabolic capacity and activities in cytochrome P450 compared toprimary porcine hepatocytes. For these reasons, hepatocytes isolated from the liver of a normal 25-year old male donor were transfected with pcDNA3.1(-) recombined vector containing the genes encoding Simian Virus 40 large T antigen(SV40LTag ). One of the resulting hepatocyte clone, has shown highly differentiated liver function and immortalized characteristics. Materials and MethodsThe reagents were purchased as follows: SV40 viral DNA(strain 776), lipofectAMINE 2000, collagenase IV, G418, hepatoZYME-SFM, pcDNA3.1(-) vector and DMEM medium from Invitrogen company(USA); fetal bovine serum from Hyclone company(USA ); QIAquick gel extraction kit, QIAprep spin miniprep kit and QIAquick DNA purify kit from QIAGEN company(USA); restriction endonuclease BamH, BsfX I and T4 DNA ligase from Promega company(USA); cell culture flasks and plates from Nunc Medos Company (Australia). The hepatocytes were isolated from the liver of 25-year old normal male donor by the standard two-step collagenase perfusion technique.SV40LTag DNA was obtained from the complete sequence of SV40 viral DNA (strain 776) by the method of polymerase chain reaction (PCR). The recombined vector containing pcDNA3.1(-) vector and SV40LTag DNA was constructed with T4 DNA ligase and expressed in the competence cells of E. coli DH5a. The SV40LTag-pcDNA3.1(-) vector was characterized by DNA sequencing with Ganger's dideoxy chain termination composition method and compared with nucleic acid sequences in the GenBank. Hepatocytes from human liver were transfected by a lipofection method with the recombined vector containing both SV40LTag and the bacterial neomycin phosphotransferase gene, which confers resistance to geneticin G418.ResultsThe sequence of SV40LTag DNA of the recombined vector extracted from positive bacteria is similar to shown in GenBank. One line of surviving cells after 42 day' selection of 700-300Mg/mL G418, grew steadily in the chemically defined serum-free hapatoZYME-SFM medium. The immortalized hepatocyte appeared epithelial and displayed morphologic characteristics of liver parenchymal cells, such as a large round nucleus with a few nucleoli and many cytoplasmic granules. The speed of cell proliferation of hepatocyte cultured in the lower sugar DMEM medium containing 15% FBS is faster than in hepatozYME-SFM medium. Similarly, the time of 3 to 4 days of cell passage cultured in DMEM medium is shorter than cultured in hepatoZYME-SFM medium. The new establ...