Int J Med Sci 2015; 12(3):248-255. doi:10.7150/ijms.11002
Establishment and Characterization of an Immortalized Human Hepatic Stellate Cell Line for Applications in Co-Culturing with Immortalized Human Hepatocytes
1. State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, China.
2. Collaborative Innovation Center for the Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
Pan X, Wang Y, Yu X, Li J, Zhou N, Du W, Zhang Y, Cao H, Zhu D, Chen Y, Li L. Establishment and Characterization of an Immortalized Human Hepatic Stellate Cell Line for Applications in Co-Culturing with Immortalized Human Hepatocytes. Int J Med Sci 2015; 12(3):248-255. doi:10.7150/ijms.11002. Available from http://www.medsci.org/v12p0248.htm
Background and objective. The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro.
Methods. Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells.
Results. The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-β), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-β1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes.
Conclusions. We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems.
Keywords: human hepatic stellate cells, simian virus 40 large T antigen, immortalization, immortalized human hepatocytes, co-culture, bioartificial liver