title: Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes.
abstract: 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.
authors: 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.
authors: XiaoPing
authors: X
authors: Pan
authors: 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.
authors: Yini
authors: Y
authors: Wang
authors: 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.
authors: XiaoPeng
authors: X
authors: Yu
authors: 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.
authors: JianZhou
authors: J
authors: Li
authors: 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.
authors: Ning
authors: N
authors: Zhou
authors: 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.
authors: WeiBo
authors: W
authors: Du
authors: 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.
authors: YanHong
authors: Y
authors: Zhang
authors: 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.
authors: HongCui
authors: H
authors: Cao
authors: 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.
authors: DanHua
authors: D
authors: Zhu
authors: 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.
authors: Yu
authors: Y
authors: Chen
authors: 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.
authors: LanJuan
authors: L
authors: Li
mesh_terms: D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D015870:Gene Expression; D055166:Hepatic Stellate Cells / Q000166:cytology*; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D008099:Liver / Q000166:cytology; D061251:Primary Cell Culture
chemical_list: D000952:Antigens, Polyomavirus Transforming
keywords: bioartificial liver; co-culture; human hepatic stellate cells; immortalization; immortalized human hepatocytes; simian virus 40 large T antigen