This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.

The aim of this study was to assess the efficacy of the combination of endoscopic variceal ligation (EVL) and partial splenic embolization (PSE) compared with EVL alone in cirrhosis patients with thrombocytopenia. In a prospective study, 84 cirrhosis patients with esophageal varices and thrombocytopenia (platelet count < 50,000/mm(3)) underwent EVL plus PSE (N = 42) or EVL alone (N = 42). Primary end points assessed during the follow-up period included the recurrence of varices, progression to variceal bleeding, and death. Comparison between combined treatment and variceal ligation alone by multivariate analysis showed a hazard ratio of 0.44 for the recurrence of varices (P = 0.02), 0.19 for progression to variceal bleeding (P = 0.01), and 0.31 for death (P = 0.04). These results suggest that the combination of EVL plus PSE can prevent the recurrence of varices, progression to variceal bleeding, and death in cirrhosis patients with esophageal varices and thrombocytopenia.

Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-β receptor (PDGF-βR), α-smooth muscle actin (α-SMA), and type I collagen (α1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-γ or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (α1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-γ (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (α1), PDGF-βR, and α-SMA expression and suppressed TGF-β1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

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