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.

Hepatocyte transplantation (HTX) has received great expectation for the treatment of a wide spectrum of liver diseases. Considering the severe shortage of human livers for hepatocyte isolation, porcine hepatocytes are an attractive alternative to normal human hepatocytes. To develop such therapy, establishment of an efficient hepatocyte isolation and transplantation model that enables accurate assessment of safety and efficacy of HTX is extremely important. Porcine hepatocytes were isolated from a surgically removed liver segment with a four-step retrograde perfusion using dispase and collagenase. The resultant hepatocytes of > 84% viability were used for transplantation experiment in a pig model of acute liver failure induced by intravenous administration of D-galactosamine (D-gal) (0.5 mg/kg). Twenty-four hours after D-gal injection, transplantation of freshly isolated porcine hepatocytes (1 x 10(9)) was safely conducted and prolonged the survival of D-gal-treated pigs. We describe an efficient porcine hepatocyte isolation and subsequent cell transplantation in pigs with D-gal-induced liver failure.

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 µg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 µg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 µg/ml) would be a useful cold preservation means for the development of cell therapies.Key words: Hepatocytes; University of Wisconsin solution; Ascorbic acid-2 glucoside; Cold preservation INTRODUCTIONcryopreservation method, and 3) establishment of a useful gene transfer technique for the isolated and/or cultured hepatocytes. Therefore, in the present study we inThe use of normal human hepatocytes in liver-targeted cell therapies is severely limited by worldwide donor vestigated an efficient procedure for isolating hepatocytes from a surgically resected segment of the pig liver. Deliver shortage. Considering the present status, it is critical to efficiently utilize such scarce donor livers for cell velopment of an effective preservation solution allows transportation of primarily isolated hepatocytes while therapies. What remains to be seen for the resolution are the following: 1) establishment of an excellent isolation maintaining adequate quality between one center where hepatocyte isolation and/or culture can be performed and procedure for functional and uniform hepatocyte preparation, 2) development of an effective cold storage and the other where such cells are actually utilized. Thus, we The resultant hepatocytes with more than 90% viabilCo., LTD., Tokyo, Japan) was intramuscularly used to ity assessed by a trypan blue exclusion test were subput the animals under sedation. An auricular vein was jected to a cold storage experiment. The following four secured to give droperidol 0.5 mg/kg (Sankyo Co.) and preservation s...

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G 1 phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirusbased p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immunofluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G 1 checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.