Yoshikazu Kosaka

Cryopreservation of primarily isolated porcine hepatocytes with UW solution.

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: Dulbeccos modified Eagles 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.

Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside.

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) Dulbeccos modified Eagles 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.

Lentiviral vector: A useful tool for transduction of human liver endothelial cells

Endothelial cells play multiple roles in pathophysiologic processes and are increasingly being recognized as target cells of gene therapy. Lentiviral vectors derived from human immunodeficiency virus type 1 have an ability to infect both dividing and nondividing cells and currently receive a great deal of attention as an innovative tool for transduction of target cells. The purpose of the present work was to evaluate the efficacy of a lentiviral vector for transducing human liver endothelial cells (HLECs) in vitro. For the present study, a pseudotyped lentiviral vector encoding a green fluorescent protein (GFP) gene, LtV-GFP, was generated by means of FuGENE 6 method and allowed to infect HLECs. Approximately 95% of HLECs were positive for GFP expression after LtV-GFP infection at a multiplicity of infection of 10. Notably, LtV-GFP transduced HLECs had stable and long term GFP expression, showed gene expression of endothelial markers including CD 34, factor VIII, flt-1, KDR/flk-1 and HGF, and maintained in vitro angiogenic potential in a Matrigel assay to the same extent as primarily cultured HLECs. These findings provide evidence that lentivirus based gene delivery is an efficient tool for transduction of endothelial cells that could be considered for cell and gene therapies and hybrid artificial organs.

Improvement in the differentiated hepatic phenotype of immortalized human hepatocytes by adenovirus mediated p21 gene transfer.

The p21 molecule, a potent cyclin dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle and is involved in terminal cellular differentiation. The overexpression of p21 has been shown to induce differentiation in various cell lines. We have made an effort to establish a reliable human hepatocyte cell line as a source of hepatic function in bioartificial liver (BAL) therapy. In this work, we investigated the effect of p21 on the differential phenotype of simian virus 40 large T antigen (SV40Tag) immortalized human hepatocytic NKNT-3 cells. A recombinant adenoviral vector expressing a p21 gene under control of the cytomegalovirus (CMV) promoter (Ad-p21) was used to efficiently transfer genes into NKNT-3 cells. The morphologic alterations, the cell cycle progression, and the expression of p-450 associated enzymes (CYPs) were carefully examined in NKNT-3 cells that had been infected with Ad-p21. Adenovirus mediated gene delivery of p21 was efficiently achieved in NKNT-3 cells without affecting cellular structure. After Ad-p21 infection, NKNT-3 cells were G0/G1 arrested in cell cycle analysis. NKNT-3 cells that had been infected with Ad-p21 showed differentiated hepatic phenotypes in morphology and improvement in protein expression of CYP 3A4 and CYP 2C9. In the present work, we demonstrate that the exogenous expression of p21 enhances the differential phenotype of immortalized hepatocytic NKNT-3 cells.