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Cell populations derived from adult tissue and stem cells possess a great expectation for the treatment of several diseases. Great efforts have been made to generate cells with therapeutic impact from stem cells. However, it is clear that the development of systems to deliver such cells to induce efficient engraftment, growth, and function is a real necessity. Biologic and artificial scaffolds have received significant attention for their potential therapeutic application when use to form tissues in vitro and facilitate engraftment in vivo. Ultimately more sophisticated methods for decellularization of organs have been successfully used in tissue engineering and regenerative medicine applications. These decellularized tissues and organs appear to provide bioactive molecules and bioinductive properties to induce homing, differentiation, and proliferation of cells. The combination of decellularized organs and stem cells may dramatically improve the survival, engraftment, and fate control of transplanted stem cells and their ultimate clinical utility, opening the doors to a new era of organ engineering.

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter-based cell sorting. ES cell-derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell-derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell-derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

Development of a subcutaneously implantable bioartificial pancreas (BAP) with immunoisolatory function could have a great impact on the treatment of diabetes mellitus. We have developed an implantable BAP device with an ethylene vinyl alcohol (EVAL) membrane. In the present study, we used basic fibroblast growth factors (bFGF), which was incorporated in a carrier for sustained release, in order to induce neovascularization when the device was implanted subcutaneously. To maintain the vasculature thus formed, a cell infusion port was attached to the BAP device, through which the device was filled with human liver vascular endothelial cell line TMNK-1, and the vasculature could be adequately maintained. Mice were divided into the following three groups. In group 1, a bFGF-free BAP device was implanted subcutaneously. In group 2, a sustained-release bFGF-impregnated BAP device was implanted. In group 3, a sustained-release bFGF-impregnated BAP device was implanted, and 3 x 10(6) TMNK-1 cells were infused into the implanted device every week. Neovascularization induced in the subcutaneous tissue around the implanted BAP device was macroscopically examined and histologically evaluated. In addition, the tissue blood flow was measured using a laser blood flow meter. In mice in group 3, neovascularization was significantly induced and maintained until week 8 postimplantation. It was confirmed by scanning electron microscopy that infused TMNK-1 cells adhered to the inner polyethylene surface of the device. It was demonstrated that the use of bFGF and vascular endothelial TMNK-1 cells induced and maintained adequate vasculature and tissue blood flow surrounding the implantable bag-type BAP device. We believe that the present study will contribute to BAP development for the treatment of diabetes.

Since Berry and Friend developed methods to isolate hepatocytes from the liver by a collagenase digestion technique in 1969, studies in laboratory animals have demonstrated that hepatocyte transplantation could potentially be used for the treatment of liver failure and inborn errors of liver-based metabolism. Healthy human hepatocytes are an ideal source for hepatocyte transplantation; however, their relative scarcity is one of the major drawbacks, further compounded by the competing demands of liver transplantation. Notably, most of the hepatocytes are isolated from discarded livers that are not suitable for organ transplantation for a variety of reasons, including excessive fat content. Importantly, the hepatocyte isolation procedure itself exerts major stress on hepatocytes by the disruption of cell-to-cell and cell-to-matrix contacts, resulting in hepatocytic apoptosis. Prevention of apoptosis would maximize yield of healthy cells and maintain hepatocyte differentiated function in culture. In this review, we describe methods to prevent apoptosis by utilizing both antiapoptotic molecules and matrices. We also introduce a new type of liver tissue engineering, hepatocyte sheet transplantation, which utilizes unwoven cloth having a cellular adhesive property.

Recently, much attention has been drawn to liver-targeted regenerative medicine for the treatment of liver failure. Researchers in various fields consider that the following cells can be used for such therapy: human embryonic stem (ES) cells, somatic stem cells, hepatic stem cells, small hepatocytes, bone marrow- and cord blood-derived hepatic progenitor cells, and human hepatocyte cell lines. The Cre/loxP-based reversible immortalization of human hepatocytes is also introduced in this review article. Here we describe the candidates that can contribute to hepatic regeneration. This reversible immortalization system allows us to establish a highly safe human hepatocyte line that is capable of differentiation at a lower cost and on a larger scale.

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 microg/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 microg/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 microg/ml) would be a useful cold preservation means for the development of cell 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 degrees 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.