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We have developed a novel bioreactor based on the observation that isolated porcine hepatocytes rapidly and spontaneously aggregate into spheroids under oscillation conditions. The purpose of this study was to characterize the influence of oscillation frequency (0.125 Hz, 0.25 Hz), cell density (1-10 ؋ 10 6 cells/mL), and storage condition (fresh, cryopreserved) of porcine hepatocytes on the kinetics of spheroid formation. The viability and metabolic performance of spheroid hepatocytes was also compared to monolayer culture. We observed that both fresh and cryopreserved porcine hepatocytes began formation of spheroids spontaneously at the onset of oscillation culture. Spheroid size was directly related to cell density and time in culture, though inversely related to oscillatory frequency. Spheroid formation by fresh porcine hepatocytes was associated with decreased cell death (lactate dehydrogenase release, 1.3 ؎ 1.0 vs. 3.1 ؎ 0.7 U/mL, P < 0.05) and increased metabolic performance (albumin production, 14.7 ؎ 3.3 vs. 4.6 ؎ 1.4 fg/c/h, P < 0.0001; ureagenesis from ammonia, 267 ؎ 63 vs. 92 ؎ 13 mol/ L/h, P < 0.001) compared with monolayer culture. In conclusion, based on the favorable properties of rapid spheroid formation, increased hepatocellular function, and ease of scale-up, the spheroid reservoir bioreactor warrants further investigation as a bioartificial liver for support of liver failure. (Liver Transpl 2005;11:901-910.)

Cryopreserved porcine hepatocytes are a ready source of metabolic function for use in a bioartificial liver (BAL). However, cryopreservation is associated with a loss of hepatocyte viability. The mechanism of cell death during cryopreservation is incompletely understood, but may involve apoptosis through caspase activation. This study evaluates the cytoprotective effect of a global caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVADfmk) during cryopreservation of porcine hepatocytes. Freshly isolated porcine hepatocytes (viability, 97.4% ؎ 0.9%) were cryopreserved in 60 mol/L ZVAD-fmk (؉ZVAD group) or without ZVAD-fmk (؊ZVAD group) for 24 to 72 hours. Apoptotic and necrotic death were both observed after thawing and after 24 hours of culture. Caspase 3-like activity was significantly reduced by ZVADfmk, and was associated with improved viability and reduced apoptotic death of porcine hepatocytes after cryopreservation. Mitochondrial membrane potential (MMP) was increased in cultures of porcine hepatocytes that were cryopreserved in ZVAD-fmk. These results demonstrate the following: 1) Caspase 3-like protease activation and apoptosis occurs in porcine hepatocytes during cryopreservation; and 2) mitochondrial injury in this process is reduced by caspase inhibition. (HEPATOLOGY 2001;33:1432-1440.)A number of liver-assist devices have been developed, including the extracorporeal use of isolated mammalian hepatocytes to provide metabolic function in a bioartificial liver (BAL). 1,2 Criteria for successful application of a BAL include bridging patients to liver transplantation, reduction of intracranial hypertension and cerebral edema, and avoidance of liver transplantation in cases of reversible hepatic failure. 3-5 To achieve these goals, hepatocytes in the BAL provide metabolic functions to the patient in liver failure. The extent to which these goals can be achieved is dependent on several variables, including the number of viable and metabolically active hepatocytes in the device. 6 Unfortunately, hepatocyte viability declines over time following isolation, and this decline limits the effectiveness and duration of BAL therapy. Cryopreservation, which allows cells to be used weeks and months after isolation, may also contribute to premature cell death. 7,8 Several BAL systems have been designed to maintain hepatocyte viability ex vivo. One such BAL system involves the entrapment of hepatocytes in cylindrical collagen gels located in the fibers of a hollow fiber bioreactor. 9,10 An in vitro model of this BAL system using gel-entrapped hepatocytes has been developed for static culture experimentation, such as the study of mechanisms of hepatocyte death. 11 The in vitro model avoids the expenses of largescale testing, and multiple experiments can be performed simultaneously. Potential immune interactions, present with in vivo models of BAL testing, are also avoided with in vitro testing.Our recent work has shown that cell death of freshly isolated hepatocytes occurs, in part, by apoptosis. 12...

Gene therapy has many potential applications in central nervous system (CNS) disorders, including the selective killing of tumor cells in the brain. A rat brain tumor model was used to test the herpes simplex virus (HSV)-thymidine kinase (TK) gene for its ability to selectively kill C6 and 9L tumor cells in the brain following systemic administration of the nucleoside analog ganciclovir. The HSV-TK gene was introduced in vitro into tumor cells (C6-TK and 9L-TK), then these modified tumor cells were evaluated for their sensitivity to cell killing by ganciclovir. In a dose-response assay, both C6-TK and 9L-TK cells were 100 times more sensitive to killing by ganciclovir (median lethal dose: C6-TK, 0.1 microgram ganciclovir/ml; C6, 5.0 micrograms ganciclovir/ml) than unmodified wild-type tumor cells or cultured fibroblasts. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to kill established brain tumors in rats as quantified by both stereological assessment of brain tumor volumes and studies of animal survival over 90 days. Rats with brain tumors established by intracerebral injection of wild-type or HSV-TK modified tumor cells or by a combination of wild-type and HSV-TK-modified cells were studied with and without ganciclovir treatments. Stereological methods determined that ganciclovir treatment eliminated tumors composed of HSV-TK-modified cells while control tumors grew as expected (p < 0.001). In survival studies, all 10 rats with 9L-TK tumors treated with ganciclovir survived 90 days while all untreated rats died within 25 days. Curiously, tumors composed of combinations of 9L and 9L-TK cells could be eliminated by ganciclovir treatments even when only one-half of the tumor cells carried the HSV-TK gene. While not completely understood, this additional tumor cell killing appears to be both tumor selective and local in nature. It is concluded that HSV-TK gene therapy with ganciclovir treatment does selectively kill tumor cells in the brain and has many potential applications in CNS disorders, including the treatment of cancer.

Pore size, membrane composition, and duration of exposure influenced the transfer of PERV across HFCs. Some HFCs decrease the risk of viral exposure to patients during bioartificial liver therapy.