Sam Coward

Alginate-encapsulated HepG2 Cells in a Fluidized Bed Bioreactor Maintain Function in Human Liver Failure Plasma

Alginate-encapsulated HepG2 cells cultured in microgravity have the potential to serve as the cellular component of a bioartificial liver. This study investigates their performance in normal and liver failure (LF) human plasma over 6-8 h in a fluidized bed bioreactor. After 8 days of microgravity culture, beads containing 1.5 x 10(9) cells were perfused for up to 8 h at 48 mL/min with 300 mL of plasma. After exposure to 90% LF plasma, vital dye staining showed maintained cell viability, while a 7% increase in lactate dehydrogenase activity indicated minimal cell damage. Glucose consumption, lactate production, and a 4.3-fold linear increase in alpha-fetoprotein levels were observed. Detoxificatory function was demonstrated by quantification of bilirubin conjugation, urea synthesis, and Cyp450 1A activity. These data show that in LF plasma, alginate-encapsulated HepG2 cells can maintain viability, and metabolic, synthetic, and detoxificatory activities, indicating that the system can be scaled-up to form the biological component of a bioartificial liver.

Fat‐loaded HepG2 spheroids exhibit enhanced protection from Pro‐oxidant and cytokine induced damage

The mechanisms by which steatosis renders hepatocytes susceptible to damage in non‐alcoholic steatohepatitis (NASH) are unclear although fat accumulation is believed to increase hepatocyte susceptibility to inflammatory cytokines and oxidative stress. We therefore investigated the susceptibility of steatotic, hepatocyte‐derived cells to TNFα and the pro‐oxidant, t‐butylhydroperoxide (TBH). HepG2 spheroids rendered steatotic by fat‐loading with 0.15 mM oleic or palmitic acid for 48 h and treated with TNFα or TBH for 18 h exhibited surprisingly lower levels of cytotoxicity, and increased anti‐oxidant activity (superoxide dismutase (SOD)) compared with non fat‐loaded controls. The protective effect of steatosis was significantly reversed by the inhibition of AMP‐activated kinase (AMPK) since spheroids transfected with a kinase‐dead AMPKα2 subunit, exhibited a significant increase in TBH‐induced cytotoxicity when fat‐loaded. In conclusion, our findings suggest that fat‐loaded hepatocyte‐derived cells are surprisingly less susceptible to cytokine and pro‐oxidant induced damage via an adaptive mechanism dependent, in part, on AMPK activity. J. Cell. Biochem. 101: 723–734, 2007.

Putative human liver progenitor cells in explanted liver

Background/Aims: Hepatocyte progenitors have frequently been cultured from rodents but reports from human liver are rare. Methods: Non-parenchymal cell fraction isolated from 19 explant livers (removed at orthotopic liver transplantation for acute or chronic liver disease) and histologically normal human liver was cultured. Results: Proliferating epithelioid colonies were identifiable after 2–3 weeks culture as a very rare event (<1 per million cells plated) expressing mRNAs and protein antigens of mixed hepatocytic/biliary phenotype. Colony survival could be prolonged by transduction of the catalytic sub-unit of telomerase. Hepatocyte growth factor, epidermal growth factor and oncostatin M did not further enhance hepatocytic differentiation. The expression of markers associated with hepatocyte precursor status was investigated by flow cytometry. Cells expressing the stem cell-associated markers CD133 and CD117 were identified at low frequency. The proportion of cells expressing the integrin CD49f was higher in diseased liver than in normal liver, but the proportion expressing the hepatocyte growth factor receptor c-met was lower. Successful enrichment of plated populations for progenitors was not achieved. Conclusion: Although there is clear histological evidence of hepatocyte precursors in human explant livers, predictable culture of such cells with differentiation toward mature hepatocyte phenotype remains elusive.