The variety of methods for measuring bioactive mass and functionality of bioartificial livers (BAL) is confusing and prevents accurate comparison of reported data. Here we present a comparison of different hepatocyte quantification methods and propose that estimation of cell pellet volume after centrifugation generates a reliable, useful and fast method. In addition a correlation is made between several function tests performed in 26 bioreactors to assess their predictive value. The ammonia eliminating capacity was found to be most predictive for other liver functions, except for lidocaine elimination as a measure of mixed function oxidase activity, which should therefore be determined separately. The oxygen consumption test proved to be an easy and predictive parameter as well. The first generation of our BAL system needed further development to assure optimal treatment of acute liver failure (ALF) patients. Changes in the porcine hepatocyte isolation method and bioreactor loading as well as changes in bioreactor configuration, including use of different materials, resulted in a significantly improved level and maintenance of in vitro BAL function. A fourfold increase in ammonia eliminating capacity, which is only reduced to 75% after seven days of culturing, offers promising prospects for further clinical application. Conclusion The current second generation of our BAL and improvement of hepatocyte isolation and testing protocols have led to a significant increase in the level as well as the maintenance of hepatocyte specific function in our BAL. Finally, consensus on definition of the bioactive mass to be loaded in the bioreactor and insight in the variation and reliability of the functional and metabolic parameters enhances comparison of the different types of bioartificial livers presented in literature.

Long-term culturing of primary porcine hepatocytes (PPH) inside the Academic Medical Center (AMC)-bioartificial liver is characterized by increased anaerobic glycolysis. Recommendations to increase oxygen availability were proposed in a previous numerical study and were experimentally evaluated in this study. Original bioreactors as well as new configuration bioreactors with 2.2-fold thinner nonwoven matrix and 2-fold more capillaries were loaded with PPHs and oxygenated with different gas oxygen pressures resulting in medium pO(2) (pO(2-med)) of either 135-150 mm Hg or 235-250 mm Hg. After 6 days culturing, new configuration bioreactors with pO(2-med )of 250 mm Hg showed significantly reduced anaerobic glycolysis, 60% higher liver-specific functions, and increased transcript levels of five liver-specific genes compared to the standard bioreactor cultures. Changed bioreactor configuration and increasing pO(2-med) contributed equally to these improvements. Histological examination demonstrated small differences in cell organization. In conclusion, higher metabolic stability and liver-specific functionality was achieved by enhanced oxygen availability based on a prior modeling concept.

The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)-BAL during a 7-day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2-fold more ammonia and produced 3-fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5-fold higher than MPHs and 6.6-fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue-like structures inside the AMC-BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading. Liver Transpl 13:589-598, 2007.

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