Design studies in the development of a bioreactor for artificial liver support

There has been much interest in recent years in the development of an hepatocyte bioreactor for artificial liver support, or bioartificial liver (BAL). Although considerable advances have been made in hepatocyte culture, the BAL environment is unlikely to be comparable to conventional culture conditions. For example: • the culture medium is likely to be human serum or plasma, as opposed to a specifically prepared culture medium, • very high density cultures will be required for the BAL, which may result in a rapid build-up of cell waste products (eg. bile), • in contrast to conventional stagnant cultures, oxygen gradients may exist within a bioreactor. Investigations were therefore undertaken to determine the effect of serum, bile and oxygen concentration on cell viability and function, as well as the effect of oxygen partial pressure (pO2) and cell density on oxygen consumption. Hep G2 human hepatoma cells were frequently used as a model of hepatocyte behaviour. When exposed to increasing concentrations of newborn calf serum (NCS) and human serum, cell growth and intracellular reduced glutathione (GSH) levels were well maintained, provided that the supply of amino acids was not limiting. Sheep bile was shown to be toxic to growing cultures of Hep G2 cells at concentrations as low as 0.1% (v/v). The evidence of toxicity was not as marked for confluent cultures of Hep G2 cells exposed to increasing concentrations of bile. Rat hepatocytes were shown to be extremely sensitive to oxygen concentration, with considerable differences in cytochrome P450 content, associated ethoxyresorufin O-dealkylase (EROD) activity, GSH content, 3H-leucine incorporation into protein and cell morphology when cultured in the presence of 5,12, 20, 28 or 35% 02- By contrast, Hep G2 cells did not display this sensitivity. Hep G2 cells were, however, observed to respond to linear decreases in both cell density and pO2 with exponential increases in oxygen consumption. This information has been applied to the design considerations of the BAL, and the techniques and equipment implemented may be used in further investigations of cell behaviour within the bioreactor environment.

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