Comparison of two size variants of a miniaturized multi-compartment three-dimensional (3D)-bioreactor system for primary human hepatocyte cultivation

Co-cultivation of primary human hepatocytes (pHH) with nonparenchymal liver cells in a perfused 3D-membrane capillary bioreactor (BR) enables more efficient long-term cell cultivation with maintenance of the physiological cell functions in comparison to standard 2D culture systems. Due to the restricted availability of primary human liver cells, miniaturization of the culture system while providing sufficient material for extra- and intracellular analyses is desirable for in vitro studies on hepatocyte physiology and metabolism. In this study two size variants of a miniaturized multi-compartment BR technology consisting of two or four capillary layers, each composed of medium perfusion and oxygenation capillaries were compared with respect to their metabolic activity. pHH from the same donors, (n=3) respectively, were cultivated in parallel in two- or four-layer BRs for 10 days with regular measurement of metabolic parameters. Cytochrome P450 (CYP) activities, including CYP1A2, CYP2B6, CYP2C9 and CYP3A4/5, as well as urea production showed no significant differences in the two different BR systems during the cultivation period. Equally aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release, measured to assess cellular injury, showed comparative concentrations in both systems. In addition, immunochemically assessed expression patterns of cytokeratin (CK) 19 as biliary marker, CK18 as hepatocyte/biliary marker and the mesenchymal marker vimentin confirmed the preservation of cellular identity within the two size variants of the BR technology. In conclusion both miniaturized BR systems are suitable for extended pHH culture while preserving specific metabolism. While the smaller model is useful for studies with small cell numbers, experiments requiring larger cell numbers for complex analyses should be performed with the four-layer model.

3D culture - bioreactors - human hepatocytes - in vitro model - liver cell culture