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Unlabelled: Primary cultures of human hepatocyte spheroids are a promising in vitro model for long-term studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver-specific functionality, have hampered a widespread adoption of this three-dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3-4 weeks in serum-free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug-metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n = 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3-4 weeks of long-term culture confirmed the presence of the liver-specific markers, hepatocyte nuclear factor 4α, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3-4 weeks of culture., Conclusion: Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver-specific activity and architecture and are thus suitable for drug testing in a long-term, repeated-dose format., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Primary cultures of human hepatocytes are a reference cellular model, because they maintain key features of liver cells in vivo, such as expression of drug-metabolizing enzymes, response to enzyme inducers, and generation of hepatic metabolites. However, there is a restricted availability of primary hepatocytes, and they show phenotypic instability in culture. Thus, different alternatives have been developed to overcome the culture limitations and to mimic in vivo tissue material. Herein, culture conditions, such as medium composition, impeller type, and cell inoculum concentration, were optimized in stirred culture vessels and applied to a three-dimensional (3D) bioreactor system. Cultures of rat hepatocytes as 3D structures on bioreactor, better resembling in vivo cellular organization, were compared to traditional monolayer cultures. Liver-specific functions, such as albumin and urea secretion, phase I and phase II enzyme activities, and the capacity to metabolize diphenhydramine and troglitazone, were measured over time. Hepatocyte functions were preserved for longer time in the 3D bioreactor than in the monolayer system. Moreover, rat hepatocytes grown in 3D system maintained the ability to metabolize such compounds, as well as in vivo. Our results indicate that hepatocytes cultured as 3D structures are a qualified model system to study hepatocyte drug metabolism over a long period of time. Moreover, these cultures can be used as feeding systems to obtain cells for other tests in a short time.

We report a study on neural metabolism of long-term three-dimensional cultures of rat embryonic brain cells in stirred vessels. Our experimental setup was optimized to keep viable aggregate cultures with neuronal maintenance for up to 44 days. Results show that aggregate size and shape could be hydrodynamically controlled depending on the impeller design, avoiding necrotic centers or significant losses in cell viability. Aggregates were composed mainly of neurons until day 16, whereas an effective growth of the glial population was observed after day 21. Cell metabolic status was evaluated by quantification of several metabolites in the culture medium; amino acid metabolism was used as a marker of metabolic interrelationships between neural cell types. Furthermore, (13)C-NMR spectroscopy was used on day 31 to explore specific metabolic pathways: incubation with [1-(13)C]glucose for 45 hr produced an increase in label incorporation in extracellular alanine, lactate, and glutamine, reflecting mainly astrocytic metabolism. The contribution of anaplerotic vs. oxidative pathways for glutamine synthesis was determined: a 92% reduction in the pyruvate carboxylase flux during the first 41 hr of incubation suggested a decrease in the need for replacing tricarboxylic acid cycle intermediates. We believe that our data corroborate the aggregating cultures as an attractive system to analyze brain cell metabolism being a valuable tool to model metabolic fluxes for in vitro brain diseases.