In-depth physiological characterization of primary human hepatocytes in a 3D hollow-fiber bioreactor

Abstract

As the major research focus is shifting to three-dimensional (3D) cultivation techniques, hollow-fiber bioreactors, allowing the formation of tissue-like structures, show immense potential as they permit controlled in vitro cultivation while supporting the in vivo environment. In this study we carried out a systematic and detailed physiological characterization of human liver cells in a 3D hollow-fiber bioreactor system continuously run for > 2 weeks. Primary human hepatocytes were maintained viable and functional over the whole period of cultivation. Both general cellular functions, e.g. oxygen uptake, amino acid metabolism and substrate consumption, and liver-specific functions, such as drug-metabolizing capacities and the production of liver-specific metabolites were found to be stable for > 2 weeks. As expected, donor-to-donor variability was observed in liver-specific functions, namely urea and albumin production. Moreover, we show the maintenance of primary human hepatocytes in serum-free conditions in this set-up. The stable basal cytochrome P450 activity 3 weeks after isolation of the cells demonstrates the potential of such a system for pharmacological applications. Liver cells in the presented 3D bioreactor system could eventually be used not only for long-term metabolic and toxicity studies but also for chronic repeated dose toxicity assessment.

Real-time in situ viability assessment in a 3D bioreactor with liver cells using resazurin assay

“…As such a noninvasive and real time assay is required. We recently demonstrated long-term cultivation of primary human hepatocytes in a 3D bioreactor system (Mueller et al 2011b) for 3 weeks. We used biochemical parameters such as galactose consumption and lactate production as well as production of the liver-specific markers albumin and urea for an indirect estimation of cellular viability since a direct, microscopic observation of the cells is not possible in this bioreactor.…”

“…Tissue collection was done according to the institutional guidelines and with the patient's written consent. Isolation and purification of non-tumor cells was performed as described previously (Mueller et al 2011b). …”

“…1b). They consist of three interwoven hollow fiber capillary bundles that form four different compartments which are integrated into a polyurethane housing as recently described (Mueller et al 2011b). …”

“…Liver cells form three-dimensional aggregates around the hollow-fibers including the formation of liver-specific structures (Zeilinger et al 2004). It was shown that the 3D bioreactors are suitable for long-term functional maintenance of primary human hepatocytes indicating high potential of these systems particularly for the assessment of chronic druginduced effects (Mueller et al 2011b). One main disadvantage of this hollow-fiber bioreactor is its black-box character, since direct cell counting methods as well as microscopic observation of cells is not possible.…”

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology

“…Many such devices for in vitro applications have been reported (Mueller et al, 2011b;Darnell et al, 2011;Hoekstra et al, 2011;tostoes et al, 2012;Zeilinger et al, 2011). Cultivation in a perfused bioreactor provides a continuous mass exchange of culture media and decentralized controlled oxygenation.…”