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

in Cytotechnology (2013), 65(2), 297-305

Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is ... [more ▼]

Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is very difficult in such systems. Commonly applied indirect methods such as glucose consumption, albumin or urea production are greatly affected by culture conditions, stress and time of cultivation and do not reflect the real time viability of the cells. In this study we established a real-time in situ viability assay namely; resazurin assay, in a 3D hollow-fiber bioreactor using human liver cells. Resazurin assay is based on the conversion of resazurin to a fluorescent dye by cytoplasmatic and mitochondrial enzymes. We show that the resazurin reagent in concentrations used in this study is non-toxic and could be rapidly removed out of the system. Moreover, we observed that dead cells do not affect the results of the assay. We optimized the assay on HepG2 cells and tested it with primary human hepatocytes. Moreover, we maintained primary human hepatocytes in the 3D bioreactor system in serum-free conditions and also assessed viability before and after the exposure to amiodarone using the resazurin assay. We show that this approach is applicable during long-term cultivation of cells in bioreactors under different conditions and can moreover be applied to pharmacological studies, e.g. investigation of chronic drug effects in such 3D bioreactors. [less ▲]

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

in Journal of tissue engineering and regenerative medicine (2011), 5(8), 207-18

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 ... [more ▼]

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. [less ▲]