Journal
HEPATOLOGY
Volume 55, Issue 4, Pages 1227-1236Publisher
WILEY
DOI: 10.1002/hep.24760
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Funding
- FCT [SFRH/BD/35296/2007, SFRH/BD/37102/2007, PTDC/EBB-BIO/112786/2009]
- European Commission [HEALTH-2007-1.4-7]
- Fundação para a Ciência e a Tecnologia [SFRH/BD/37102/2007, PTDC/EBB-BIO/112786/2009, SFRH/BD/35296/2007] Funding Source: FCT
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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 4a, 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. (HEPATOLOGY 2012)
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