Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing

Authors


  • Potential conflict of interest: Dr. Björquist owns stock in Cellartis.

  • This work was supported by FCT fellowships SFRH/BD/35296/2007 (to R.T., under the scope of the MIT-Portugal program) SFRH/BD/37102/2007 (to S.L.), project PTDC/EBB-BIO/112786/2009, and by the European Commission under the scope of the Hyperlab project (High Yield and Performance Stem Cell Lab) (HEALTH-2007-1.4-7).

Abstract

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. (HEPATOLOGY 2012)

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