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

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)