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.


CONCLUSIONS
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.
ITQB-UNL, Oeiras, Portugal.
Rui M
RM
Tostões
Sofia B
SB
Leite
Margarida
M
Serra
Janne
J
Jensen
Petter
P
Björquist
Manuel J T
MJ
Carrondo
Catarina
C
Brito
Paula M
PM
Alves
D000418:Albumins / Q000378:metabolism; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D064420:Drug-Related Side Effects and Adverse Reactions; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053538:Keratin-18 / Q000378:metabolism; D010477:Perfusion / Q000379:methods*; D018874:Spheroids, Cellular
D000418:Albumins; D051557:Hepatocyte Nuclear Factor 4; D053538:Keratin-18; D051544:Cytochrome P-450 CYP3A