Figure 6
From: Generation of proliferating human adult hepatocytes using optimized 3D culture conditions
3D cultured PHH exhibit optimal drug detoxification capabilities. (A) Heatmap showing the expression of the main phase I and II enzymes and drug transporters in FIH (black), 2D cultures (white) and in PHH cultured in 3D at d15 (yellow). (B) LC/MS–MS assays of the activities of the major CYPs isoforms involved in the liver detoxication of xenobiotics in 3D cultured PHH at d15. CYP1A2 was induced by 3-methylcholantrene (5 μm, 24 h), CYP3A4 and CYP2C8 were induced by rifampicin (50 μM, 72 h) and CYP2B6 was induced by phenobarbital (3.2 mM, 72 h) (Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test). (C) Quantification of mRNA levels of phase II (UGT1A1, NAT1, GSTA1/2), hepatic transporters (MRP2, OCT1, BSEP) and CYPs regulators (CAR, PXR, AhR) in PHH cultured in 3D at d4, d15 and d28. The results are from at least three independant experiments and are expressed according to the 2D culture level (Mean ± SD, *p < 0.05, one-way ANOVA test). (D) Immunostaining and functional analysis of the MRP2 drug transporter in spheroids revealed bile canaliculi from the beginning to the late stages of the culture (d4, d15 and d28). MRP2 (red), albumin (green) and nuclei (blue) are detected by immunostaining. Functionality of the transporter was assessed by CDFDA processing (green). Scale bar = 25 μm.
