Cytochrome P450 induction response in tethered spheroids as a three‐dimensional human hepatocyte in vitro model

Abstract

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules

“…5A) and was about 20% of the level produced by PHHs [40]. Urea production in SM-Hep was 62 ± 11 pg/cell/48 hours which was significantly higher than that of GF-Hep (15 ± 0.36 pg/cell/48 hours) and 57% of PHHs (110 pg/cell/48 hours [40]) (Fig. 5B).…”

“…The functions were compared to PHHs data generated in our lab from three different hepatocyte donors [40]. SM-Hep produced 0.21 ± 0.067 pg/cell/48 hours albumin which was similar, to that of GF-Hep (Fig.…”

“…5E). The fold induction of CYP1A2, although lower than GF-Hep (21 ± 1.8 fold) for CYP1A2 was approximately 35% of PHHs (PHHs fold induction=20; average of 3 donors [40]). The fold induction for CYP3A4 was higher in the SM-Hep (27 fold ± 3.3) when compared to GFHep (2.8 ± 1.8 fold) and was approximately 54% of PHHs (PHHs fold induction=50; average of 3 lots [40]).…”

“…The fold induction of CYP1A2, although lower than GF-Hep (21 ± 1.8 fold) for CYP1A2 was approximately 35% of PHHs (PHHs fold induction=20; average of 3 donors [40]). The fold induction for CYP3A4 was higher in the SM-Hep (27 fold ± 3.3) when compared to GFHep (2.8 ± 1.8 fold) and was approximately 54% of PHHs (PHHs fold induction=50; average of 3 lots [40]). The fold inductions measured by mRNA levels were confirmed by functional activity M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9 (metabolite production in basal and induced samples were quantified by LCMS).…”

“…The metabolite production by CYP3A4 for SM-Hep and GF-Hep was 0.029 ± 0.0074 and 0.041 ± 0.020 pmol/million cells/min respectively. These values are about 8-10 % of the metabolite production by PHHs (3.75 pmol/million cells/min for CYP1A2 activity and 0.21 pmol/million cells/min for CYP3A4; average of 3 donors [40]). …”