Preliminary Evaluation of Three-Dimensional Primary Human Hepatocyte Culture System for Assay of Drug-Metabolizing Enzyme-Inducing Potential

Abstract

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids). Hepatocyte spheroids could be used up to 21 d after seeding (pre-culture for 7 d and exposure to inducer for up to 14 d), based on preliminary evaluation of basal activities of CYP subtypes and mRNA expression of the corresponding transcription factor and xenobiotic receptors (aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR)). After 2 d exposure of hepatocyte spheroids to omeprazole, phenobarbital and rifampicin (typical inducers of CYP1A2, 2B6 and 3A4, respectively), CYP1A2, 2B6 and 3A4 mRNA expression levels were significantly increased. The mRNA induction of CYP2B6 remained reasonably stable between days 2 and 14 of exposure to inducers, while induction of both CYP1A2 and 3A4 continued to increase up to day 14. These enzyme activities were all significantly increased compared with the control until day 14. Our findings indicate that our 3D hepatocyte spheroids system would be especially suitable for long-term testing of enzyme activity induction by drugs, either to predict or to verify clinical events.

Evaluation of the metabolic capability of primary human hepatocytes in three‐dimensional cultures on microstructural plates

“…We reported the metabolic activities of Non‐Cultured Hepatocytes (using the same lot as in the present study) as 22.2, 36.7 and 82.7 pmol/min/10 6 cells for CYP1A2, 2B6 and 3A4, respectively (Arakawa et al, ). Although the activities of CYP1A2, 2B6 and 3A4 reduced to 1.79 (8.1%), 0.19 (0.51%), 12.7 (15.3%) pmol/min/10 6 cells after 4‐days culture on NCP, they showed detectable activities and induction potential.…”

“…Although the activities of CYP1A2, 2B6 and 3A4 reduced to 1.79 (8.1%), 0.19 (0.51%), 12.7 (15.3%) pmol/min/10 6 cells after 4‐days culture on NCP, they showed detectable activities and induction potential. Although the metabolic activities of hepatocytes on NCP are much lower than those on Cell‐able in our previous study (Arakawa et al, ) and Berger et al (), they are cultured with feeder cells. The usage of feeder cells might also enhance the metabolic activities on the NCP.…”

Utility of Three-Dimensional Cultures of Primary Human Hepatocytes (Spheroids) as Pharmacokinetic Models

“…However, as already discussed, this method is not suitable for long-term exposure studies. Therefore, we investigated whether the induction of drug-metabolizing enzymes could be evaluated using PHH spheroids, and we compared the results with those from 2D culture [ 55 ].…”

“…Cultured human cancer-derived hepatocytes have long been used for human-specific toxicity assessments, but they generally have lower expression levels of drug-metabolizing enzymes than PHHs [ 39 , 40 ]. Various applications of PHH spheroids as pharmacokinetic models [ 41 , 42 , 43 , 44 ] or hepatotoxicity detection models [ 16 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ] have already been reported, and it has become clear that this model is extremely versatile and offers a variety of advantages over 2D cultures ( Table 2 ) [ 16 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. PHH spheroids have also been used as disease models [ 58 , 59 , 60 ].…”

Utility of human hepatocyte spheroids without feeder cells for evaluation of hepatotoxicity

“…We previously established that three-dimensionally (3D) cultured hepatocytes (hepatocyte spheroids) are suitable for long-term metabolic assays (Ohkura et al, 2014;Arakawa et al, 2017). These spheroids remained active for at least 21 days, exhibiting well-maintained secretion of albumin, a stable leakage level of aspartate aminotransferase (AST) and unchanged morphology.…”