Liver Grafts Preserved in Celsior Solution as Source of Hepatocytes for Drug Metabolism Studies: Comparison With Surgical Liver Biopsies

Abstract

Suitability of human liver grafts preserved in Celsior solution (CS) for preparing metabolically competent hepatocyte cultures has been examined. To this end, basal and induced activity and mRNA levels of major hepatic cytochrome P450 (P450) enzymes have been measured. By 24 h in culture, measurable levels of the 10 P450 mRNAs studied were found in all hepatocyte preparations examined, with CYP2E1, CYP2C9, and CYP3A4 mRNAs being the most abundant. Compared with hepatocytes obtained from surgical liver resections (SLRs), lower content of each P450 mRNA was found in hepatocytes from the CS group; however, the relative distribution of individual P450 mRNAs was similar. Similar results were observed after measuring P450 activities. CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 activities in hepatocytes from CS-flushed grafts were lower than but comparable with those of cultures prepared from SLRs. No differences in the metabolite profile of testosterone were found. Treatment of hepatocytes from CS-preserved grafts with model P450 inducers shows that 2 microM methylcholanthrene only increased CYP1A1 and CYP1A2 mRNAs (>100-fold over control), 1 mM phenobarbital markedly increased CYP2A6, CYP2B6, and CYP3A4 mRNA content (>7-fold), and 50 microM rifampicin highly increased CYP3A4 mRNA levels (>10-fold), whereas minor effects (<3-fold) were observed in CYP2A6, CYP2B6, and CYP2C9 mRNAs. This induction pattern of P450s was similar, in terms of magnitude, reproducibility, and specificity, to that shown in primary hepatocytes from surgical biopsies. Overall, our results indicate that, cold-preserved in CS, liver grafts constitute a valuable source of human hepatocytes for drug metabolism studies.

Gene Expression in Human Hepatocytes in Suspension After Isolation Is Similar to the Liver of Origin, Is Not Affected by Hepatocyte Cold Storage and Cryopreservation, but Is Strongly Changed After Hepatocyte Plating

“…Because intact cells more closely reflect the environment to which drugs are exposed in the liver, isolated primary human hepatocytes are a well accepted system for evaluating pharmacological and toxicological effects in humans (Gomez-Lechon et al, 2004;O'Brien et al, 2004;Liguori et al, 2005) and have been described as one of the best in vitro models for the prediction of in vivo metabolism in humans (Lave et al, 1999;Li, 2001). This has prompted research groups, including ours, to optimize the isolation and culture conditions of human hepatocytes from both surgical liver resections and nontransplantable livers (Richert et al, 2004;Donato et al, 2005;LeCluyse et al, 2005).…”

In Vitro and in Vivo Induction of Cytochrome P450: A Survey of the Current Practices and Recommendations: A Pharmaceutical Research and Manufacturers of America Perspective

“…Whereas the AhR is a ligand-activated transcription factor belonging to the basic helix-loop-helix/Per-Arnt-Sim family, CAR and PXR are members of the nuclear hormone receptor superfamily. Target genes for these receptors of interest to the study of drug metabolism include various Phase I (e.g., members of the CYP1A, -2B, -2C, and -3A subfamilies) and Phase II enzymes [e.g., uridine diphosphate glucuronosyl transferases (UGTs), glutathione S-transferases, and sulfotransferases] and drug transporters including Pglycoprotein (Kast et al, 2002;Maglich et al, 2002;Madan et al, 2003;Donato et al, 2005;Girault et al, 2005;Hartley et al, 2006;Jigorel et al, 2006). Activation of other nuclear receptors such as the farnesyl X receptor (FXR), liver X receptor, peroxisome proliferatoractivated receptors (PPARs), and the vitamin D receptor (VDR) has been associated with induction of genes relevant to drug metabolism.…”

Functional Assessment of the Quality of Human Hepatocyte Preparations for Cell Transplantation

“…In addition Banked hepatocytes can be used on demand for programmed infusions and are available for immediate use to intrinsic donor characteristics, however, other factors related to the procurement of liver sample and hepatoin urgent cases. Different cryopreservation protocols have been proposed, but a universal consensus about the cyte isolation procedure can contribute to the observed variability in drug-metabolizing activities (11,15). Drugmost appropriate freezing procedure has not been reached (1,2,17,20,42).…”