New Hepatocyte In Vitro Systems for Drug Metabolism: Metabolic Capacity and Recommendations for Application in Basic Research and Drug Development, Standard Operation Procedures

Abstract

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity...

Increased hepatotoxicity of tumor necrosis factor–related apoptosis-inducing ligand in diseased human liver

“…Because liver explants retain an intact structure that presumably avoids dedifferentiation of hepatocytes and induction of apoptosis-relevant stress genes, our ex vivo model should more truly reflect the physiological situation than would isolated hepatocytes. It must be emphasized that we observed no background cytotoxicity in the liver explants within an 8-hour incubation period, suggesting adequate oxygen and nutrient exchanges, which is in line with studies using similar liver slices (reviewed in Elaut et al, 33 Berry et al, 34 and Gebhardt et al 35 ).…”

“…33 Furthermore, in organotypic culture, the complexity of liver architecture-cellular heterogeneity, cell-cell interactions and bipolarity of hepatocytes-is largely maintained. 34,35 Using caspase and LDH measurements, we first verified that liver explants can be incubated in vitro for at least 8 hours without a significant loss of cell viability (data not shown).…”

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

“…One model found to be useful in the prediction of drug metabolism is the culture of primary human hepatocytes (4,5). In current practice, isolated hepatocytes are cultured in suspension, a configuration shown to maintain high levels of cytochrome P450 (CYP450) activity up to 6 h in vitro.…”

Chip-based liver equivalents for toxicity testing – organotypicalness versus cost-efficient high throughput

“…42,43 Use of primary hepatocytes combined with a change of cell culture format to 3D and, more importantly, co-cultivation with non-parenchymal liver cells, such as endothelial cells, hepatic stellate cells or Kupffer cells, might improve their cellular and metabolic functions towards in vivo-like levels. 22,18,44,45 Induced stem cell technologies, recently awarded with the Nobel Prize in Medicine, [46][47][48][49] might provide new solutions to the well described but restricted access to human liver tissue. 50,51 Random dynamic monocultures A number of microfluidic liver culture devices exposing hepatocytes in random 3D culture at a lower organisational level have been engineered and will be discussed in this section.…”