HepaChip-MP - a twenty-four chamber microplate for a continuously perfused liver coculture model.

HepaChip microplate (HepaChip-MP) is a microfluidic platform comprised of 24 independent culture chambers with continuous, unidirectional perfusion. In the HepaChip-MP, an automated dielectrophoresis process selectively assembles viable cells into elongated micro tissues. Freshly isolated primary human hepatocytes (PHH) and primary human liver endothelial cells (HuLEC) were successfully assembled as cocultures aiming to mimic the liver sinusoid. Minimal quantities of primary human cells are required to establish micro tissues in the HepaChip-MP. Metabolic function including induction of CYP enzymes in PHH was successfully measured demonstrating a high degree of metabolic activity of cells in HepaChip-MP cultures and sufficient sensitivity of LC-MS analysis even for the relatively small number of cells per chamber. Further, parallelization realized in HepaChip-MP enabled the acquisition of dose-response toxicity data of diclofenac with a single device. Several unique technical features should enable a widespread application of this in vitro model. We have demonstrated fully automated preparation of cell cultures in HepaChip-MP using a pipetting robot. The tubeless unidirectional perfusion system based on gravity-driven flow can be operated within a standard incubator system. Overall, the system readily integrates in workflows common in cell culture labs. Further research will be directed towards optimization of media composition to further extend culture lifetime and study oxygen gradients and their effect on zonation within the sinusoid-like microorgans. In summary, we have established a novel parallelized and scalable microfluidic in vitro liver model showing hepatocyte function and anticipate future in-depth studies of liver biology and applications in pre-clinical drug development.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Long term cultures of primary human hepatocytes as an alternative to drug testing in animals.

Due to species differences, primary human hepatocytes are still the in vitro system of choice to analyse liver specific processes and functions. Human hepatocytes were cultured for several weeks in a serum-free two-dimensional culture system, which was used to study the effects of acetaminophen (APAP) on hepatocellular functions and vitality. Non-invasive determinations of albumin, urea and lactate dehydrogenase concentrations in cell culture supernatants allowed continuous monitoring for at least two weeks. APAP was applied every 4 days for 24 h. Each application reduced urea production by 25% and albumin synthesis by approximately 70% without any effects on cellular viability. After removal of the substance, hepatocellular functions returned to control levels within one (urea) to three (albumin) days. The repetitive analyses of APAP-mediated effects on cellular metabolism led to identical results for up to five cycles. The drug also caused reversible and repetitive ultrastructural modifications, in particular an almost complete replacement of rough endoplasmic reticulum by smooth endoplasmic reticulum and a massive degradation of glycogen stores. The data demonstrate the suitability of the culture system to serve as a model for repetitive testing of drug-mediated changes on hepatocellular functions, thereby reducing animal studies during drug development.

Anticancer drug cis-4-hydroxy-L-proline: Correlation of preclinical toxicology with clinical parameters of liver function.

cis-4-Hydroxy-L-proline (CHP) is being clinically evaluated as an anticancer drug. Since this compound targets the production of L-proline-rich proteins and critical L-proline residues, its impact on long-term cultures of human hepatocytes and toxicity in rats was studied to investigate possible effects on hepatic function, previously reported in rat hepatocytes. In the HEPAC2 human hepatocyte culture system, concentrations of CHP below 3.2 mg/ml had no significant effects on the release of lactate dehydrogenase (LDH), albumin, and urea. In rats, continuous administration of three different doses of CHP were tested for 28 days and resulted in signs of liver damage, as indicated by elevations of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) at a dose of 903 mg/kg, corresponding to a plasma concentration of approximately 200 µg/ml. Data from a clinical study of CHP in bladder and prostate cancer patients showed no adverse effects of administration of 8 g CHP/day, 4 days/week for 3 weeks in liver parameters ALAT, ASAT, γ-glutamyltransferase (γ-GT) and alkaline phosphatase (AP). In conclusion, the HEPAC2 human hepatocyte culture system correlates well with clinical results of a Phase II study of CHP, whereas a previous rat hepatocyte culture system predicted the compound would have toxic effects. The HEPAC2 system therefore constitutes a valuable tool for the preclinical screening of the hepatotoxicity of chemotherapeutic and other drugs, thereby reducing the need for experimental animals.

In vitro system for the prediction of hepatotoxic effects in primary hepatocytes.

Prediction of liver toxicity and compound responses continues to be a major challenge for the pharmaceutical industry. In vitro studies on liver cells have been developed to reduce or replace animal experiments. However, most of the tests in use are based on cell lines which do not necessarily represent normal cell physiology. We compared the response of primary human hepatocytes from two donors with primary rat hepatocytes and the cell line HepG2 to the test compound acetaminophen (AAP) by measuring oxygen consumption, extracellular acidification and cell adhesion as dynamic parameters of cell metabolism. Primary human hepatocytes were cultured on collagen pre-coated sensor chips or in conventional two-dimensional cultures in chemically defined Human Hepatocyte Maintenance Medium. This medium allows cultivation of functionally differentiated hepatocytes for several weeks. Sensor chip based results were compared with conventional assays for hepatocytes like albumin release and urea release. The hepatocytes were exposed to AAP (50-2815 mg/l) for 24 h. Cell respiration was inhibited by AAP concentrations of 500 mg/l and more in all three cell types, whereas only the cellular acidification rates and cell adhesion of the rat hepatocytes and the HepG2 cells were affected by AAP. In conventional cultures of human hepatocytes, AAP had no effect on cellular viability. Whereas high doses of AAP (2815 mg/l) diminished albumin secretion by 70-80%.

Use of a standardised and validated long-term human hepatocyte culture system for repetitive analyses of drugs: repeated administrations of acetaminophen reduces albumin and urea secretion.

Human hepatocytes are the in vitro system of choice to study drug-induced processes in man. Here, we present HEPAC(2): a standardised and validated culture system in which human hepatocytes are maintained in HHMM (Human Hepatocyte Maintenance Medium) with HGF (hepatocyte growth factor) and EGF (epidermal growth factor). Cellular viability and hepatocellular functions were monitored daily. Albumin and urea production remained on a relatively constant level for up to 2-3 weeks. Based on this, a standard protocol was established that allows repeated exposure of hepatocytes to study drug metabolism. We used acetaminophen (AAP) to assay the feasibility of this system. Hepatocytes were exposed to AAP (100-2815 mg/l) for 24 h. Subsequently, the culture medium was replaced by medium without AAP and the same exposure scenario was repeated at intervals of 4 days. High doses of AAP (2815 mg/l) diminished urea production by 15-30% and albumin secretion by 70-80%. These effects were reversible. After removal of AAP, secretion of urea and albumin returned to control levels. AAP hepatotoxicity is caused by its biotransformation to the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) mediated by CYP2E1 and CYP1A2. The AAP activating enzymes were active for at least 21 days and their activity was maintained during at least four repeated cycles of exposure to AAP. In conclusion, these data demonstrate the suitability of our long-term culture system to serve as a tool for repetitive screening of drug-mediated changes of hepatocellular functions. This culture technique may help to overcome the sparse availability of human hepatocytes for testing drugmediated responses in man.