Cytochrome P450-dependent metabolism in HepaRG cells cultured in a dynamic three-dimensional bioreactor.

Reliable and stable in vitro cellular systems maintaining specific liver functions important for drug metabolism and disposition are urgently needed in preclinical drug discovery and development research. The cell line HepaRG exhibits promising properties such as expression and function of drug-metabolizing enzymes and transporter proteins, which resemble those found in freshly isolated human hepatocytes. In this study, HepaRG cells were cultured up to 68 days in a three-dimensional multicompartment capillary membrane bioreactor, which enables high-density cell culture under dynamic conditions. The activity of drug-metabolizing cytochrome P450 (P450) enzymes was investigated by a cocktail of substrates for CYP1A1/2 (phenacetin), CYP2C9 (diclofenac), CYP2B6 (bupropion), and CYP3A4 (midazolam). The model P450 substrates, which were introduced to the bioreactor system mimicking in vivo bolus doses, showed stable metabolism over the entire experimental period of several weeks with the exception of bupropion hydroxylase, which increased over time. Ketoconazole treatment decreased the CYP3A4 activity by 69%, and rifampicin induced the CYP3A4- and CYP2B6-dependent activity 6-fold, which predicts well the magnitude of changes observed in vivo. Moreover, polarity of transporter expression and formation of tissue-like structures including bile canaliculi were demonstrated by immune histochemistry. The long-lasting bioreactor system using HepaRG cells thus provides a promising and stable liver-like in vitro model for continuous investigations of the hepatic kinetics of drugs and of drug-drug interactions, which well predict the situation in vivo in humans.

Interwoven four-compartment capillary membrane technology for three-dimensional perfusion with decentralized mass exchange to scale up embryonic stem cell culture.

We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent 'arteriovenous' flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 x 10(9) cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining 'stemness' markers during cell expansion.