Dose-response in a high density three-dimensional liver device with real-time bioenergetic and metabolic flux quantification.

Real-time dose-response curves for fructose have been non-invasively determined in primary rat hepatocyte alginate spheroids cultured in a NMR-compatible fluidized-bed bioreactor. Using 13C-labeled glucose and glycine culture medium, fructose dose was compared to glucose uptake and glycogen synthesis rate using 13C NMR spectroscopy, and to ATP and fructose-1-phosphate concentration using 31P NMR spectroscopy. A highly efficient multicoaxial perfusion system maintains high density 3-D hepatocyte cultures, permitting 13C and 31P NMR spectral time courses with 1min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16min, glycogen plummeted, lactate became the largest 13C-glucose metabolite via anaerobic glycolysis, while glutathione was the largest 13C-glycine metabolite. ATP depletion and fructose-1-phosphate formation demonstrated a dose response with a 3h EC50 of 19mM±8.9mM and 17.4mM±3.7mM, respectively. Computational modeling of mass transfer corroborated experimental results and helped determine the optimal bioreactor loading densities, oxygen concentration, and perfusion rates to maintain physiologically-relevant nutrient levels. The total bioreactor plus perfusion loop has a dead volume of 2ml, and contains 5 million hepatocytes. Due to the non-invasive measurements, there is a reduction of animal tissue by an order-of-magnitude, depending on the number of time points in an experiment. This dynamic flux approach may have generic utility for dose-response studies monitoring multiple metabolic reactions in other primary mammalian cells, such as human, that have strict oxygen demands.

Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies.

Accurate prediction of metabolism is a significant outstanding challenge in toxicology. The best predictions are based on experimental data from in vitro systems using primary hepatocytes. The predictivity of the primary hepatocyte-based culture systems, however, is still limited due to well-known phenotypic instability and rapid decline of metabolic competence within a few hours. Dynamic flow bioreactors for three-dimensional cell cultures are thought to be better at recapitulating tissue microenvironments and show potential to improve in vivo extrapolations of chemical or drug toxicity based on in vitro test results. These more physiologically relevant culture systems hold potential for extending metabolic competence of primary hepatocyte cultures as well. In this investigation, we used computational fluid dynamics to determine the optimal design of a flow-based hepatocyte culture system for evaluating chemical metabolism in vitro. The main design goals were (1) minimization of shear stress experienced by the cells to maximize viability, (2) rapid establishment of a uniform distribution of test compound in the chamber, and (3) delivery of sufficient oxygen to cells to support aerobic respiration. Two commercially available flow devices - RealBio® and QuasiVivo® (QV) - and a custom developed fluidized bed bioreactor were simulated, and turbulence, flow characteristics, test compound distribution, oxygen distribution, and cellular oxygen consumption were analyzed. Experimental results from the bioreactors were used to validate the simulation results. Our results indicate that maintaining adequate oxygen supply is the most important factor to the long-term viability of liver bioreactor cultures. Cell density and system flow patterns were the major determinants of local oxygen concentrations. The experimental results closely corresponded to the in silico predictions. Of the three bioreactors examined in this study, we were able to optimize the experimental conditions for long-term hepatocyte cell culture using the QV bioreactor. This system facilitated the use of low system volumes coupled with higher flow rates. This design supports cellular respiration by increasing oxygen concentrations in the vicinity of the cells and facilitates long-term kinetic studies of low clearance test compounds. These two goals were achieved while simultaneously keeping the shear stress experienced by the cells within acceptable limits.