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 1 min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16 min, 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 3 h EC50 of 19 mM ± 8.9 mM and 17.4 mM ± 3.7 mM, 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 2 ml, 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.