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

Abstract

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.

Keywords: (31)P and (13)C NMR spectroscopy; Alginate-hepatocyte encapsulation; Dose response; Drug safety; Fluidized-bed bioreactor; Fructose; NMR-compatible.