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

• Published in: Toxicology in vitro Vol. 45; no. Pt 1; pp. 119 - 127
• Main Authors: Tikunov, Andrey P., Shim, Yoo-Sik, Bhattarai, Narayan, Siler, Scott Q., Soldatow, Valerie, LeCluyse, Edward L., McDunn, Jonathan E., Watkins, Paul B., Macdonald, Jeffrey M.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2017; Elsevier Science Ltd
• Subjects: 31P and 13C NMR spectroscopy; Alginate-hepatocyte encapsulation; Alginates; Alginic acid; Animal tissues; Animals; Artificial Organs; ATP; Bioenergetics; Bioreactors; Biosynthesis; Carbon 13; Cell culture; Computational Biology; Computer applications; Density; Dosage; Dose response; Drug safety; Fluidized bed reactors; Fluidized beds; Fluidized-bed bioreactor; Fructose; Glucose; Glutathione; Glycine; Glycogen; Glycolysis; Hepatocytes; Hepatocytes - physiology; Lactic acid; Liver; Liver - physiology; Magnetic resonance spectroscopy; Mammalian cells; Mass transfer; Membrane reactors; Metabolic flux; Metabolites; NMR spectroscopy; NMR-compatible; Nutrient concentrations; Nutrient loading; Oxygen; Perfusion; Phosphates; Rats; Rats, Wistar; Real time; Spectroscopy; Spheroids; Fluidized-bed bioreactor; NMR-compatible; Alginate-hepatocyte encapsulation; Dose response; Drug safety; 31P and 13C NMR spectroscopy; Fructose; P and C NMR spectroscopy
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2017.08.021

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. An NMR-compatible 5mm glass bioreactor with a multicoaxial perfusion system has been established using alginate encapsulated hepatocytes to non-invasively obtain kinetic parameters, in response to fructose. Using 31P NMR spectroscopy the EC50 of fructose was determined for ATP depletion. Using 13C NMR spectroscopy the rates of 13C-glucose uptake and glycogen incorporation were plotted versus fructose dose, from which EC50s can be determined. [Display omitted] •Real-time fluxomics in rat hepatocytes in a NMR-compatible fluidized-bed bioreactor.•A new multicoaxial life support device is described and validated.•The dose response of fructose on glycogen, lactate, and glutathione synthesis rate.•Determined glucose uptake & glycogen rate, & ATP and fructose-1-phosphate levels.•There is a reduction of animal tissue by an order-of-magnitude.