Effect of Oxygen Concentration on Viability and Metabolism in a Fluidized-Bed Bioartificial Liver Using P-31 and C-13 NMR Spectroscopy

Many oxygen mass-transfer modeling studies have been performed for various bioartificial liver (BAL) encapsulation types; yet, to our knowledge, there is no experimental study that directly and noninvasively measures viability and metabolism as a function of time and oxygen concentration. We report the effect of oxygen concentration on viability and metabolism in a fluidized-bed NMR-compatible BAL using in vivo P-31 and C-13 NMR spectroscopy, respectively, by monitoring nucleotide triphosphate (NTP) and C-13-labeled nutrient metabolites, respectively. Fluidized-bed bioreactors eliminate the potential channeling that occurs with packed-bed bioreactors and serve as an ideal experimental model for homogeneous oxygen distribution. Hepatocytes were electrostatically encapsulated in alginate (avg. diameter, 500 mu m; 3.5 x 10(7) cells/mL) and perfused at 3 mL/min in a 9-cm (inner diameter) cylindrical glass NMR tube. Four oxygen treatments were tested and validated by an in-line oxygen electrode: (1) 95: 5 oxygen: carbon dioxide (carbogen), (2) 75: 20: 5 nitrogen: oxygen: carbon dioxide, (3) 60: 35: 5 nitrogen: oxygen: carbon dioxide, and (4) 45: 50: 5 nitrogen: oxygen: carbon dioxide. With 20% oxygen, beta-NTP steadily decreased until it was no longer detected at 11 h. The 35%, 50%, and 95% oxygen treatments resulted in steady beta-NTP levels throughout the 28-h experimental period. For the 50% and 95% oxygen treatment, a C-13 NMR time course (similar to 5 h) revealed 2-C-13-glycine and 2-C-13-glucose to be incorporated into [2-(13)-Cglycyl]glutathione (GSH) and 2-C-13-lactate, respectively, with 95% having a lower rate of lactate formation. P-31 and C-13 NMR spectroscopy is a noninvasive method for determining viability and metabolic rates. Modifying tissue-engineered devices to be NMR compatible is a relatively easy and inexpensive process depending on the bioreactor shape.