An NMR-compatible bioartificial liver for metabolomic investigation of drug action.

摘要

NMR-compatible bioartificial liver (BAL) studies have been performed for thirty years and still have not been maintained beyond 8hrs. This doctoral work describes the engineering efforts in creating a long-term NMR-compatible BAL. Four general types of BALs have been reported: suspension, microcarrier, membrane, and entrapment. Reasons and efforts toward establishing a fluidized-bed entrapment bioreactor, which maintains hepatocytes entrapped in alginate for 30 hrs, and likely for long-term, are described. The electrostatically-encapsulated cells generate 1.5 mls of 500 mum diameter spherical encapsulates, containing about 10,000 cells each, in about 5 minutes. These encapsultates containing entrapped cells are then incolulated into a 10 mm glass NMR tube and are percholate in the bottom of the glass tube forming the fluidized-bed.;To demonstrate the power of the NMR-compatible BAL in toxicity studies using in vivo 31P and 13C NMR spectroscopy, a rat hepatoma cell line, JM1, was used. The encapsulated cells were maintained overnite (16hrs) with 3-13C-cysteine and u-13C-glucose replaced in the perfused media, and production rates for glutathione, the body's primary antioxidant, and lactate, an anaerobic glycolytic end-product common in cancer, were determined. The next day (16-20 hrs) when [3-13C-cysteinyl]glutathione was at 13C isotopic steady-state and the JM1 cells were at metabolic steady-state, the effects of two doses of bromobimane, a glutathione depleting agent, and three different doses of acetaminophen on the in vivo 31P and 13C NMR spectra were determined. The application of this time series data to toxicodyanamics and toxicokintetics is discussed. This is the first study demonstrating with 1 minute temporal resolution, the non-steady-state real-time toxicokinetics of glutathione.;Once the NMR-compatible BAL was demonstrated with a relatively easy liver cell-type to culture, a cell line (i.e., JM1), the fluidized-bed bioreactor was established with primary rat hepatocytes. Liver is exquisitely sensitive to oxygen tension and ranges from 8% to 3% across its capillary-bed, yet all previous NMR-compatible BAL studies have all gasified the perfusion media with 95% oxygen. Therefore, the effect of four oxygen concentrations (20%, 35%, 55%, and 95%) on viability was monitored by in vivo 31P NMR. Only the 35% and 55% oxygen treatments maintained hepatocytes viability for 28 hours and likely beyond with no change in beta-nucleotide triphosphate levels. Analysis of the in vivo 13C NMR data for the 55% oxygen treatment revealed synthetic rates for lactate and glutathione demonstrating differentiated functions were present and quantifying the function. This is the first demonstration of any primary hepatocyte culture being beyond 8 hrs in a NMR-compatible BAL.