Metabolomics footprinting of three dimensional bioreactors with applications to in-cell NMR Public Deposited

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  • March 21, 2019
Creator
  • Seagle, Christopher M.
    • Affiliation: School of Medicine, UNC/NCSU Joint Department of Biomedical Engineering
Abstract
  • Electrostatic devices for the manufacturing of cell encapsulations were constructed and used to form spherical alginate encapsulations containing hepatocytes, hepatic progenitor cells, mixed hepatic populations, and Escherichia coli. The encapsulations were tested for cell viability, cell functionality, respiration, and encapsulation architecture. A novel high through-put NMR method was used to test serial media extractions from multi-coaxial bioreactors to determine metabolic function of human hepatic cell encapsulations over a 30 day period. These tests revealed that by day five, lactate decreased below detection limits while glucose concentrations did not significantly change indicating aerobic metabolism. Evidence of system perturbation and recovery following ammonia spiking of superfused media was also observed along with glutamine metabolism indicating urea cycle activity. These NMR results are the first demonstration of an assay to monitor the full spectrum of metabolites found in ex vivo encapsulated hepatic cell culture media for determination of consumption and production rates. Improvements to the NMR method were introduced to accommodate for evaporative loss and allow for more precise determination of actual metabolite consumption of media constituents and production of metabolites. This new approach was used to compare metabolism differences between three-dimensional closed loop bioreactors and classic two-dimensional cultures to test the hypothesis that the more efficient 3D bioreactor will facilitate aerobic metabolism compared to 2D cultures. This will be reflected by consumption of aerobic nutrients, such as glutamine and alanine, and anaerobic production of lactate. Based upon differences in metabolism of glucose and other energy sources, these studies seek to also test the hypothesis that traditionally observed oxygen levels (95%) in 3D culture studies are excessive for hepatocytic function. The tools of encapsulation based metabolomics were adapted to in-cell protein conformation studies using Escherichia coli. Preliminary findings, while inconclusive, suggest that encapsulation based studies can eliminate confounding extracellular signals in NMR studies.
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  • In Copyright
Advisor
  • Macdonald, Jeffrey
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Open access
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