Towards Dry Preservation of Mammalian Cells at Ambient Temperature: Modulating Solution Effects Injury

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ProQuest, 2008 - 132 pages
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Ultimately, to achieve a stable matrix suitable for long-term storage, the viscosity needs to approach a level that renders the sample 'glassy' or solid-like at the given storage temperature. For room temperature storage, a trehalose-based sample requires dehydration to below 0.1 gH2O/gdw. Because water acts as a plasticizer in these storage formulations, wetter samples will have a lower glass transition temperature. The goal of this work was to advance towards dry preservation at room temperature by first achieving a level of dehydration that can enable storage at -80C, a temperature that can be achieved with electric freezers, thus avoiding the need for expensive cryogens for long-term bio-banking. Advances were made that improved the limits of dehydration that can be tolerated by mammalian cells during processing for dry preservation. A cumulative osmotic stress model of drying injury was proposed and evaluated to enable the rationale design of drying protocols. Using this model as a guide, osmometric modifications were made to existing dry-preservation protocols to reduce the magnitude of cumulative osmotic stress experienced by cells. This improved the overall outcome, enabling full cell functionality to be retained at lower moisture contents. A novel microwave-based drying technique for mammalian cells was also developed to enable rapid dehydration of biological samples, thus reducing cumulative chemical stresses that occurred during processing. Finally a multiscale analysis of drying metrics was undertaken. Gravimetry based macroscale metrics were compared to the microscale measures obtained with time-resolved fluorescence spectroscopy techniques. The difference between these metrics highlighted the need to study drying characteristics at a microscopic level.
  

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Contents

DEHYDRATION INJURY IN MAMMALIAN CELLS
11
OSMOTIC INJURY IN MAMMALIAN CELLS THRESHOLD AND RATE
20
PREPROCESSING MAMMALIAN CELLS PRIOR TO DRYING
41
A ROLE FOR MICROWAVE PROCESSING IN THE DRPRESERVATION
56
MICROENVIRONMENT IN A DRYING DROPLET LOCAL MOISTURE
88
CONCLUSION
106
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