Towards Dry Preservation of Mammalian Cells at Ambient Temperature: Modulating Solution Effects Injury
ProQuest, 2008 - 132 pages
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 -80°C, 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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DEHYDRATION INJURY IN MAMMALIAN CELLS
OSMOTIC INJURY IN MAMMALIAN CELLS THRESHOLD AND RATE
PREPROCESSING MAMMALIAN CELLS PRIOR TO DRYING
A ROLE FOR MICROWAVE PROCESSING IN THE DRPRESERVATION
MICROENVIRONMENT IN A DRYING DROPLET LOCAL MOISTURE
1.17 Osm trehalose active heating periods anhydrous preservation apoptosis apoptotic cell membrane cell samples dried cells dried cellular chemical potential cooling rates Corning Incorporated coverslips cryobiology cryopreservation cumulative osmotic stress curve datasets dehydration desiccation desiccation tolerant detached cells dried from isotonic dried in drybox droplets dried dry weight drybox drying droplet drying process drying rate drying solution drying techniques endocytosis extracellular osmolality final moisture content fluorescence fluorescence anisotropy freeze drying gH2O/gdw glassy grams of dry grams of water hyperosmotic hyperosmotic exposure hypertonic images incubated indicate injury intermittent microwave exposure intracellular trehalose mammalian cells microwave drying microwave processing microwave technique moisture level molecular mobility mOsm mouse macrophage cells Osm trehalose solution Osm/kg extracellular solution osmolality osmolytes Propidium Iodide protein protocol recalcitrant seeds rotational correlation solution in drybox Spray drying step change studies surface total number total viability trehalose-PBS solution Viability of mouse viability response viability score viability trend volume water per grams