Cryopreservation is the technology used to stabilize cells at subzero temperature for a variety of applications including diagnosis and treatment of disease, and the production of therapeutic proteins. Current theories of cell damage during freezing were developed in the 1960’s, and little has changed since then. However, our understanding of cell biology as well as tools to interrogate cell responses during freezing has improved in the last 50 years. Low temperature Raman spectroscopy has been used to verify, for the first time using chemical spectra, the presence of ice inside the cell during freezing. With this tool, it is possible to internally observe frozen cells, and identify specific chemical and morphological changes that result in cell life or death. In this work, we propose to use this powerful tool to test two hypotheses to enhance our understanding of the mechanism of cell damage during freezing and thawing, and the manner by which the damage can be mitigated to improve cryopreservation outcome. For the first part of this work, we hypothesize that not all intracellular ice formation (IIF) is lethal and the conditions of cell membrane, cytoskeleton and mitochondria play an important role in determining IIF and cryopreservation outcome. Freezing responses of single cells as well as multi-cellular system cryopreserved and thawed in dimethyl sulfoxide (DMSO) solution will be examined to test this hypothesis. DMSO-free cryopreservation has attracted much recent interest due to the toxicity of DMSO. For the second part of this work, we hypothesis that non-DMSO multicomponent osmolyte solutions can be used to preserve cell viability and one component, disaccharide, acts to protect the cell through multiple interactions. Freezing responses of cells cryopreserved in a combination of non-DMSO cryoprotectants such as sugars, sugar alcohols, and amino acids will be examined. Interactions among sucrose (a typical disaccharide), water and cell membrane at low temperature will be also be investigated in order to test the second hypothesis. Enhancing our understanding of freezing damage and strategies to mitigate damage will improve the methods of preserving cell therapy products and therefore enable the treatment to reach the patients who could benefit from them.
University of Minnesota Ph.D. dissertation. September 2018. Major: Mechanical Engineering. Advisor: Allison Hubel. 1 computer file (PDF); xv, 143 pages.
Characterizing Freezing and Thawing Responses of Multiple Types of Cells Cryopreserved in Both DMSO and non-DMSO Cryoprotectants.
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