Cell preservation is accomplished primarily by two methods: cryopreservation and dehydration, with the former being the standard technique used. In order to optimize and develop cell preservation protocols for cells that are difficult to preserve or whose end application is incompatible with current cell preservation protocls and to advance preservation by dehydration, a better understanding of the freeze- and dehydration-induced changes to the cell membrane is required. Despite a large body of literature on the topic, the mechanisms of damage to cells during slow freezing and dehydration are still ambiguous. The objective of this study is to investigate the mechanisms of damage to the cell membrane during slow freezing and dehydration and expand our outlook beyond the cell membrane to its underlying support, the cytoskeleton. In this study, we used several model systems to investigate slow freezing and dehydration. We used a liposome model to gather basic information on changes that can occur to a simple membrane system during freezing. This study revealed that eutectic formation was capable of dehydrating the membrane at low temperatures which may be contribute to alteration of the post-thaw membrane structure. We used a bacteria model to investigate the role of the phase transition and immediate versus slow osmotic stress on post-rehydration viability. This study revealed that going through a lyotropic membrane phase transition was detrimental to post-rehydration viability. This study also demonstrated that a rapidly applied osmotic stress was more detrimental to the structure/ organization of the membrane than gradual osmotic stress. We then subjected a model mammalian cell to both hyperosmotic stress and freeze-thaw and investigated both the membrane and cytoskeletal responses. Osmotic stress experiments suggested that alterations in membrane structure (i.e., surface defects and lipid dissolution) were directly dependent on the change in the chemical potential of water. These experiments also suggest that cell shrinkage and the resulting formation of membrane protrusions negatively affect viability upon return to isotonic conditions. It was found that membrane morphology in the dehydrated state and post-hyperosmotic viability was dependent on the stiffness of the cytoskeleton. Freeze/ thaw experiments suggested that ice-cell interaction decreases post-thaw viability. However, similar to osmotic stress experiments, cell shrinkage and cytoskeletal stiffness negatively impact post-thaw viability. We suggest the resulting membrane morphology due to cell shrinkage is also responsible for damage during freeze/ thaw. The various mechanisms discovered and the models proposed can be used in developing new protocols for cell preservation and for cell destruction (e.g. cryosurgery).
University of Minnesota Ph.D. dissertation. October 2010. Major: Mechanical Engineering. Advisor: Alptekin Aksan. 1 computer file (PDF) xviii, 199 pages.
Mechanisms and models of dehydration and slow freezing damage to cell membranes.
Retrieved from the University of Minnesota Digital Conservancy,
Content distributed via the University of Minnesota's Digital Conservancy may be subject to additional license and use restrictions applied by the depositor.