Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007. / Includes bibliographical references (leaves 142-149). / Inspired by the variety of organisms that are naturally desiccation tolerant, anhydrobiotic preservation potentially furnishes a means of processing and storing mammalian cells in a state of "suspended animation" at ambient conditions in carbohydrate glasses. Although there have been promising applications of this technique, especially when employing the disaccharide trehalose, the ultimate goal of room temperature long-term storage has thus far not been achieved -- at least in part owing to an incomplete understanding of the fundamental cellular damage mechanisms. Although there have been many studies examining the thermodynamics of relevance to anhydrobiotic preservation, particularly with regard to lipid phase and the effect of carbohydrates thereupon, comparatively little attention has been paid to the effect of transport kinetics on preservation success. Further, although cells are typically dried in carbohydrate solutions on a solid support, there are few studies on the role played by the support. This work seeks to help remedy such deficiencies. First, considering damage mechanisms at the individual cell level, giant liposomes were employed as a model cell system, given that the cell membrane is a key damage site. / (cont.) The influence of solid surface - lipid bilayer interactions was investigated in the presence and absence of trehalose. Two lipids were chosen in order to determine the effect of lipid phase on surface interactions: gel-phase 1,2-distearoyl-sn -glycero-3-phosphocholine (DSPC) and liquid-crystalline phase 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). In the absence of trehalose, DSPC liposomes adsorbed to the polystyrene support surface, producing irreversible structural changes and apparent leakage of all intravesicular solute upon drying and re-hydration. Addition of trehalose significantly reduced vesicle adsorption with only transitory intravesicular solute leakage for the re-hydrated vesicles, likely owing to a transient osmotic imbalance; however, at very low moisture contents, the vesicles underwent permanent structural changes. In contrast to the results with DSPC vesicles, DLPC vesicles largely evaded adsorption and exhibited high intravesicular solute retention when dried and re-hydrated even in the absence of trehalose, despite significant internal structural changes. Next, taking a more macroscopic view, the influence of the solid support and desiccation kinetics was analyzed at the whole droplet level. / (cont.) During desiccation, sessile droplets of glass-forming carbohydrate solutions exhibit complex dynamic phenomena, including fluid flow, droplet deformation and crack formation, all of which may alter cell preservation efficacy. Two factors were identified that strongly influenced the features of the preserved giant liposome suspension droplets: the underlying surface and the liposome concentration. In particular, the surface altered the droplet shape as well as the microflow pattern - and in turn the moisture conditions encountered by the liposomesr during desiccation. A ring deposit formed when the droplets were dried on polystyrene -- as would be expected owing to the capillary flow that generally occurs in pinned droplets. In contrast, when dried on the more hydrophilic glass slide, the resulting droplets were thinner and the liposomes accumulated near their centers -- an unexpected result likely owing to the glass-forming nature of the trehalose solutions. As might be anticipated given the variations in liposome distribution, the choice of surface also influenced crack formation upon continued drying. In addition to providing a preferential path for drying, such cracks are relevant because they could inflict mechanical damage on cells. / (cont.) Liposome concentration had an even more profound effect on crack formation; indeed, while cracks were found in all droplets containing liposomes, in their absence few of the droplets cracked at all, regardless of the surface type. Given the experimentally-determined non-uniform distribution of liposomes within the sessile droplets, a finite element method model was formulated to assess the moisture content variation within desiccating trehalose solution microdroplets - both unsupported and sessile. In the unsupported droplet, a thin glassy skin was found to form at the droplet surface, which significantly hampered further evaporation owing to the extremely low diffusivity of water in trehalose glasses. Thus, residual water was essentially trapped in the droplet core for long times, preventing a transition to the glassy state there. This is significant for anhydrobiotic preservation because most liposomes, or cells, would be located in the droplet core rather than in the thin glassy skin. The sessile droplet provided another degree of complexity in that the moisture concentration was inhomogeneous not only in the direction perpendicular to the interface, but along it as well, since the glassy skin did not form uniformly, instead progressing inward from the contact line. / (cont.) In summary, surface interactions were found to play a significant role in anhydrobiotic preservation, both at the cellular level through adsorption and at the whole droplet level through their effect on distribution of suspended liposomes (or cells) and crack formation. Further, kinetic phenomena had a strong influence, again at the cellular level through transient osmotic imbalances and at the whole droplet level in the form of inhomogeneous moisture distributions. Such effects clearly merit further investigation in the development of anhydrobiotic preservation protocols. / by Dana R. Adams. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/38986 |
| Date | January 2007 |
| Creators | Adams, Dana R. (Dana Renée) |
| Contributors | Robert S. Langer., Massachusetts Institute of Technology. Dept. of Chemical Engineering., Massachusetts Institute of Technology. Dept. of Chemical Engineering. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 149 leaves, application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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