Thesis (M. Eng.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references (leaves 48-49). / Hepatocytes are widely used in the pharmaceutical and medical fields for drug metabolism studies, bioartificial liver devices, and repopulation of damaged livers as an alternative to transplantation. However, these cells are scarce and difficult to maintain in culture for prolonged periods of time. Banks of cryopreserved liver cells would significantly alleviate issues of hepatocyte availability, and efforts are being made to improve the viability and functionality of frozen hepatocytes. Previously, most work on improving post-thaw viability has hinged on limiting the physical damage of freezing by adding cryoprotective agents and optimizing cooling rates. Membrane-permeable cryoprotectants, such as dimethyl sulfoxide, though widely used, can be extremely toxic to the cell. More natural, non-membrane-permeable cryoprotectants, inspired by freeze-tolerant animals have also been used. A non-metabolizable glucose analog, 3-0-methyl- glucose (30MG), has shown promise with hepatocytes and was used in this study. Kinetics of the rGLUT2 cellular transporter used for 30MG uptake were quantified; Km and Vmax were determined to be 27.6 mM and 1.38 mM/s, respectively, by Lineweaver-Burk analysis and 70.0 mM and 1.82 mM/s, respectively, by Eadie-Hofstee analysis. This study also aimed to investigate the role of mitochondria in cell death induced by freezing. In particular, mitochondrial membrane potential (MMP) was investigated as a predictor of a cell's likelihood to avoid apoptosis from freeze-induced stress. Cells were sorted into high and low MMP subpopulations, frozen, thawed, and cultured for 24 hours. / (cont.) Cell cultures were analyzed for attachment yield, viability of attached cells and overall viability, which were 87%, 68% and 59%, respectively for the high MMP subpopulation, and 68%, 53% and 35%, respectively for the low MMP subpopulation. Morphological differences such as extent of membrane blebbing were observed as well, verifying that cells with a high MMP are more likely to survive the cryopreservation process. These results demonstrated that MMP is a determinant of both frozen hepatocyte adherence efficiency and viability; a high MMP yields a significant advantage in both. Our understanding of the role of MMP in freeze-thaw death and of the characteristics of the rGLUT2 transporter will lead to the development of more successful cryopreservation protocols. / by Margaux E. Daly. / M.Eng.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/33087 |
| Date | January 2005 |
| Creators | Daly, Margaux E. (Margaux Erin) |
| Contributors | Mehmet Toner and Gregory Stephanopoulos., Harvard University--MIT Division of Health Sciences and Technology., Harvard University--MIT Division of Health Sciences and Technology. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 61 leaves, 3031371 bytes, 3033020 bytes, application/pdf, application/pdf, 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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