Influence of mitochondrial membrane potential on the cryopreservation survival of hepatocytes

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.

Department dc:contributor.department

Harvard University--MIT Division of Health Sciences and Technology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Daly, Margaux E. (Margaux Erin)

Advisor dc:contributor.advisor

• Mehmet Toner and Gregory Stephanopoulos.

• Harvard University--MIT Division of Health Sciences and Technology.

Statement dc: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.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/33087

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/33087