Micropatterned cell arrays for detecting DNA damage

Numerous agents are capable of interacting with DNA and damaging it. Permanent changes in the DNA structure can be both mutagenic and cytotoxic; therefore, methods to measure the susceptibility of cells to mutations are important for risk assessment and identifying therapeutic interventions. One classical method for assessing DNA damage at a global level is the COMET assay, based on electrophoretic extension of the nuclear DNA in single cells embedded in agarose. In this assay, the size and shape of the extended 'comet tail' can be correlated to breaks in the DNA. This assay was first developed in the mid 1980s for non adherent cells such as lymphocytes; however, it has been plagued by technical difficulties, low throughput, and lab-to lab variation. These challenges have been exacerbated in adhesion-dependent cells as DNA damage accrues variably over time as they are enzymatically detached from their microenvironment. This thesis explores whether the COMET assay can be improved by micropatterning adherent cells prior to agarose embedding. Hepatocytes were chosen as a model cell type and x-ray radiation was chosen as a model DNA damaging agent. In order to establish the feasibility of measuring x-ray induced damage on hepatocyte DNA, standard curves were first generated for hepatocytes suspended in agarose. These experiments revealed a minimum detectable threshold of 1 Gy and displayed a monotonic increase in DNA damage in response to exposures up to 10 Gy. In comparison, adherent hepatocytes overlaid with agarose and irradiated in situ displayed similar levels of mean damage but lower levels of variability than suspended cells. We hypothesize that the decreased variability could be due to a reduction in programmed cell death incurred by detachment of adherent cells.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Electrical Engineering and Computer Science.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Mittal, Sukant

Advisor dc:contributor.advisor

• Sangeeta N. Bhatia.

• Electrical Engineering and Computer Science.

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/1721.1/44734

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/44734