Micropatterned cell arrays for detecting DNA damage

Abstract

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.

(cont.) Finally, we explored the feasibility of performing the comet assay by in situ irradiation of a micropatterned array of adherent cells. Single cell hepatocyte patterning was achieved by photolithographic patterning of collagen I on glass and optimization of seeding conditions. Gradiations of x-ray exposure were achieved by employing localized domains of lead shielding between the cells and the source. As a proof of principle, we obtained two domains of differential x-ray exposure and the resulting DNA damage was similar in the micropatterned format to the randomly-organized adherent format. Several challenges emerged from these experiments including potential interactions of DNA with the glass surface leading to 'streaking' artifacts. Nonetheless, with increased resolution of x-ray exposure, and further technical improvements, this assay has the potential to offer both reduced variability for adherent cells as well as assay multiplexing due to spatial encoding of x-ray dosage.