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Modification of the alkaline comet assay with human mesenchymal stem cells

Authors

  • Robert Fuchs,

    Corresponding author
    1. Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria
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  • Ingeborg Stelzer,

    Corresponding author
    1. Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria
    2. Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria
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  • Christoph M. P. Drees,

    1. Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria
    2. Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria
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  • Christian Rehnolt,

    1. Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria
    2. Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria
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  • Elisabeth Schraml,

    1. Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria
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  • Anton Sadjak,

    1. Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria
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  • Wolfgang Schwinger

    1. Division of Pediatric HematologyOncology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Auenbruggerplatz 30, 8036 Graz, Austria
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These authors contributed equally to this study.

To whom correspondence should be addressed (email robert.fuchs@medunigraz.at).

Abstract

MSCs (mesenchymal stem cells) are planned foruse in regenerative medicine to offset age-dependent alterations. However, MSCs are affected by replicative senescence associated with decreasing proliferation potential, telomere shortening and DNA damage during in vitro propagation. To monitor in vitro senescence, we have assessed the integrity of DNA by the alkaline comet assay. For optimization of the comet assay we have enhanced the stability of comet slides in liquid and minimized the background noise of the method by improving adhesion of agarose gels on the comet slides and concentrating cells on a defined small area on the slides. The modifications of the slide preparation increase the overall efficiency and reproducibility of the comet assay and minimize the image capture and storage. DNA damage of human MSCs during in vitro cultivation increased with time, as assessed by the comet assay, which therefore offers a fast and easy screening tool in future efforts to minimize replicative senescence of MSCs in vitro.

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