In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

CEITEC Central European Institute of Technology Mendel University in Brno Zemědělská 1 CZ 613 00 Brno Czech Republic

Department of Chemistry University of Wyoming 1000 E University Ave WY 82071 Laramie USA

Department of Molecular Cellular and Biomedical Sciences University of New Hampshire 46 College Road Durham NH 03824 USA

Faculty of Mathematics and Physics Charles University Prague Ke Karlovu 5 Prague 2 CZ 121 16 Czech Republic

Institute of Chemistry and Biochemistry Faculty of Agronomy Mendel University in Brno Zemědělská 1 CZ 613 00 Czech Republic

ISCARE IVF a s Jankovcova 1692 CZ 160 00 Praha 6 Czech Republic

The Czech Academy of Sciences Institute of Biophysics Královopolská 135 CZ 612 65 Brno Czech Republic

The Czech Academy of Sciences Institute of Physics Na Slovance 2 CZ 182 21 Prague 8 Czech Republic

VFN Gynekologicko porodnická klinika Apolinářská 18 CZ 120 00 Czech Republic

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