1 SCIENTIFIC REPORTS | (2018) 8:14694 | DOI:10.1038/s41598-018-32939-5 www.nature.com/scientificreports Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are diferentially controlled by cryoprotectants Martin Falk 1 , Iva Falková 1 , Olga Kopečná 1 , Alena Bačíková 1 , Eva Pagáčová 1 , Daniel Šimek 2 , Martin Golan 2,3 , Stanislav Kozubek 1 , Michaela Pekarová 1 , Shelby E. Follett 4 , Bořivoj Klejdus 5,6 , K. Wade Elliott 7 , Krisztina Varga 7 , Olga Teplá 8,9 & Irena Kratochvílová 2 In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the frst time, we describe replicating cell-specifc DNA damage and higher-order chromatin alterations after freezing and thawing. We identifed 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. Application of cryopreservation to living cells and tissues has revolutionized biotechnology and modern med- icine 1,2 . However, extensive damage occurs to a percentage of frozen and thawed cells and tissues. Tough the freeze-thaw process can be greatly afected by the use of cryoprotective additives to improve cell viability 3,4 , the efects of freezing and cryoprotectants per se on the complex status of cell nuclei (and the genetic information contained therein) remain controversial 4–7 . Contradictory results in the literature have prevented a consensus on the fundamental question of the extent of DNA and chromatin fragmentation that occurs during freezing and 1 The Czech Academy of Sciences, Institute of Biophysics, Královopolská 135, CZ-612 65, Brno, Czech Republic. 2 The Czech Academy of Sciences, Institute of Physics, Na Slovance 2, CZ-182 21, Prague 8, Czech Republic. 3 Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, Prague 2, CZ-121 16, Czech Republic. 4 Department of Chemistry, University of Wyoming, 1000 E. University Ave, WY 82071, Laramie, USA. 5 Institute of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemědělská 1, CZ-613 00, Czech Republic. 6 CEITEC-Central European Institute of Technology, Mendel University in Brno, Zemědělská 1, CZ-613 00, Brno, Czech Republic. 7 Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, 46 College Road, Durham, NH, 03824, USA. 8 ISCARE IVF a.s, Jankovcova 1692, CZ-160 00, Praha 6, Czech Republic. 9 VFN Gynekologicko-porodnická klinika, Apolinářská 18, CZ-120 00, Czech Republic. Correspondence and requests for materials should be addressed to M.F. (email: falk@ibp.cz) or I.K. (email: krat@fzu.cz) Received: 17 April 2018 Accepted: 17 September 2018 Published: xx xx xxxx OPEN