Critical defects in cryopreserved cell nuclei: DNA
structure changes
Martin Falk1, Iva Falková1, Eva
Pagáčová1, Olga Kopečná1, Alena Bačíková1,
Daniel Šimek2, Martin Golan2,4, Stanislav Kozubek1,
Michaela Pekarová1, Shelby E. Follett3, Bořivoj Klejdus5,6,
K. Wade Elliott7, Krisztina Varga7, Olga Teplá8,9 and
Irena Kratochvílová2
1The Czech Academy of
Sciences, Institute of Biophysics, Královopolská 135, CZ-612 65 Brno, Czech
Republic
2The Czech Academy of
Sciences, Institute of Physics, Na Slovance 2, CZ-182 21, Prague 8, Czech
Republic
3Department of
Chemistry, University of Wyoming, 1000 E. University Ave Laramie, WY 82071, USA
4Faculty of Mathematics
and Physics, Charles University in Prague, Ke Karlovu 5, CZ-121 16 Prague 2,
Czech Republic
5Institute of Chemistry
and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemědělská 1, CZ-613 00
6CEITEC-Central
European Institute of Technology, Mendel University in Brno,
Zemědělská 1, CZ-613 00 Brno, Czech Republic
7Department of
Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, 46
College Road, Durham, NH 03824, USA
8ISCARE IVF a.s. Jankovcova
1692, CZ-160 00 Praha 6
9VFN
Gynekologicko-porodnická klinika, Apolinářská 18, CZ-120 00, Czech Republic
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 [1].
1.
M. Falk, I. Falková, E. Pagáčová, O. Kopečná, A.
Bačíková, D. Šimek, M. Golan, S. Kozubek, M. Pekarová, S. E. Follett, B.
Klejdus, K. W. Elliott, K. Varga, O. Teplá, I. Kratochvílová, Scientific
Reports, 8/14694, 1-18