Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants

Martin Falk,Irena Kratochvílová,Eva Pagáčová,Shelby E Follett,Alena Bačíková,Martin Golan,Daniel Šimek,Olga Teplá,Bořivoj Klejdus,Olga Kopečná,K Wade Elliott,Krisztina Varga,Iva Falková,Stanislav Kozubek,Michaela Pekarová

doi:10.1038/s41598-018-32939-5

Abstract

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.

Highlights

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells
In our previous work[3], we focused on the formation of ice during freezing as an important parameter that strongly influences cellular destruction and examined specific properties of selected cryoprotectant solutions during freezing, including dimethyl sulfoxide (DMSO), trehalose and a recombinant antifreeze fusion protein (AFP) that was originally isolated from the Anatolica polita desert beetle[2,3]
We identified a new type of DNA lesion that is associated with freezing and thawing, that appears in replicating (S-phase) cells and that can be labelled with γH2AX and 53BP1 antibodies

Summary

Introduction

We shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. 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. In our previous work[3], we focused on the formation of ice during freezing as an important parameter that strongly influences cellular destruction and examined specific properties of selected cryoprotectant solutions during freezing, including dimethyl sulfoxide (DMSO), trehalose and a recombinant antifreeze fusion protein (AFP) that was originally isolated from the Anatolica polita desert beetle[2,3] Building on this knowledge, here, we used these cryoprotectants to investigate the importance and extent of chromatin damage in freeze-thawed cells, fragmentation and structural changes of chromatin. We described the post-freeze-thaw status of cells from two major perspectives: (i) the widely debated damage to DNA integrity, which can directly lead to death or genetic defects in cryopreserved cells, and (ii) the previously unexplored, less prominent alterations in the functional status of the higher-order chromatin structure and its impact on the viability of freeze-thawed cells

Methods

Results

Conclusion