Freezing process in human cell nuclei: determination of cryoprotectant parameters strongly influencing the cell condition

Irena Kratochvílová1*, Martin Falk2, David Řeha3,5,  Jakub Šebera2, Martin Golan2,6, Krisztina Varga4*

1Institute of Physics of the Czech Academy of Sciences, Na Slovance 2, CZ-182 21, Prague 8, Czech Republic

2Institute of Biophysics,  Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, CZ-612 65 Brno, Czech Republic

3 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Zámek 136, CZ-373 33 Nové Hrady, Czech Republic

4Department of Chemistry, University of Wyoming, 1000 E. University Ave Laramie, WY 82071, USA

5Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zámek 136, 373 33 Nové Hrady, Czech Republic

6Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, CZ-121 16 Prague 2, Czech Republic

*Corresponding authors


In this work, the freezing process and cryoprotective function of three principally different cryoprotectants (antifreeze protein, trehalose and DMSO) were investigated in living cells as well as the phase transition processes of the cryoprotectants solutions. In order to gain deeper insight to the behaviour of the complex cell nucleus, the changes in the genome and nuclear envelope integrity were investigated simultaneously with the higher-order chromatin structure. Cells were cultured in standard medium and compared to cells that were treated with cryoprotectants at the initial unfrozen state, after freezing, and after melting. Analysis was performed after results were obtained from ab-initio modelling, molecular dynamics, Raman spectroscopy, differential scanning calorimetry and X-ray diffraction for each of the cryoprotectants solutions. Our results provide valuable experimental and computational data and help to design novel cryoprotective substances and develop more efficient cryoprotection protocols.