Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization

Abstract

Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth.

Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing

“…Apart from the cooling rate, the warming rate also represents an important parameter Recent studies (16,30,31) have shown that recrystallization of vitreous water and crystal growth during the warming process can inflict lethal damage on the cells. The transient simulation of the heat transfer for the warming process was also performed (SI Appendix, Fig.…”

“…These CCRs are experimentally unattainable values for droplets of a diameter >10 μm (as, e.g., in cells). Fortunately, complete vitrification is not necessary for cryopreservation, i.e., cells seem to tolerate well the presence of small ice crystals (less than 1 μm) (16). Since the tolerance depends on the CPAs, the tolerance in CPA-free cryopreservation can be expected to be very low.…”

Rapid freezing versus Cryotop vitrification of mouse two-cell embryos

“…Therefore, by Fourier's law, the rate of warming through the critical zone of ice nucleation and formation will be slower than the rate of cooling through this zone [32]. Previously, it was presumed that CPAs exerted their main effects by preventing ice crystallization during cooling.…”

Viability of embryonic axes of Araucaria angustifolia after freezing using two cryopreservation methods

“…Direct immersion in liquid nitrogen (LN), or flash-cooling, induces intracellular freezing earlier in the process, which prevents rapid cell dehydration. Furthermore, the ice crystals that form within the cell are minuscule and do not cause any damage (Engelmann & Dussert, 2013;Huebinger et al, 2016). As such, the EA with high water concentration that are flash-cooled could survive cryopreservation (Normah & Makeen, 2008;Berjak & Pammenter, 2013).…”

“…At this temperature, water molecules form "ice embryos" that are of a critical size and thermodynamically capable of growing and forming into crystals (Thomson et al, 2015;Lo et al, 2017). Ice crystals affect the structural and osmotic integrity of cells causing physical ruptures and mechanical damage (Pammenter & Berjak, 2014;Huebinger et al, 2016).…”