Predictive correlation and mechanistic understanding of ice nucleation in cryoprotective solutions

Abstract

Ice crystallization remains a fundamental barrier to successful cryopreservation, with cryoprotective agents (CPAs) employed to suppress ice nucleation. However, CPA development has been largely empirical, hindered by a limited understanding of the molecular mechanisms governing nucleation inhibition. Here, we present a parameterized correlation that predicts the nucleation temperature based on CPA type, concentration, sample volume, and cooling rate, providing a quantitative guideline for controlling ice nucleation. Through microscopic-level analysis, we show that nucleation inhibition of CPAs is rooted in their ability to alter water structure and dynamics. Structurally, CPAs disrupt water’s tetrahedral arrangement and increase tetrahedral entropy; dynamically, they form strong hydrogen bonds that impede the translational and rotational motion of water molecules, thereby increasing the activation energy barrier for the rearrangement of the hydrogen bond network. These findings elucidate the physicochemical basis of cryoprotection and provide rational design insights for next-generation CPAs.