Do Inhibition Studies of Ice Crystallization Indicate Cryoprotectant Efficacy?

Common cryoprotectants (CPs) (dimethyl sulfoxide, glycerol, trehalose) require high concentrations (>10 wt %) and can cause adverse effects to cells and DNA. Alternative CPs are actively being investigated, and ice recrystallization inhibition (IRI), dynamic ice-shaping (DIS), and nucleation temperature (Tn) assays are being utilized in the discovery and down selection of new CP materials. We performed a three-pronged study with IRI, DIS, and Tn as metrics applied to three classes of molecules saccharides (mono- and di-), small molecules, and polymers (synthetic and biological). The results presented indicate that these metrics do not correlate to the efficacy of known CPs. Select polymeric materials, including antifreeze protein (AFP) and polyvinyl alcohol (PVA), showed positive results across all three metrics even at low concentrations (≤10 μM). At 1 mM, most saccharides and small molecules we studied yielded no positive IRI or DIS activity and did not change Tn significantly. Surprisingly, out of all the molecules studied, cysteamine induced nucleation of ice by elevating Tn by >50% over pure (supercooled) water. These results raise the question of whether the assays provide data that allows for discovery and down selection of new CP materials, or if other metrics should be formulated.