Ice Recrystallization Inhibition Is Insufficient to Explain Cryopreservation Abilities of Antifreeze Proteins

Antifreeze proteins (AFPs) and glycoproteins (AFGPs) are exemplary at modifying ice crystal growth and at inhibiting ice recrystallization (IRI) in frozen solutions. These properties make them highly attractive for cold storage and cryopreservation applications of biological tissue, food, and other water-based materials. The specific requirements for optimal cryostorage remain unknown, but high IRI activity has been proposed to be crucial. Here, we show that high IRI activity alone is insufficient to explain the beneficial effects of AF(G)Ps on human red blood cell (hRBC) survival. We show that AF(G)Ps with different IRI activities cause similar cell recoveries of hRBCs and that a modified AFGP variant with decreased IRI activity shows increased cell recovery. The AFGP variant was found to have enhanced interactions with a hRBC model membrane, indicating that the capability to stabilize cell membranes is another important factor for increasing the survival of cells after cryostorage. This information should be considered when designing novel synthetic cryoprotectants.