Ice recrystallization occurs during cryopreservation and is correlated with reduced cell viability after thawing.

Ice recrystallization inhibition (IRI) activity is a very desirable property for an effective cryoprotectant. This property was first observed in biological antifreezes (BAs), which cannot be utilized in cryopreservation due to their ability to bind to ice. To date, potent IRI active compounds have been limited to BAs or synthetic C-linked AFGP analogues (1 and 2), all of which are large peptide-based molecules. This paper describes the first example of low molecular weight carbohydrate-based derivatives that exhibit potent IRI activity. Non-ionic surfactant n-octyl-β-D-galactopyranoside (4) exhibited potent IRI activity at a concentration of 22 mM, whereas hydrogelator N-octyl-D-gluconamide (5) exhibited potent IRI activity at a low concentration of 0.5 mM. Thermal hysteresis measurements and solid-state NMR experiments indicated that these derivatives are not exhibiting IRI activity by binding to ice. For non-ionic surfactant derivatives (3 and 4), we demonstrated that carbohydrate hydration is important for IRI activity and that the formation of micelles in solution is not a prerequisite for IRI activity. Furthermore, using solid-state NMR and rheology we demonstrated that the ability of hydrogelators 5 and 6 to form a hydrogel is not relevant to IRI activity. Structure–function studies indicated that the amide bond in 5 is an essential structural feature required for potent IRI activity.

In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at −80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells.

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.