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.

Significant cell damage occurs during cryopreservation resulting in a decreased number of viable and functional cells post-thawing. Recent studies have correlated the unsuccessful outcome of regenerative therapies with poor cell viability after cryopreservation. Cell damage from ice recrystallization during freeze-thawing is one cause of decreased viability after cryopreservation. We have assessed the ability of two C-AFGPs that are potent inhibitors of ice recrystallization to increase cell viability after cryopreservation. Our results indicate that a 1-1.5 mg/mL (0.5-0.8 mM) solution of C-AFGP 1 is an excellent alternative to a 2.5% DMSO solution for the cryopreservation of human embryonic liver cells.

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