Abstract
During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.
Highlights
IntroductionIce recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures
During cryopreservation, ice recrystallization is a major cause of cellular damage
Several of these small molecules are effective additives for the freezing of human red blood cells (RBCs) resulting in significantly higher numbers of intact RBCs post-thaw while using greatly reduced quantities of glycerol with slow freezing rates[16]. These small molecule inhibitors of ice recrystallization constitute a novel class of cryoprotective agents that may meet the increasing needs for long-term storage of important biological materials for emerging cell therapeutics in the field of regenerative medicine and tissue engineering. We demonstrate that these ice recrystallization inhibitors (IRIs) are capable of mitigating ice growth in vitro with reduced quantities of glycerol and this ability is correlated to increased post-thaw viability using annucleate human RBCs as an appropriate model
Summary
Ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming. Work in our laboratory identified a class of carbon-linked (C-linked) antifreeze glycoprotein (AFGP) analogues (Fig. 1) that possess custom-tailored antifreeze activity[8,9] While these compounds are very effective inhibitors of ice recrystallization and excellent cryoprotectants for human liver cell lines in the absence of DMSO, www.nature.com/scientificreports/
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