Department of Chemistry and Biomolecular Sciences , University of Ottawa , Ottawa , Ontario K1N 6N5 , Canada.
Canadian Blood Services , Centre for Innovation , Edmonton , Alberta T6G 2R8 , Canada.
Langmuir. 2019 Jun 11;35(23):7452-7458. doi: 10.1021/acs.langmuir.8b02126. Epub 2018 Sep 7.
Ice formation remains central to our understanding of the effects of low temperatures on the biological response of cells and tissues. The formation of ice inside of cells and the net increase in crystal size due to recrystallization during thawing is associated with a loss of cell viability during cryopreservation. Because small-molecule ice recrystallization inhibitors (IRIs) can control the growth of extracellular ice, we sought to investigate the ability of two aryl-glycoside-based IRIs to permeate into cells and control intracellular ice recrystallization. An interrupted graded freezing technique was used to evaluate the IRI permeation into human red blood cells (RBCs) and mitigate cell damage during freezing and thawing. The effect of IRIs on the intracellular growth of ice crystals in human umbilical vein endothelial cells (HUVECs) was visualized in real time under different thawing conditions using fluorescence cryomicroscopy. Adding an aryl glycoside to 15% glycerol significantly increased post-thaw RBC integrity by up to 55% during slow cooling compared with the 15%-glycerol-only control group. The characteristics of the cryobiological behavior of the RBCs subjected to the interrupted graded freezing suggest that the aryl-glycoside-based IRI is internalized into the RBCs. HUVECs treated with the IRIs were shown to retain a large number of small ice crystals during warming to high subzero temperatures and demonstrated a significant inhibition of intracellular ice recrystallization. Under slow thawing conditions, the aryl glycoside IRI p-bromophenyl-β-d-glucoside was shown to be most effective at inhibiting intracellular ice recrystallization. We demonstrate that IRIs are capable of internalizing into cells, altering the cryobiological response of cells to slow and rapid freezing and controlling intracellular ice recrystallization during freezing. We conclude that IRIs have tremendous potential as cryoprotectants for the preservation of cells and tissues at high subzero temperatures.
冰的形成仍然是我们理解低温对细胞和组织生物反应影响的核心。细胞内冰的形成以及解冻过程中由于再结晶导致的晶体尺寸净增加与细胞在冷冻保存过程中的活力丧失有关。由于小分子冰重结晶抑制剂 (IRI) 可以控制细胞外冰的生长，我们试图研究两种基于芳基糖苷的 IRI 渗透进入细胞并控制细胞内冰重结晶的能力。采用间断分级冷冻技术评估 IRI 渗透进入人红细胞 (RBC) 的能力，并减轻冷冻和解冻过程中的细胞损伤。在不同的解冻条件下，使用荧光冷冻显微镜实时观察 IRI 对人脐静脉内皮细胞 (HUVEC) 内冰晶生长的影响。与仅含 15%甘油的对照组相比，在缓慢冷却过程中，在 15%甘油中添加芳基糖苷可使解冻后 RBC 的完整性提高多达 55%。RBC 经历间断分级冷冻的冷冻生物学行为特征表明，基于芳基糖苷的 IRI 被内化到 RBC 中。用 IRI 处理的 HUVEC 在升温至高亚零温度时保留了大量小冰晶，并表现出对细胞内冰重结晶的显著抑制。在缓慢解冻条件下，芳基糖苷 IRI p-溴苯基-β-d-葡萄糖苷在抑制细胞内冰重结晶方面最为有效。我们证明 IRI 能够渗透进入细胞，改变细胞对缓慢和快速冷冻的冷冻生物学反应，并控制冷冻过程中的细胞内冰重结晶。我们得出结论，IRI 作为高亚零温度下细胞和组织保存的冷冻保护剂具有巨大的潜力。
