Red Blood Cell Cryopreservation with Minimal Post-Thaw Lysis Enabled by a Synergistic Combination of a Cryoprotecting Polyampholyte with DMSO/Trehalose.
Red Blood Cell Cryopreservation with Minimal Post-Thaw Lysis Enabled by a Synergistic Combination of a Cryoprotecting Polyampholyte with DMSO/Trehalose.
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DOI：
10.1021/acs.biomac.1c00599
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发表时间：
2022-02-14
影响因子：
6.2
通讯作者：
Gibson MI
中科院分区：
文献类型：
--
作者：
Murray A;Congdon TR;Tomás RMF;Kilbride P;Gibson MI
从创伤病房到化疗，红细胞在现代医学中至关重要。目前储存红细胞的方法通常使用甘油（40重量%）作为冷冻保护剂。尽管非常有效，但解冻后的脱甘油过程是耗时的，并且在洗涤程序期间导致红细胞的一些损失。在这里，我们证明了两性聚电解质，大分子冷冻保护剂，协同增强绵羊红细胞冷冻保存在混合冷冻保护剂系统。DMSO和海藻糖混合物的筛选确定了最佳条件，其中细胞毒性最小化，但冷冻保护益处最大化。补充两性聚电解质允许97%的解冻后恢复（3%溶血），即使在极具挑战性的缓慢冷冻和解冻条件下。细胞耐受冷冻保护剂的解冻后洗涤，这对于任何应用都是至关重要的，并且优化的混合物可以直接应用于细胞，在暴露1小时后不会引起溶血。该程序也按比例使用血袋，显示了与应用相关的规模上的实用性。流式细胞术和三磷酸腺苷测定证实了解冻后血细胞的完整性。显微镜检查证实，完整的红细胞被回收，但有一些收缩，这表明解冻后洗涤的优化可以进一步改善这种方法。这些结果表明，大分子冷冻保护剂可以与小分子冷冻保护剂一起提供用于储存必需细胞类型的协同益处，以及在加工/处理方面的潜在实际益处。
From trauma wards to chemotherapy, red blood cells are essential in modern medicine. Current methods to bank red blood cells typically use glycerol (40 wt %) as a cryoprotective agent. Although highly effective, the deglycerolization process, post-thaw, is time-consuming and results in some loss of red blood cells during the washing procedures. Here, we demonstrate that a polyampholyte, a macromolecular cryoprotectant, synergistically enhances ovine red blood cell cryopreservation in a mixed cryoprotectant system. Screening of DMSO and trehalose mixtures identified optimized conditions, where cytotoxicity was minimized but cryoprotective benefit maximized. Supplementation with polyampholyte allowed 97% post-thaw recovery (3% hemolysis), even under extremely challenging slow-freezing and -thawing conditions. Post-thaw washing of the cryoprotectants was tolerated by the cells, which is crucial for any application, and the optimized mixture could be applied directly to cells, causing no hemolysis after 1 h of exposure. The procedure was also scaled to use blood bags, showing utility on a scale relevant for application. Flow cytometry and adenosine triphosphate assays confirmed the integrity of the blood cells post-thaw. Microscopy confirmed intact red blood cells were recovered but with some shrinkage, suggesting that optimization of post-thaw washing could further improve this method. These results show that macromolecular cryoprotectants can provide synergistic benefit, alongside small molecule cryoprotectants, for the storage of essential cell types, as well as potential practical benefits in terms of processing/handling.
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影响因子：
16.6
作者：
Biggs CI;Bailey TL;Ben Graham;Stubbs C;Fayter A;Gibson MI
通讯作者：
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作者：
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