保护液的玻璃化状态对红细胞冷冻干燥保存后回收率的影响

为了研究保护液的玻璃化状态对红细胞冷冻干燥 (简称冻干 )保存后回收率的影响 ,采用含有 7%二甲亚砜 (v/v)和 2 0 %、30 %、4 0 %或 5 0 %聚乙烯吡咯烷酮 (PVP) (w/v)的缓冲液作为保护液进行保护液的玻璃化测试和红细胞的冻干保存实验。首先检测溶液的玻璃化状态 ,如果冷冻和解冻过程中任一过程出现白色冰晶即为非玻璃化溶液 ;再将浓集红细胞和不同的保护液按比例混匀 ,预冻后移入冻干机内进行冻干处理 ;冻干完毕后 ,快速水化样品 ,测定红细胞回收率、血红蛋白回收率和上清游离血红蛋白浓度 ,然后对冻干后红细胞形态进行电镜观察。结果表明 :2 0 %PVP +7%DMSO和 30 %PVP +7%DMSO在冷冻和解冻过程中都出现白色冰晶 ;4 0 %PVP +7%DMSO在冷冻过程中无冰晶出现 ,但在解冻过程中出现冰晶 ;而 5 0 %PVP +7%DMSO在冷冻和解冻过程中均无冰晶出现。冻干红细胞再水化后 ,4 0 %PVP +7%DMSO的细胞回收率和血红蛋白回收率分别为 (81.36±14 94 ) %和 (77.5 4± 12 .86 ) % ,显著高于其它 3组 (P <0 .0 1) ;另外 4 0 %PVP +7%DMSO的上清游离血红蛋白浓度也显著低于其它 3组 (P <0 .0 1)。研究表明 :随着溶液中PVP浓度的升高 ,溶液的玻璃化程度也随之增加 ,同时冻干 再水化后红细胞的各项指标也随之改善 ,当溶液中PV

Effect of Vitrification State of Protective Solutions on Recovery of Red Blood Cells after Lyophilization Preservation

To study effect of vitrification state of protective solutions on recovery of red blood cells after lyophilization, four protective solutions composed of isotonic buffers containing 7% DMSO (v/v) and 20%, 30%, 40% or 50% polyvinylpyrrolidone (PVP) (w/v) were adopted. Vitrification state of protective solutions was examined first when white ice crystal appeared in any protective solution during freezing or thawing, if the used solution was not a vitrification solution. Red blood cells were lyophilized in MINILYO45 freeze-dryer after washing, mixing with protective solutions and prefreezing. After lyophilization, the samples were quickly rehydrated by 37 degrees C rehydration solution. The results showed that in vitrification and devitrification experiments, white ice crystal appeared in solution of 20% PVP + 7% DMSO and 30% PVP + 7% DMSO during freezing and thawing; vitrification appeared in solution of 40% PVP + 7% DMSO during freezing, but devitrification appeared during thawing; vitrification appeared in solution of 50% PVP + 7% DMSO during freezing and thawing. After rehydration, the recoveries of red blood cells and hemoglobin in 40% PVP + 7% DMSO group were (81.36 +/- 14.94)% and (77.54 +/- 12.86)%, which were significantly higher than that in 20% PVP + 7% DMSO, 30% PVP + 7% DMSO and 50% PVP + 7% DMSO groups (P < 0.01). The concentration of free hemoglobin in 40% PVP + 7% DMSO group was also significantly lower than that in other three groups (P < 0.01). With increase of PVP concentration in protective solutions, vitrification state and protective effect of these solutions also increased; when concentration of PVP in protective solution was 40% though it was not a vitrification solution, the effect of lyophilization was the best; but when concentration of PVP further increased to 50%, though it was a vitrification solution, the effect decreased. It is concluded that excessive vitrification state could not benefit lyophilization of red blood cells.