冷冻干燥保存人脐血实验研究  

Freeze-drying of mononuclear cells and whole blood of human cord blood

作　　者：李军[1] 华泽钊[1] 谷雪莲[1] 肖洪海[1] 王欣[1] 吴直江[2] 孟绿蓉[2] 陈健[2] 高奇蓉[2] 

机构地区：[1]上海理工大学低温生物医学与技术研究所,上海200093 [2]中国科学院上海生物化学与细胞生物学研究所,上海200031

出　　处：《低温工程》2005年第4期41-44,共4页Cryogenics

基　　金：国家自然科学基金重点项目(50436030);上海市教委重点学科资助

摘　　要：尝试用冷冻干燥法来长期保存脐血和有核细胞,并对冻干效果进行比较分析。选用聚乙烯吡咯烷酮(PVP)、蔗糖和甘露醇等作为冻干保护剂,先将样品冻结到-38℃,随后在-30℃搁板温度下进行升华干燥,最后在15℃搁板温度下进行解吸干燥。冻干后的样品用磷酸盐缓冲液复水,然后用血细胞计数板、流式细胞仪和扫描电镜进行检测。实验结果表明:在400 g/L PVP+200 g/L蔗糖+100 g/L甘露醇作为保护剂时细胞数目恢复率最高,为(75.0±4.1)%(P<0.01),PI染色鉴定测得单个核细胞拒染率在400 g/L PVP+200 g/L蔗糖+10%胎牛血清作为保护剂时最高,为(98.57±0.68)%(P<0.01),CD34+细胞回收率最高为35.59%。最终确定有核细胞的冻干效果比全血的要好,初步判定冷冻干燥法保存脐血是可行的。To preserve the mononuclear cell of human cord blood by freeze-drying, firstly, the samples were frozen to -38 ℃ by different cooling protocols in the presence of PVP, sucrose and mannitol. Then they were vacuum-dried at a selected shelf temperature of -30 ℃ for main drying stage, and finally at 15 ℃ for second drying. The dried samples were rehydrated with PBS and the results were evaluated by haemacytometer, flow cytometer （FCM） and scanning electron microscope （SEM）. The results are as follows： The highest cell numerical count recovery of （75.0±4. 1 ）% （P〈0.01） of MNC was obtained in the protocol of 40% PVP ＋ 20% Sucrose ＋ 10% Mannitol. The highest livability of the nucleated cells using PI dyeing and the highest recovery of CD34＋ were （98.57±0.68）% （P〈0.01） and 35.59% respectively in different protocols.

关 键 词：冷冻干燥法 胎血 造血干细胞 

分 类 号：TB69[一般工业技术—制冷工程] Q256[生物学—细胞生物学]