脐带血造血干细胞的采集、浓缩与低温冷冻保存

目的 :探讨脐带血造血干细胞库建立中的脐带血采集、分离、低温冻存的方法。方法 :自 1997年 7月至2 0 0 0年 7月已冻存经过HLA配型的脐带血 (cordblood ,CB) 3 74 4份。CB采自经检查符合要求的足月顺产或剖宫产新生儿 ,经 (citratephosphateadeninedextnse ,CPD A)抗凝 ,用密闭式血袋采集法采集。采集后置于室温保存 ,18h内处理。用羟乙基淀粉 (hydroxythylstarch ,HES)沉降后一次分离方法去除绝大多数红细胞及大部分血浆。采用以二甲亚砜 (dimethylsulfoxide ,DMSO)为主的冷冻保剂 ,冷冻保存CB。DMSO的终质量分数为 10 %。经序控降温后 ,置于 - 196℃液氮中保存。结果 :分析了 3 74 4份冻存CB ,平均采集量为 (93± 2 2 )ml(45～ 198ml)。浓缩后平均体积为 (3 8± 8)ml(3 0～ 60ml) ;浓缩前后有核细胞数 .浓缩前平均有核细胞数为 11.2× 10 8± 5 .3× 10 8(4.5× 10 8～ 4 5 .1× 10 8) ;采用HES沉降一次离心浓缩去除红细胞后的平均有核细胞数为 9.7× 10 8± 4 .6× 10 8(3 .1× 10 8～ 3 0 .5× 10 8) ;平均有核细胞回收率为 79%。质控检测 4 0份标本细胞活率为 88.2 % ,CFU GM回收率和CD3 4 +细胞计数分别为 97.6%和 86.4 %。结论 :本研究采用的CB采集、冻存及造血干细胞和病原生物检测的方法是安

Collection, processing and cryopreservation of placental cord blood hematopoietic stem cells

OBJECTIVE: To establish methods of collection, processing and cryopreservation of placental cord blood units. METHODS: Between July 1997 and July 2000, 3,744 placental cord blood (CB) units were stored in Beijing Cord Blood Bank. All of CB units had been HLA-typed with molecular (sequence specific primer/sequence specific oligonucleotide, SSP/SSO) and serological methods. CB units were collected by trained midwives while the placentas remained in utero. Collected CB units were temporarily stored at room temperature and processed within 18 hours after collection. Depletion of red blood cells was performed using hydroxythyl starch with one time centrifuge. CB units were cryopreserved with DMSO and hydroxythyl starch. RESULTS: The mean volume collected from 3,744 CB units was (93 +/- 22) ml (mean +/- s, range 45-198 ml). The mean total nucleated cell count per unit was 11.2 x 10(8) +/- 5.3 x 10(8) (rang 4.5 x 10(8)-45.1 x 10(8)). After depletion of red blood cells, the mean nucleated cell count was 9.7 x 10(8) +/- 4.6 x 10(8) (rang 3.1 x 10(8)-30.5 x 10(8)) with mean nucleated cell recovery of 79%. Forty CB units were thawed for testing, and trypan blue viability of nucleated cells was 88.2%. The recoveries of CFU-GM and CD34+ cells (ProCOUNT) were 97.6%, 86.4% respectively. CONCLUSION: We have established a CBB, and have shown that our system of the whole procedure and methods is functional for supplying qualified cord blood units in transplantation.