脐带血造血干细胞和祖细胞的分析与冷冻保存

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood.

作者信息

Meyer T P H, Hofmann B, Zaisserer J, Jacobs V R, Fuchs B, Rapp S, Weinauer F, Burkhart J

机构信息

Blood Donor Service, Bavarian Red Cross, Herzon-Heinrich-Strasse 4, 80336 Munich, Germany.

出版信息

Cytotherapy. 2006;8(3):265-76. doi: 10.1080/14653240600735685.

DOI:10.1080/14653240600735685

PMID:16793735

Abstract

BACKGROUND

Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB.

METHODS

Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed.

RESULTS

The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%.

DISCUSSION

Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.

摘要

背景

脐带血（UCB）是临床移植后造血系统重建的重要造血干细胞和祖细胞（HSC/HPC）来源。这些细胞的冷冻保存对于脐带血库和移植以及通过提供随时可用的标本进行研究应用至关重要。本研究的目的是优化脐带血中CD34+HSC/HPC的冷冻保存条件。

方法

在研究规模的设置中测试了单核细胞（MNC）制备后脐带血中CD34+HSC/HPC的冷冻保存。实验变量包括冷冻保护剂的浓度、蛋白质添加剂和细胞浓度。此外，将涉及缓慢、连续添加和去除二甲基亚砜（DMSO）的方案与标准方案（快速添加和去除DMSO）进行比较，以避免冷冻保存细胞受到渗透压应激。计算MNC、CD34+细胞和总集落形成单位（CFU）的活力和回收率作为读数。此外，在进一步处理之前对收集的脐带血单位进行无菌检测。

结果

MNC制剂中CD34+HPC冷冻保存的最佳条件是在高细胞浓度（5×10⁷MNC/mL）下使用10%DMSO和2%人白蛋白，并快速添加和去除DMSO。使用计算机控制的冷冻机进行冷冻保存后，观察到CD34+细胞的高活力（89%）和回收率（89%）以及CFU的回收率（88%）。收集的脐带血样本的微生物污染率降至6.4%。