Almici C, Carlo-Stella C, Wagner J E, Rizzoli V
Department of Hematology, University of Parma, Italy.
Acta Haematol. 1996;95(3-4):171-5. doi: 10.1159/000203873.
The clonogenic capacity of human umbilical cord blood (UCB) has been evaluated in several studies, which have shown high numbers of primitive hematopoietic progenitor cells. Recently, UCB progenitor cells were demonstrated to possess significant advantages over bone marrow (BM) in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been considered an attractive source of hematopoietic stem cells for both research and clinical applications. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving UCB samples with minimal loss of progenitor cells. We compared separation over three different densities of Percoll (1.069, 1.077 and 1.084 g/ml), sedimentation over poligeline (Emagel), and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBCs (hematocrit of the final cellular suspension 0.4 +/- 0.1%), while maintaining a high recovery of CD34+ cells (85.3%) and total recovery of CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units after 5 weeks of longterm culture. Moreover, the presence of Stem cell factor significantly increased CFU-GEMM (14 +/- 4 vs. 49 +/- 5, p < or = 0.0005) and CFU-GM (112 +/- 18 vs. 178 +/- 19, p < or = 0.025), but not B FU-E (42 +/- 7 vs. 53 +/- 7, p < or = 0.375) growth. In conclusion, RBC depletion of UCB can be accomplished with minimal loss of committed and primitive hematopoietic progenitors. This procedure may have important implications in the large-scale banking of UCB and in ex vivo expansion/gene therapy protocols.
多项研究对人脐带血（UCB）的克隆形成能力进行了评估，这些研究表明存在大量原始造血祖细胞。最近，已证明UCB祖细胞在增殖能力和免疫反应性方面比骨髓（BM）具有显著优势。因此，UCB已被视为用于研究和临床应用的有吸引力的造血干细胞来源。先前的报告记录了除冷冻保存外的任何操作都会导致祖细胞大量损失。我们评估了对UCB样本进行分级分离和冷冻保存且使祖细胞损失最小的可行性。我们比较了在三种不同密度的Percoll（1.069、1.077和1.084 g/ml）上的分离、在聚明胶肽（Emagel）上的沉降以及在聚明胶肽上沉降后再在Ficoll/Hypaque（F/H）上分离的效果。对分离后的样本（n = 25）分析CD34+细胞和祖细胞（CFU-GEMM、BFU-E、CFU-GM）的回收率。在聚明胶肽上沉降后再进行F/H分离可使红细胞消耗最多（最终细胞悬液的血细胞比容为0.4 +/- 0.1%），同时保持CD34+细胞的高回收率（85.3%）以及CFU-GEMM、BFU-E和CFU-GM的总回收率。冷冻保存后，长期培养5周后CFU-GEMM的克隆形成祖细胞回收率为82%，BFU-E为94%，CFU-GM为82%，集落形成单位为90%。此外，干细胞因子的存在显著增加了CFU-GEMM（14 +/- 4对49 +/- 5，p≤0.0005）和CFU-GM（112 +/- 18对178 +/- 19，p≤?0.025）的生长，但未增加BFU-E（42 +/- 7对53 +/- 7，p≤0.375）的生长。总之，UCB的红细胞去除可在已定向的和原始造血祖细胞损失最小的情况下完成。该程序可能对UCB的大规模储存以及体外扩增/基因治疗方案具有重要意义。
