Development Of Continuous Density Gradient Cell Separati

应用连续密度梯度细胞分离法分离人外周血和脐血的造血祖细胞。并将该方法应用于人外周血和黄褐色大衣的分离实验。从10ml外周血中分离大量红细胞(&gt；1010)中的有核细胞(&gt；107)约100分钟。淋巴细胞密度为1.065~1.070，中性粒细胞密度为1.080。流式细胞仪分析表明，CD34阳性细胞主要集中在细胞密度为1.060的部分。台盼蓝拒染试验表明，分离的细胞存活率在95%以上。为了验证分离细胞的功能潜能，对分离的细胞进行了外周血和脐带血的集落形成细胞实验。用无菌等渗Percoll培养液和PBS配制5种密度分别为1.060、1.065、1.070、1.075和1.080的分离液进行分离，分离后的细胞在含有hSCF、hGM-CSF、hIL-3和hEPO的甲基纤维素基上培养。在这两个样本中，最高频率是红系集落形成单位(BFU-E)，大约71%的集落形成在外周血中，大约49%的集落在脐带血中。其次是粒细胞巨噬细胞集落形成单位(CFU-GM)，分别约占24%和46%。集落形成单位粒细胞、红系、巨噬细胞、巨核细胞(CFU-GEMM)似乎是由CD34阳性干细胞发展而来的，其比例分别约为3%和4%。来自有核细胞的集落百分比约为0.05%，密度为1.070的部分来自外周血，密度约为1.4%的部分来自脐血，密度为1.065的部分来自脐血。这种新方法似乎可以在不丧失增殖能力的情况下采集造血祖细胞，并可以防止CD34阴性祖细胞的潜在损失。将粒细胞从干细胞中排除将对减少输血医学中产生的副作用具有重要意义。

The application of the continuous density gradient cell separation method was applied to isolate hematopoietic progenitor cells from human peripheral and cord blood. The performance of this method was examined on the separation of human buffy coat and peripheral blood. Nucleated cells (>107) among a large population of erythrocytes (>1010) were separated from 10ml of peripheral blood for about 100 minutes. Lymphocytes with densities of 1.065 to 1.070 were well separated from neutrophils with a density of 1.080. Flow cytometric analyses on the separation of stem cells from human buffy coat revealed that CD34 positive cells were concentrated in the fraction with a density of 1.060. The viability of separated cells was over 95% by the trypan blue dye exclusion test. In this paper, in order to prove the functional potentiality of separated cells, the colony-forming cell assay was performed on them from peripheral blood and umbilical cord blood. Five separation media with densities of 1.060, 1.065, 1.070, 1.075 and 1.080, prepared with sterile isotonic Percoll media and PBS, were used for the separation, and the fractionated cells were cultured in a methylcellulose-based medium containing hSCF, hGM-CSF, hIL-3, and hEPO. The highest frequency was Burst-forming unit-erythroid (BFU-E) in both the samples, with approximately 71% of the formed colonies in peripheral blood and approximately 49% in umbilical cord blood. The second highest frequency was Colony-forming unit-granulocyte, macrophage (CFU-GM) with approximately 24% and approximately 46%, respectively. The frequency of Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), which seemed to be developed from CD34 positive stem cells, was approximately 3% and approximately 4%, respectively. The percentage of colonies from nucleated cells was approximately 0.05% in a fraction with the density of 1.070 from peripheral blood and approximately 1.4% in a fraction with the density of 1.065 from umbilical cord blood. It appears that this new method could allow for harvesting of hematopoietic progenitor cells without losing their ability to proliferate and could prevent a potential loss of CD34 negative progenitor cells. The exclusion of granulocytes from stem cells would be of significance for the decrease in side effects which are produced in transfusion medicine.