新しい連続細胞分離法により採取した造血系前駆細胞のコロニーアッセイ

抄録

A novel cell separation method which can continuously separate the cells according to their densities has been developed for application to the transfusion medicine. In the past the performance of this method was examined on separation of human buffy coat, peripheral blood and co-cultured cell suspensions. Flow cytometry analysis on separation of human buffy coat revealed that CD34-positive cells, which were assumed to be hematopoietic progenitor cells, were distributed around density = 1.065. Recently, the colony-forming cell assay was performed on human hematopoietic progenitor cells separated from peripheral blood by the present method. Five polymer 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. Colony-forming unit-erythroid (CFU-E) were appeared on day 7. Burst-forming unit-erythroid (BFU-E), CFU-GM (granulocyte, macrophage) and CFU-GEMM (granulocyte, erythroid, macrophage, megakaryocyte) were observed on day 14. BFU-E, CFU-GM and CFU-GEMM were colonies that were found in the fourth fraction (density = 1.075), among those no CD-34 positive cells being detected by flow cytometry analysis. These results suggest that the method might enable to harvest many types of human hematopoietic progenitor cells according to minute differences in their densities. [J Physiol Sci. 2006;56 Suppl:S76]