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.
