Isolation of colony-forming cells from human blood and evaluation of population purity by flow cytometry

Endothelial colony-forming cells (ECFCs) represent a rare population of circulating endothelial progenitor cells (EPCs), which is of interest for tissue engineering, cell therapy, and studying diseorders associated with endothelial dysfunction. Advances in this field imply isolation and pure culturing of endothelial colony-forming cells, which may be obtained from the mononuclear fraction of human peripheral blood. Flow cytometry may provide rather accurate phenotyping and checking purity of the cell cultures. The aim of this study was to obtain a population of colony-forming endothelial cells evaluated by flow cytometry in order to confirm the phenotype and assess the purity of the cell populations. Heparinized donor blood from peripheral vein was used to obtain ECFCs. The mononuclear cell fraction was isolated on a Histopaque 1077 density gradient. The obtained cells were cultured in complete nutrient medium EGM-2 MV with 5% FBS. On the 11^th, 15^th and 18^th days of culture, the cells were detached with trypsin. Some cell aliquots were taken for flow cytometry, and the remaining cells were transferred to a plate for further cultivation until 70% confluency was reached. The phenotype of colonies obtained was evaluated by flow cytometry. Sample preparation of whole peripheral blood, mononuclear fraction and cell culture was performed using two panels of fluorochrome-labeled monoclonal antibodies. Colony growth was visually evaluated by phase-contrast microscopy. Statistical processing of the obtained results was performed using the applied program package “Statistica 6.0”. Up to 9 days, the cell colonies were not detectable visually. During further cultivation, the proliferating colonies of the “cobblestone” type were formed. Two populations were detected by flow cytometry: CD45⁺ and CD45^-. In the course of our study, it was revealed that, with increasing cultivation terms, a decrease of CD45⁺ population was observed, along with progressive increase of CD45^- cells, which may be associated with the gradual displacement of hematopoiesis cells by endothelial colony-forming cells. As a result of our study, the detected CD45⁺ population had a phenotype of hematopoietic lineage cells, whereas the CD45^- population exhibited endothelial phenotype: CD31⁺CD309⁺vWF⁺CD146⁺. By 18 days, the purity of culture reached 97.6%.