Isolation, culture and identification of bone marrow-derived endothelial progenitor cells in mice

Abstract

Objective To explore a practical and feasible method for isolation, culture and identification of mouse bone marrow endothelial progenitor cells(EPCs). Methods Bone marrow-derived mononuclear cells isolated by density gradient centrifugation were cultured in endothelial cell growth medium-2 MV medium. Growth and morphological changes of the cells were observed under inverted microscopy. Cell proliferation was observed by cell counting kit-8 assay. Surface markers of the EPCs were detected by flow cytometry. Angiogenic tube formation was determined by Matrigel tube formation assay. Fluores cein isothiocyanat e-ulex europaeus agglutinin-1(FITC-UEA-1) binding and Dil-Ac-LDL uptake capabilities were observed by fluorescent microscopy. Results In the early stage, the cells were round and spindle-shaped after induced culture for 4 days. After 7 days, the cells grew in colony arrangement and gradually increased in number. After 14 days, the cells were differently shaped, such as short shuttle and triangle. After 21 days, the typical paving stone appearance of the cells was observed. The cells were positive for endothelial markers in flow cytometry: CD34+ (84.3%), vascular endothelial growth factor receptor 2+ (74.1%), but CD45+ (4.04%). The cells were capable of forming capillary-like tubes, up-taking Dil-Ac-LDL and binding FITC-UEA-1 in Matrigels. Conclusions A reliable method for isolation, culture and identification of mouse bone marrow EPCs may be improved on the basis of previous experiences. Since the EPCs obtained by this method may be capable of good proliferation, large in number, and stable in biological characteristics, they can serve as ideal seed cells for related subsequent studies. Key words: Bone marrow; Cell culture techniques; Monocytes; Endothelial progenitor cells; Cell identification