Laboratory Research
Isolation, culture and identification of bone marrow-derived endothelial progenitor cells in mice
Wei Luo, Xianghe Li, Xianteng Yang, Senlei Li, Yuanzheng Wang, Yi Zhang, Xiaobin Tian, Li Sun
Published 2018-06-15
Cite as Chin J Orthop Trauma, 2018, 20(6): 523-528. DOI: 10.3760/cma.j.issn.1671-7600.2018.06.012
Abstract
ObjectiveTo explore a practical and feasible method for isolation, culture and identification of mouse bone marrow endothelial progenitor cells(EPCs).
MethodsBone 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.
ResultsIn 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.
ConclusionsA 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
Contributor Information
Wei Luo
Graduate School of Guizhou Medical University, Guiyang 550000, China
Xianghe Li
Graduate School of Guizhou Medical University, Guiyang 550000, China
Xianteng Yang
Department of Orthopaedics, People's Hospital of Guizhou Province, Guiyang 550002, China
Senlei Li
Department of Orthopaedics, People's Hospital of Guizhou Province, Guiyang 550002, China
Yuanzheng Wang
Department of Orthopaedics, People's Hospital of Guizhou Province, Guiyang 550002, China
Yi Zhang
Department of Orthopaedics, People's Hospital of Guizhou Province, Guiyang 550002, China
Xiaobin Tian
Department of Orthopaedics, People's Hospital of Guizhou Province, Guiyang 550002, China
Li Sun
Department of Orthopaedics, People's Hospital of Guizhou Province, Guiyang 550002, China