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

doi:10.3969/j.issn.2095-4344.2013.14.015

Abstract

Abstract:

BACKGROUND: Endothelial progenitor cells have a very broad application prospect, but the isolation and culture methods differ greatly and therefore are hard to repeat.
OBJECTIVE: To investigate the isolation and culture methods of bone marrow-derived endothelial progenitor cells.
METHODS: Bone marrow mononuclear cells were isolated from 4-week-old Sprague-Dawley rats by density gradient centrifugation methods and cultured by EGM-2 MV medium. Then the cells were identified by morphological observation, FITC-UEA-1 binding and Dil-Ac-LDL uptake assay, and fluorescent immunocytochemistry for detection CD133 and VEGFR2 expression. In addition, angiogenic tube formation was determined by Matrigel tube formation assays.
RESULTS AND CONCLUSION: (1) Morphology: After induced culture, the isolated bone marrow mononuclear cells exhibited a spindle-shaped, triangular, and round appearance in the early stage (about the 8th day) and round and short spindle-shaped appearance in the late stage (about the 15th day). (2) FITC-UEA-1 binding and Dil-Ac-LDL uptake assay: Cells were positive on days 8 and 21. (3) Fluorescent immunocytochemistry: on day 8, cells expressed CD133 and VEGFR2. (4) Matrigel tube formation assays: Capillary-like structures formed at 15 hours on Matrigel. These findings suggest that rat bone marrow mononuclear cells isolated by density gradient centrifugation method and cultured by EGM-2MV medium correspond to the characteristics of endothelial progenitor cells. This method is simple, quick, and reliable to harvest enough endothelial progenitor cells.

Key words: stem cells, stem cell culture and differentiation, endothelial progenitor cells, mononuclear cells, density gradient centrifugation, cell culture, cell identification, tube formation, National Natural Science Foundation of China, stem cell photographs-containing paper

CLC Number:

R394.2

Wei Xiao-yan, Zhang Li, Lin Ming, Chen Huan, Peng Bo, Peng Yuan-yuan, Li Fu-yun, Hong Pei-xin, Fan Yi-fan. Isolation, culture and identification of rat bone marrow-derived endothelial progenitor cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(14): 2570-2577.

Figures/Tables 4

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