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Isolation， Cultivation and Identification of Canine Bone Marrow Mesenchymal Stem Cells in Vitro
Shi-Gen Zhong1, Zhi-Gang Wang1, Zhi-Yu Ling2*, Yue-Hui Yin2, Qiao Li1, Jie Luo1, Xing-Sheng Li3
1Institute of Ultrasound Imaging of Chongqing Medical University; 2Department of Cardiology, 3Department of Geriatrics of the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
Abstract: To explore the approaches of isolation， culture and identify the canine bone marrow mesenchymal stem cells (BMMSCs) in vitro. Bone marrow mononuclear cells were extracted from canine bone marrow and purified via a density gradient centrifugation on Percoll (density 1.073g/ml)， then proliferated in vitro. Cellular immunohistochemistry study and flow cytometry detection were performed identify the cells and determine the cycle for surface marker protein of canine BMMSCs. Immunohistochemistry detection showed the expression of CD29 and CD44 was positive， and the expression of CD31， CD34， CD45 and vWF was negative. 88.88% cells were in the G0/G1 period. Which demonstrates that the separated cells were BMMSCs. The cultured stem cells can be expanded to 107 orders of magnitude in 2 weeks， and the purity of more than 99% can be achieved. This may provide sufficient sources of cells for stem cell transplantation in the treatment of ischemic heart disease.
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