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| Combined Enzymatic Digestion Method and Explants Culture Method Used on Primary Culture and Biological Characteristic Identification of Pulmonary Artery Smooth Muscle Cells of mice |
| XIN Yi, GONG Da, XI Xin, SHI Hong-tao, LIU Sa, XU Xiu-fang, LI Na, HUANG Yi-min |
| Capital Medical University Affiliated Beijing Anzhen Hospital, Beijing Institute of Heart, Lung ＆ Blood Vessel Diseases, Beijing 100029, China |
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Abstract Objective:To investigate a technical method to isolate, cultivate and identify the mice pulmonary arterial smooth muscle cells(PASMCs). Methods:Isolation of pulmonary arterial smooth muscle cells (PASMCs) has been performed using Trypsin-typeⅠ collagen and papain digestion method and tissue explants culture method. The cell growth of PASMCs was observed under invert microscope. Cell viability rate of the different cells passage was evaluated by trypan blue staining. The PASMCs proliferation profile was analyzed by curve of growth, MTT assay and DNA cell cycle analysis respectively,and were identified by immunofluorescence as well. Result:After one day culture in the enzyme digested method, the adherent cells were observed to grow quickly with a typical "peak-valley" after 4 days under invert microscope. The cells can be passaged 7-8d after the rapid growth. But in the explants culture method, the cells started to amplify to grow gradually from the center to the periphery. Cells began to grow rapidly after 7d culture and single compact arrangement then fused 80% after 10 day. The cell viabilities from both isolation methods were more than 96% tested by trypan blue staining. There was over 90% positive expression in intracellular a-smooth muscle actin (a-SMA) in immunofluorescence staining.The shape of the second passage cell growth curves were similar in both methods ; There were also no significant differences in cell cycle and MTT assay in both methods. In cell cycle over 85% of cells were in G0/G1 phase. Conclusion:The PASMCs can be effectively isolated both by enzyme digestion method and explant methods which would provide us ideal seeding cells for pulmonary vascular diseases.
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Received: 20 March 2013
Published: 25 September 2013
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