Primary culture,identification and in vitro angiogenesis of mouse pulmonary microvascular endothelial cells |
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Authors: | GAO Run-di CAO Jie LU Shan YING Lin GAO Xuan LU Guo-hua LI He-quan ZHOU Jian-ying |
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Institution: | Department of Respiratory Diseases, The First Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou 310003, China |
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Abstract: | AIM: To establish a simple and reliable method for primary culture of mouse pulmonary microvascular endothelial cells (PMVECs), and to investigate the ability of angiogenesis of the cells in vitro. METHODS: PMVECs from C57BL/6 new born mice were cultured with peripheral pulmonary tissue pasted method. The cells were observed under inverted phase-contrast microscope and identified by immunocytochemical and immunofluorescent methods. PMVECs were seeded at the density of 20 000 cells per well in 96-well plate on top of growth factor-reduced matrigel and incubated for 24 h at 37 ℃ in 5% CO2. The plates were examined every 2 h for tube formation under an inverted microscope. RESULTS: The primary PMVECs showed fusiform shape or polygonal, regular cobblestone morphology after fusion to monolayer, and expressed factor Ⅷ -related antigen (ⅧF-Ag). Under fluorescence microscope, those cells demonstrated positive staining for uptaking acetylated low-density lipoprotein (DiI-Ac-LDL) and binding Bandeiraea simpli-cifolia lectin I (FITC-BSI). The purity of PMVECs was above 90%. PMVECs formed capillary-like structures within 4 h to 6 h after plated on matrigel and fully developed a "honeycomb" appearance at 10~12 h, and then the capillary tubes began to break apart. CONCLUSION: The tissue block pasted method for primary cultivation of PMVECs is easy to handle, with high yield and purity, and will further facilitate related studies of well-established tube-formation assay in vitro. |
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Keywords: | Mice Pulmonary microvascular endothelial cells Primary culture Angiogenesis |
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