Primary cultivation and identification of human placental microvascular endothelial cells

doi:10.3969/j.issn.2095-4344.2014.11.011

Abstract

Abstract:

BACKGROUND: Establishment of in vitro culture system of human placental microvascular endothelial cells with high purity is very important. In recent studies, some scholars have successfully obtained a large number of placental microvascular endothelial cells by three-stepenzyme digestion and magnetic separation method, but the procedures were extremely complex and it had great damage to the cells. Therefore, how to separate human placental microvascular endothelial cells easily and obtain high-purified cells has become a research hotspot.

OBJECTIVE: To investigate an efficient method to isolate and purify human placental microvascular endothelial cells from early villus microvessels, observe the cell growth and identify the cells.

METHODS: The villi from normal early pregnancies (6-8 weeks) after artificial abortion were collected aseptically. Using two-step digestion procedure and discontinuous Percoll density gradient centrifugation method, human placental microvascular endothelial cells were obtained. Then the cells were identified by trypsin digestion method and repeated adherence method.

RESULTS AND CONCLUSION: Human placental microvascular endothelial cells were isolated successfully from early villi. The primary cells adhered to the walls after inoculated for 24 hours and entered logarithmic phase at 10 days. 80% of the cells achieved a confluence at 12-13 days after inoculating. The subculture cells grew swiftly with the typical cobblestone appearance. Immunofluorescence staining showed that, cultured human placental microvascular endothelial cells demonstrated a strong positive reaction to von Willebrand factor antigen and CD31, accounting for 100%. MTT assay results showed that, human placental microvascular endothelial cells at passage 5 exhibited an S-shaped growth curve. High-purity human placental microvascular endothelial cells can be obtained by proteolytic enzymes digestion and discontinuous Percoll density gradient centrifugation method, and the purity is detected by trypsin digestion method and repeated adherence method.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: placenta, endothelial cells, blood vessels, cells, fluorescent antibody technique

CLC Number:

R394.2

Zhang Hui-li, Du Pei-li, Fang Yuan-long, Zhang Jing, He Yu-tian, Sun Bin, Xiao Xue, Sun Wen, Zhou Yan-mei, Chen Dun-jin. Primary cultivation and identification of human placental microvascular endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(11): 1706-1711.

Figures/Tables 2

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