Isolation, cultivation and identification of human skin microvascular endothelial cells

doi:10.3969/j.issn.2095-4344.2016.51.012

Abstract

Abstract:

BACKGROUND: Currently, the enzymatic digestion combined with magnetic activated cell sorting for isolating microvascular endothelial cells are cumbersome and do harm to cells. Therefore, how to simplify the isolation and culture of human dermal microvascular endothelial cells to obtain highly purified endothelial cells in vitro becomes a hotspot.
OBJECTIVE: To explore a simple and effective cultivation method of microvascular endothelial cells from diabetic patient skins in vitro, and to detect the cell growth.
METHODS: Diabetic patients with chronic foot wounds after amputation were enrolled to collect the limb proximal skin and topical skin around the wound superficial dermal tissue. Human dermal microvascular endothelial cells were obtained using adherent method and trypsin method, folloewd by purified utilizing trypsin digestion and repeated attachment method when passage culture.
RESULTS AND CONCLUSION: Human dermal microvascular endothelial cells were obtained successfully, Primary cultured endothelial cells completely adhered to the wall at 24 hours, entered the logarithmic phase at the 10th day, and the cell concentration reached 80% at the 12th-13th day. While the passage cells grew more actively than primary cells, and fully covered the bottom in a “cobblestone” arrangement after 5-7 days of culture. Immunohistochemical staining showed that cultured cells were positive for FVIII and CD31-associated antigens with 100% positive rate. MTT assay showed that cell growth curves of 2, 4, and 5 generations of dermal microvascular endothelial presented the inverted "S" shape. These results suggest that abundant highly purified human dermal microvascular endothelial cells can be obtained through the adherent method and a small amount of short-term trypsin 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

Key words: Skin, Endothelial cells, Blood Vessels, Tissue Engineering

CLC Number:

R318

Wang Guang-yu, Wang Yu, Zhu Yan-ping, Kang Yu-dong, Wang Fu-sheng, Ding Yi, Dong Yu, Xu Xu-ying . Isolation, cultivation and identification of human skin microvascular endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(51): 7678-7683.

Figures/Tables 2

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