Primary cultivation and identification of human placental microvascular endothelial cells
10.3969/j.issn.2095-4344.2014.11.011
- VernacularTitle:人胎盘微血管内皮细胞的分离培养及鉴定
- Author:
Huili ZHANG
;
Peili DU
;
Yuanlong FANG
;
Jing ZHANG
;
Yutian HE
;
Bin SUN
;
Xue XIAO
;
Wen SUN
;
Yanmei ZHOU
;
Dunjin CHEN
- Publication Type:Journal Article
- Keywords:
placenta;
endothelial cel s;
blood vessels;
cel s;
fluorescent antibody technique
- From:
Chinese Journal of Tissue Engineering Research
2014;(11):1706-1711
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Establishment of in vitro culture system of human placental microvascular endothelial cel s with high purity is very important. In recent studies, some scholars have successful y obtained a large number of placental microvascular endothelial cel s by three-stepenzyme digestion and magnetic separation method, but the procedures were extremely complex and it had great damage to the cel s. Therefore, how to separate human placental microvascular endothelial cel s easily and obtain high-purified cel s has become a research hotspot.
OBJECTIVE:To investigate an efficient method to isolate and purify human placental microvascular endothelial cel s from early vil us microvessels, observe the cel growth and identify the cel s.
METHODS:The vil i from normal early pregnancies (6-8 weeks) after artificial abortion were col ected aseptical y. Using two-step digestion procedure and discontinuous Percol density gradient centrifugation method, human placental microvascular endothelial cel s were obtained. Then the cel s were identified by trypsin digestion method and repeated adherence method.
RESULTS AND CONCLUSION:Human placental microvascular endothelial cel s were isolated successful y from early vil i. The primary cel s adhered to the wal s after inoculated for 24 hours and entered logarithmic phase at 10 days. 80%of the cel s achieved a confluence at 12-13 days after inoculating. The subculture cel s grew swiftly with the typical cobblestone appearance. Immunofluorescence staining showed that, cultured human placental microvascular endothelial cel s demonstrated a strong positive reaction to von Wil ebrand factor antigen and CD31, accounting for 100%. MTT assay results showed that, human placental microvascular endothelial cel s at passage 5 exhibited an S-shaped growth curve. High-purity human placental microvascular endothelial cel s can be obtained by proteolytic enzymes digestion and discontinuous Percol density gradient centrifugation method, and the purity is detected by trypsin digestion method and repeated adherence method.
