人脐静脉血管内皮细胞培养、冻存、复苏与鉴定

The Cultivation,Refrigerated Storage,Revivification and Identification of Human Umbilical Vein Endothelial Cells

目的:探讨人脐静脉血管内皮细胞(HUVEC)体外分离、培养、冻存、复苏与鉴定的可靠方法。方法:采用改良的胶原酶灌注消化法对HUVEC体外分离、培养,按慢冻速融原则对第3代细胞进行液氮冻存、复苏。采用免疫组化Ⅷ因子相关抗原染色和活体细胞荧光D iI-Ac-LDL鉴定。结果:分离的原代HUVEC总数平均为1×106个细胞,最多达3×106个细胞;两种鉴定结果HUVEC均为95%以上阳性;冻存后分3批复苏细胞活性均超过90%。结论:成功建立了人脐静脉血管内皮细胞体外培养、冻存、复苏与鉴定方法,获得了足够数目和纯度的血管内皮细胞,保持了细胞的同源同代性。

Objective：To investigate a reliable method for cultivation, refrigerated storage, revivification and identification of human umbilical vein endothelial cells （HUVEC） in vitro. Methods ： in-vitro isolation and cultivation of HUVEC was performed using modified collagenase perfusion and digestion techniques. Based on the ＂slow-freeze and rapid-thaw＂ principle, the 3rd generation cells were stored in liquid nitrogen and then revivified. Factor Ⅷ related antigen was introduced to recognize the cells with histoimmunochemical method and the viability of the cells was identified by DiI-Ac-LDL uptake. Results： The average amounts of the endothelial cells isolated from HUVEC were 1 × 10^6 cells, with a maximum number of 3 × 10^6 cells. More than 95% cells were positive identified by factor VIII and DiI-Ac-LDL. Over 90% of the cells were viable from revivification in 3 batches after refrigerated storage. Conclusion： A method for cultivation, refrigerated storage, revivification and identification of HUVECs in vitro was established, yielding in adequate number and purity of endothelial cells as well as good cellular homology and lineage.