Isolation,cultivation,purification and identification of vascular endothelial cells of rat thoracic aorta / 解放军医学杂志

ABSTRACT

Objective To explore an effective method of isolating and culturing the vascular endothelial cells of rat thoracic aorta.MethodThe thoracic aorta was harvested under aseptic condition from the thorax of a Wistar rat.The peripheral connective tissue and fat of the thoracic aorta were stripped and disposed,and then the thoracic aorta was turned inside out to expose the intima.The thoracic aorta was ligated with silk and cauterized on both ends with heated forceps.Then the thoracic aorta was cultured in medium DMEM/F12 containing 20% newborn calf serum in a 50 ml culture bottle which was already blanketed with rat tail collagen.The thoracic aorta was discarded and the new culture medium was added into the culture bottle six days later.The migrating cells were differentially digested by 0.125% pancreatic enzyme for serial subcultivation.The cells were identified by immunohistochemical method with anti-Ⅷ factor antibody.ResultA small amount of cells were seen to migrate from the aorta and adhered to the bottom of culture bottle 6 days after cultivation;the migrating cells spread to cover most part of the bottom of culture bottle 12-14 days later.About 70% of the migrated cells were in a confluent monolayer.The confluent cells grew rapidly after being digested with pancreatic enzyme,and they showed a typical cobblestone appearance.The cells were identified as endothelial cells with 100% expression of Ⅷ factor,which was regarded as the marker of endothelial cells.ConclusionThe method established in the present study is simple and easy to handle,it does not need collagen enzyme and endothelial cell growth promoting substrate,and it is economical and applicable.It is especially suitable for isolation and cultivation of vascular endothelial cells of vessels of small caliber.