[Primary culture and identification of mouse brain microvascular endothelial cells].

ABSTRACT

Objective To develop and establish a simple and repeatable method of primary culture of mouse brain microvascular endothelial cells (BMECs) of relatively high purity. Methods Isolated and cultured from the brain tissue of ICR mice aged 2-3 weeks old, the mouse BMECs were gained through physical fragmentation methods such as cutting up and passing through cell sieve, BSA density gradient centrifugation and chemical enzyme digestion. The digestion time of 1 g/L type II collagenase was strictly controlled within 15-20 minutes. The cultured cells were identified by cell morphological observation and immunocytochemical staining to detect factor VII-associated antigen. Results In 24 hours after seeding, the spindle-shaped or polygon-shaped cells began to migrate from the micro vessel segment and gradually gathered and grew in clusters; after 7 days, the fused cells showed a typical single layer and arranged in the shape of paving stone. VII-associated antigen showed positive in the cytoplasm which was brown red. The purity of microvascular endothelial cells was above 95%. Conclusion We have successfully established a simple and repeatable primary culture method for mouse BMECs of relatively high purity.