ABSTRACT
Objective: To explore and obtain the isolation and culture methods of the highly purified primary rat brain microvascular endothelial cells (BMECs). Methods: The brains of 10 3-week-old Wistar rats were harvested by decapitation, removing the white matter and mincing into approximately 1 mm3. A highly purified brain microvascular fragments were obtained through enzymatic digestion twice, 20% bovine serum albumin (BSA) and 33% continuous Percoll density gradient centrifugation. They were incubated into gelatin-coated 35 mm dish plates and the endothelial medium containing 4 μg/ml puromycins was added. The medium was renewed after 2 days. The endothelial cell growth and its morphology were observed using an inverted microscope. The expression of BMECs markers factor VIII-related antigen (vWF) was detected by immunocytoehemical method and was identified. Results: The endothelial cells grew out around the adherent brain microvascular fragments after culturing for 24 hours, and the cells were spindle-shaped. A fusion was formed after 5 to 6 days. BMECs after the fusion showed a typical "cobblestone-like" appearance. Immunocytochemical analysis showed the vWF expression of BMECs was positive, while the glial fibrillary protein (GFAP) expression was negative. The purity of BMVECs reached more than 96%. Conclusion: This method may successfully obtain the highly purified rat primary BMECs.