Isolation and Primary Culture of Rat Cerebral Microvascular Endothelial Cells

Abstract: To establish rat cerebral microvascular endothelial cells (RCMEC) culture model， we developed a method for isolation and primary culture of RCMEC and observed the morphology of endothelial cells. After relatively pure cerebral microvessel fragments were obtained from 2-3 weeks old SD rats by careful dissection， two steps of enzyme digestions and gradient centrifugation with BSA or dextran and Percoll， they were seeded on dishes coated with the substrata. RCMEC were identified according to the morphology of the cultured cells， immunocytochemistry of factor VIII-associated antigen and transmission electron microscopy. We found that the cultured cells began to migrate from microvessel fragments after 12 hours， showed the spindle-shaped morphology and reached the monolayer confluence after 5-7 days. Attachment and growth of the cultured cells depended on the substrata provided and fibronectin/type IV collagen was superior to collagen from rat tail and gelatin. The cultured cells had factor VIII-associated antigen and showed tight junction-like cell-cell appositions at the electron microscopic level. The results indicated that relatively pure primary culture of RCMEC was successfully established using this method and the model system could be applicable to the studies of physiology， biochemistry and pharmacology of the brain endothelium and could also be used to develop an in vitro model of the rat blood-brain barrier.

CSTR: 32200.14.cjcb.2005.01.0020