title: A simple method for isolating and culturing the rat brain microvascular endothelial cells.
abstract: Brain microvascular endothelial cells (BMECs), a main component of the blood-brain barrier, play a critical role in the pathogenesis of many brain diseases. The primary culture of BMECs has been used in various models for studying cerebrovascular diseases in vitro. However, there are still several problems existing in the isolation and cultivation of primary rat BMECs, such as low yield, contamination with other cell types, and requirement of a large number of animals and expensive growth factor. In this study, we describe a simple, economical (without any growth factor) and repeatable method to obtain endothelial cells with high purity (>99%) and yield (about 2.2×10(7) per rat) from cerebral cortexes of neonatal rat, mainly from gray matter. In vitro examinations determined that the isolated cells expressed typical phenotypic markers of differentiated brain endothelium such as multiple drug resistant protein, von Willebrand factor, platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), and intercellular adhesion molecule (ICAM). These cells also possessed morphological and ultra-structural characteristics that were observed by phase contrast microscope and electric microscope. Then GFAP and α-SMA were used, respectively, to identify astrocyte and pericyte which were potential to contaminate primary culturing of BMECs. And specific reaction of endothelial cells to external stimulation was tested by culture with TNF-α for 24h. All these results of our experiments supply that our protocol provides an effective and reliable method to obtain high purity and yield of rat BMECs and offers a useful tool for studying cellular physiology, cerebrovascular diseases, brain tumors, blood-brain barrier and neurovascular units, etc.
authors: College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China; College of Traditional Chinese Medicine and Pharmacology, Southwest University, Chongqing 400715, China; Chongqing Engineering Research Center for Pharmacological Evaluation, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, TCM, Chongqing 400715, China.
authors: Yang
authors: Y
authors: Liu
authors: Qiang
authors: Q
authors: Xue
authors: Qing
authors: Q
authors: Tang
authors: Min
authors: M
authors: Hou
authors: Hongyi
authors: H
authors: Qi
authors: Gang
authors: G
authors: Chen
authors: Weihai
authors: W
authors: Chen
authors: Jifen
authors: J
authors: Zhang
authors: Yi
authors: Y
authors: Chen
authors: Xiaoyu
authors: X
authors: Xu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D048430:Cell Shape; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000098:blood supply*; D017097:Electric Impedance; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D055806:Microvessels / Q000166:cytology / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D010641:Phenotype; D051381:Rats; D013997:Time Factors
chemical_list: D015415:Biomarkers