Establishment Of An In Vitro Model Of The Blood-Brain Barrier

The blood-brain barrier (BBB) is a barrier separating the central nervoussystem (CNS) from the periphery. Since it prevents the brain uptake of＞98％of smallmolecules and～100％of large molecules, the blood-brain barrier (BBB) is the mainlimiting factor in the development of therapeutics for the central nervous system(CNS). Because of the limitation of in vivo research, the pharmacologists have beensearching for an in vitro blood-brain barrier model (BBBM) that preserves in vivotransporter functions in CNS drug discovery and development.The aim in this paper was to establish a flexible, abundantly available,reproducible and functionally characterized in vitro model of the blood-brain barrier(BBB) which comprises a co-culture of brain microvascular endothelial cells andastrocytes.Methods: In a first step, brain microvascular endothelial cells isolated from newborn SD rats were primary cultured and sub-cultured by homogenate, filtration,density centrifigution and collagenase digestion.Astrocytes were also isolated fromnew born SD rats for culturing. Subsequently, a co-culture of primary brainmicrovascular endothelial cells (BMEC) on transwells, with astrocytes on the bottomof the membrane of transwell was established. Besides, when the BBB model wasestablished, its properties were estimated by some tests such as measure oftransendothelial electrical resistance (TEER) and permeability.Results: (1) The cultured microvascular endothe-lial cells displayed monolayer growth and looked like typical "cobblestones" after about 7～9 day culture. Theywere identified as endothelial cells by morphological observation and character ofgrowth. (2) Astrocytes displayed characteristic astrocyte morphology and expressedglial fibrillary acidic protein (GFAP). (3) The co-culture of brain microvascularendothelial cells and astrocytes displayed restriction of paracellular transport and hightransendothelial electrical resistance (TEER).Conclusions: A high quality and mass-production in vitro BBB model wasestablished in which experiments with physiological and pharmacological objectivescan be reproducibly performed.