Neuropharmacology and Neurotoxicology NeuroReport 0959-4965 # Lippincott Williams & Wilkins A new model of the blood±brain barrier: co- culture of neuronal, endothelial and glial cells under dynamic conditions Kathe A. Stanness, John F. Neumaier, 1 Timothy J. Sexton, 1 Gerald A. Grant, 1 Adriana Emmi, 2 Donald O. Maris 2 and Damir Janigro CA Cleveland Clinic Foundation, Department of Neurosurgery NB20, 9500 Euclid Avenue, Cleveland, OH 44195; 1 Department of Neurological Surgery and 2 Department of Psychiatry, Harborview Medical Center, University of Washington, Seattle, WA 98104 USA. CA,3 Corresponding Author and Address DEVELOPING in vitro blood±brain barrier (BBB) models that closely mimic the natural state is important for theoretical and practical applications, including drug development. We previously developed an in vitro BBB model based on co-culturing endothelial cells with glia in the presence of ¯ow on hollow ®ber tube culture substrates. We now report that this dynamic in vitro BBB (DIV-BBB) can be successfully used to co-culture differentiated serotonergic neurons in the presence of a BBB. These neurons demonstrated ¯uoxetine-sensitive serotonin (5HT) uptake and depolarization-induced release of [ 3 H]5HT. Our results demonstrate that the DIV-BBB is a suitable model for culturing of neurons in a quasi-physiological microenvironment and in the presence of a high-resistance, stereoselective BBB. NeuroReport 10:3725±3731 # 1999 Lippincott Williams & Wilkins. Key words: Blood±brain barrier; In vitro; Model Introduction The mammalian blood±brain barrier (BBB) com- prises microvascular endothelial cells (EC) that acquire specialized properties upon exposure to yet unknown factors present in the CNS, and presum- ably secreted by neighboring glia [1]. Among the crucial properties of BBB endothelia is the capacity to act as an impenetrable barrier isolating the brain from systemic in¯uences, while simultaneously con- stituting pathways for transport of nourishment to neurons and clearance of potentially toxic substances from the brain [2]. Thus, the BBB does not consti- tute a passive cellular layer with exclusion proper- ties, but rather consists of a specialized multicellular tissue with complex physiological properties. Mod- ern understanding of many neurological disorders has pinpointed the failure of brain endothelial mechanisms in the etiology of a variety of CNS disorders [3], warranting additional insight into BBB function in health and disease. We have learned more about brain microvascular endothelial cell physiology from in vitro experi- ments than by direct observations in situ, which is almost impossible with today's techniques [3,4]. Some of the salient properties of EC in culture (e.g. tight junctions) are greatly enhanced by exposure to glial factors or glia themselves [5]. Other phenotypic aspects of the BBB have been more dif®cult to reproduce in vitro, since low trans-endothelial elec- trical resistance (TEER) values and abnormally high permeability characterized both monoculture and co-culture BBB models [3,6±8]. Surprisingly, it was not until recently that emphasis was placed on the possibility that ¯ow may constitute an important factor regulating gene expression and physiology of the BBB in addition to glial in¯uences [9]. EC in vivo are exposed to shear stress generated by the ¯ow of blood across their apical surfaces. Both short- and long-term changes occur in cerebral arterioles in response to intraluminal ¯ow [9±11]. Stanness et al. have recently developed a new dynamic in vitro model of the BBB (DIV-BBB) characterized by a tridimensional, pronectin-coated hollow ®ber structure that enables co-culturing of EC with glia [12±15]. In the hollow ®ber apparatus, EC are seeded intraluminally and exposed to ¯ow. Under these conditions, EC develop a morphology that closely resembles the endothelial phenotype in situ [9]. In the presence of glia, EC develop a BBB- speci®c phenotype including low permeability to potassium, negligible extravasation of proteins, and NeuroReport 10, 3725±3731 (1999) Vol 10 No 18 16 December 1999 3725