Anin vitroblood–brain barrier model: Cocultures between endothelial cells and organotypic brain slice cultures

Abstract

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.The microenvironment of the central nervous system (CNS) is important for neuronal function. In this context, the bloodbrain barrier (BBB) provides and maintains the extracellular medium compatible with normal neuronal activity. Through multiple studies, the characteristics of the BBB in mammals were found to be due to cerebral endothelial cells. Among these characteristics, the presence of tight junctions (TJs), the selective permeability, and the polarity were found to be essential for a functional BBB (1-3).The difficulties inherent to the use of whole animals as experimental models for studying permeability and metabolic processes at the cellular level has led to major efforts in the last decades to design suitable in vitro models. Three prototypes are noteworthy: the first consists of suspensions of isolated microvessels obtained from cerebral cortex gray matter (4-7). The second was developed following the demonstration by Panula et al. (8) that brain endothelial cells could be maintained in culture. In this model, primary passage cultures and clones (9, 10) of isolated brain endothelial cells were used as an in vitro BBB (11-13). The third was provided by the work of Stewart and Willey (14) and Janzer and Raff (15) on the importance of the cellular environment of endothelial cells. As a consequence, it has been possible to simulate a...

In Vitro Models of CNS Barriers

“…However, the complexity of the geometry (multiple hollow fi bres) in this model, and the assumptions made in calculating TEER from the current measured, make it diffi cult to compare TEER values with those from fl at fi lter confi gurations. Generation of more-complex 3-D cultures has also been reported (Duport et al 1998 ;Hatherell et al 2011 ). However, tri-culture, DIV and 3-D models are more diffi cult to set up and maintain than standard mono or co-cultured models (Fig.…”

Neuron-Glial Interactions in Blood-Brain Barrier Formation

“…The other cell types present at the BBB, including pericytes, perivascular macrophages, and neurons, also contribute to barrier induction (Duport et al 1998, Ramsauer et al 2002, Zenker et al 2003. Given the complexity of the BBB properties and the anatomical relationship of the associated cell types, it is not surprising to find synergistic inductive functions involving more that one cell type.…”

Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

“…In 1998, Duport et al (1998) reported a coculture consisting of cortical organotypic slice cultures (COSCs) and BECs and validated this model using electrophysiological recordings and electron microscopy. However, an extensive study of BBB properties under physiologic and pathophysiological conditions would benefit from the application of additional techniques, such as immunohistochemistry (IHC), fluorescent live cell imaging, and electrical cell substrate impedance sensing (ECIS, which is a validated marker for barrier integrity in real time).…”