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Background  Modelling the blood-CNS barriers of the brain and spinal cord  in vitro  continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful  in vitro  primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the  in vivo  situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise  in vitro  conditions for inducing phenotypic characteristics in these cells that were reminiscent of the  in vivo  situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies.    Methods  Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as  in vitro  blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and  in vitro  phenotyping by immunocytochemistry and western blotting.    Results  Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the  in vitro  culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for  in vitro  permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter.    Conclusions  Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers  in vitro . These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier  in vitro  and for pre-clinical drug discovery.

bmc neuroscience

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitromodels of the rat blood-brain barrier and blood-spinal cord barrier