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

Abstract

BackgroundModelling 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.MethodsBrain 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.ResultsUsing 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.ConclusionsOur 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.

Communication from the periphery to the hypothalamus through the blood–brain barrier: An in vitro platform

“…Likewise, bEnd.3 cells presented a characteristic spindle-shaped morphology (Mühlner et al, 1999;Sheibani and Frazier, 1995). Importantly, the presence of continuous and organized tight and adherens junctions, known to be required for the proper BBB functioning Martins et al, 2013;Watson et al, 2013), was observed in the interendothelial cell contact. These data support the establishment of a highly restrictive barrier.…”

“…Transendothelial flux test was adapted from a protocol described by Watson and co-workers (Watson et al, 2013). The test was performed after 11 days of bEnd.3 cells and astrocytes coculture.…”

“…As control, bEnd.3 cells monocultures were also tested. coculture and monoculture were calculated as previously described (Nakagawa et al, 2009;Watson et al, 2013). The average cleared volumes were plotted versus time in minutes.…”

“…The study of the humoral transmission of peripheral signals to the brain has been considerably expanded, either in fundamental biology and drug discovery contexts Watson et al, 2013). However, the CNS is highly protected and, before reaching the brain, the peripheral signals need to overcome the reduced permeability of the barriers that ensure the stability of its internal environment.…”

In Vitro Models of CNS Barriers

“…By contrast, all cell-based systems require specifi c growth surface coatings and cell culture media for growing BECs. Although the cell preparations and culture conditions are all based on the same principle, in order to obtain functional in vitro BBB models several small but signifi cant differences between the systems, as well as preferences between laboratories have been introduced (Garberg 1998 ;de Boer and Sutanto 1997 ), an ongoing process as shown by recent papers (Abbott et al 2012 ;Patabendige et al 2013b ;Watson et al 2013 ). In the following sections current in vitro models of the BBB are briefl y surveyed; for greater detail on specifi c systems the reader is referred to the corresponding key publications.…”