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. 2013 Jun 18:14:59.
doi: 10.1186/1471-2202-14-59.

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

P Marc D Watson  1 , Judy C Paterson, George Thom, Ulrika Ginman, Stefan Lundquist, Carl I Webster
Affiliations

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

P Marc D Watson et al. BMC Neurosci. .

Abstract

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.

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Figures

Figure 1
Figure 1
Culture and passaging schedule for rat brain and spinal cord endothelial cells with mixed glial cells. Maximum plating densities for cell culture inserts and tissue culture dishes are suggested for each cell type.
Figure 2
Figure 2
Isolation and culture of rat brain and spinal cord microvascular endothelial cells. Following BSA density centrifugation and enzymatic digestion, isolated rat brain and spinal cord microvessel fragments were plated out onto collagen 1/fibronectin coated tissue culture plates. On plating, (a) brain and (d) spinal cord microvessels exhibit a “beads-on-string” appearance with rounded endothelial cells present on the surface (20× objective magnification). After 2–3 days in culture, (b) brain and (e) spinal cord endothelial cells are clearly visible migrating from the microvessels onto the matrix-coated tissue culture dish (10× objective magnification). After 5–7 days in culture both (c) brain and (f) spinal cord endothelial cells form a pure, near confluent monolayer (10× objective magnification).
Figure 3
Figure 3
Effect of media composition on RBEC barrier formation and characteristics. (a) Comparison of the effects of the DMEM/MVGS and EBM-2/EGM-2 media formulations on the TEER of RBECs grown for 14 days on cell culture inserts. Data is presented as mean ± SEM and was analysed using an unpaired, two-tailed students t-test, ***P < 0.0001; n = 5 independent cell culture experiments in 24-well plates, with 3 inserts per experiment, equivalent to 15 inserts total . (b) Calculated permeability coefficients for the paracellular passage of 100 μM (50 μg/mL) Lucifer yellow over a 90 minute period at 37°C across RBEC monolayers on cell culture inserts cultured in DMEM/MVGS and EBM-2/EGM-2 media formulations. Data is presented as mean ± SEM and was analysed using an unpaired, two-tailed students t-test, ***P < 0.0001; n = 5 independent cell culture experiments, with 3 inserts per experiment, equivalent to 15 inserts total. Fluorescence microscope images of RBECs stained with an antibody raised against the tight junction protein claudin-5 following culture in (c) DMEM/MVGS supplement, or (d) EBM-2/EGM-2. White arrows indicate regions of discontinuous claudin-5 staining. Images are representative of 3 independent cultures, with five fields of view taken from each individual preparation of cells using the 20× objective on an Olympus IX81 microscope. (e) Western blot analysis of claudin-5 protein expression levels in RBECs cultured in DMEM/MVGS and EBM-2/EGM-2. Blots were reprobed with anti-actin antibodies as a control for equal loading of cell lysates. (f) Densitometry analysis of claudin-5 band intensity, normalised to actin levels, for RBECs grown in DMEM/MVGS vs. EBM-2/EGM-2. Data is presented as mean ± SEM and was analysed using an unpaired, two-tailed students t-test, *P < 0.01; n = 3 independent experiments.
Figure 4
Figure 4
Characterisation of RBEC monolayer barrier function induced by co-culture in optimised EBM-2/EGM-2 media. (a) Development and stabilisation of TEER for passaged RBECs grown on cell culture inserts in the optimised EBM-2/EGM-2 conditions over a two week period. TEER was measured following equilibration of the inserts in cell culture medium to room temperature, n = 5 independent cell culture experiments. (b) Relationship between pre-experimental TEER and permeability to Lucifer yellow over 90 minutes at 37°C in the EBM-2/EGM-2 media conditions. Data was fitted to a one-phase exponential decay curve, R2 = 0.78, n = 6 independent cell culture experiments, equivalent to 71 inserts in total. Peak TEER at room temperature in this experiment reached as high as 999 Ω × cm2. (c) Permeability of Lucifer yellow (50 μg/mL) and FITC-labelled dextrans (10 mg/mL) of increasing hydrodynamic radius across RBEC monolayers in the optimised EBM-2/EGM-2 media conditions. Pe was calculated over a 90 minute time-course and was plotted versus the hydrodynamic radius of each molecule. Data was fitted to a one-phase exponential decay curve, R2 = 0.86, n = 2 independent cell culture experiments, equivalent to 6 inserts in total for each molecule tested.
Figure 5
Figure 5
Characterisation of BBB protein expression in cultured RBECs by immunocytochemistry. Images show fixed and permeabilised RBECs cells grown on collagen I and fibronectin-coated 96-well plates and stained with antibodies for (a) von Willebrand Factor, (b) smooth muscle actin, (c) claudin-5, (d) ZO-1, (e) occludin, (f) clathrin heavy chain, (g) caveolin-1, (h) P-gp. Images are representative of 3 independent cultures, with five fields of view taken from each individual preparation of cells using the 20× objective [10× for 5 (b) for a wider field of view] on an Olympus IX81 microscope.
Figure 6
Figure 6
Barrier function demonstrated by RSCEC monolayers co-cultured on cell culture inserts with mixed glial cells. Relationship between pre-experimental TEER, measured at room temperature, and permeability to Lucifer yellow over 90 minutes at 37°C in the optimised EBM-2/EGM-2 media conditions. Data was fitted to a one-phase exponential decay curve, R2 = 0.91, n = 8 independent cell culture experiments, 24 cell culture inserts in total.
Figure 7
Figure 7
Characterisation of cultured RSCECs by immunocytochemistry. Images show fixed and permeabilised RSCECs cells grown on collagen I and fibronectin-coated 96-well plates and stained with (a) von Willebrand Factor, (b) smooth muscle actin and vWF, (c) claudin-5, (d) ZO-1, (e) occludin, (f) clathrin heavy chain, (g) caveolin, (h) P-gp. Images are representative of 3 independent cultures, with five fields of view taken from each individual preparation of cells using the 20× objective [10× for 7(b) for a wider field of view] on an Olympus IX81 microscope.
Figure 8
Figure 8
Characterisation of optimised RBEC and RSCEC barriers for use in small molecules drug studies. Intracellular accumulation of the fluorescent P-gp efflux transporter substrate rhodamine 123 in (a) RBECs (n = 4) and (b) RSCECs (n = 3) cultured on cell culture inserts in the absence and presence of the inhibitor verapamil. Data is presented as mean ± SEM and was analysed using an unpaired, two-tailed students t-test, *P < 0.01, **P < 0.001. Permeability coefficients for CNS-permeable and impermeable small molecule drugs were calculated across (c) RBEC (n = 5 independent cell culture experiments) and (d) RSCEC monolayers cultured on cell culture inserts (n = 3 independent cell culture experiments).
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