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Three-Dimensional Blood-Brain Barrier Model for in vitro Studies of Neurovascular Pathology.

Cho H, Seo JH, Wong KH, Terasaki Y, Park J, Bong K, Arai K, Lo EH, Irimia D - Sci Rep (2015)

Bottom Line: We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants.To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic conditions and measured the protective function of antioxidant and ROCK-inhibitor treatments.Overall, our 3D BBB model provides a robust platform, adequate for detailed functional studies of BBB and for the screening of BBB-targeting drugs in neurological diseases.

View Article: PubMed Central - PubMed

Affiliation: BioMEMS Resource Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02129, United States.

ABSTRACT
Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple. In vitro models of BBB are simpler, however they have limited functionality and relevance to disease processes. To address these limitations, we developed a 3-dimensional (3D) model of BBB on a microfluidic platform. We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants. Moreover, we verified the localization at endothelial cell boundaries of ZO-1 and VE-Cadherin, two components of tight and adherens junctions. To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic conditions and measured the protective function of antioxidant and ROCK-inhibitor treatments. Overall, our 3D BBB model provides a robust platform, adequate for detailed functional studies of BBB and for the screening of BBB-targeting drugs in neurological diseases.

No MeSH data available.


Related in: MedlinePlus

BBB model response to neuroinflammation stimulus.(a) The changes in the levels of twenty-nine cytokines were measured after stimulations of the BBB model with tumor necrosis factor alpha (TNF-α, upper row) compared to unstimulated baseline (second row). (b) Seven inflammation involving cytokines were noticeably further secreted by the BBB model with TNF-α treatment [100 ng.mL−1] for 24 hours. (c) The levels of ZO-1 decreased with increasing treatment duration and concentration of TNF-α. The levels of ZO-1 decrease to 60% of control after 6 hours of exposure to TNF-α [100 ng.mL−1]. The dotted line represents the value of ‘Control’ group (no treatment). ncytokine = 4, ncell = 20 for each condition. Data represented as mean ± s.e.m.
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f3: BBB model response to neuroinflammation stimulus.(a) The changes in the levels of twenty-nine cytokines were measured after stimulations of the BBB model with tumor necrosis factor alpha (TNF-α, upper row) compared to unstimulated baseline (second row). (b) Seven inflammation involving cytokines were noticeably further secreted by the BBB model with TNF-α treatment [100 ng.mL−1] for 24 hours. (c) The levels of ZO-1 decreased with increasing treatment duration and concentration of TNF-α. The levels of ZO-1 decrease to 60% of control after 6 hours of exposure to TNF-α [100 ng.mL−1]. The dotted line represents the value of ‘Control’ group (no treatment). ncytokine = 4, ncell = 20 for each condition. Data represented as mean ± s.e.m.

Mentions: We constructed our 3D BBB model in the form of a tube inside a single layered microfluidic platform. The tube of endothelial cells inside the platform is geometrically similar to small brain blood vessels. The single-layered platform is convenient for integrating with other functional components and assays, i.e. chemotactic transmigration (Fig. 1). Our current model is static, with no fluid flow and no shear stress on the constructed model during culturing. To achieve faster maturation of barrier function, cells were seeded at high density on both the top and bottom surfaces and were confined along the compartments through the use of surface tension at the ends (Figs 1b, 2, 3, Supplementary Fig. S1). The seeded cells formed strong attachment to the thin gel layer on microstructured PDMS substrate (Supplementary Fig. S2) and formed tight monolayers two or three days after plating. We validated the formation of endothelial monolayers on 3D surfaces and the tightness of the junctions between adjacent cells by imaging each surface (Figs 1b, 4, supplementary Fig. S3). The confocal images showed a 3D membrane (Fig. 1c, 1) and monolayers of endothelial cells on all surfaces (Figs 1c, 2). The tightness of the BBB was confirmed by immunostaining of proteins: ZO-1 and VE-Cadherin, which are known to be responsible for the integrity of the BBB2728. The tightness of the BBB was confirmed by immunostaining of proteins representative for the BBB tightness: ZO-1 and VE-Cadherin. We found ZO-1 and VE-cadherin expressed selectively along the cellular boundaries, in representative patterns (Fig. 1d). The localization of expressed ZO-1 and VE-Cadherin on our model was not as clear as other endothelial monolayers on thin gel-coated or flat substrates, probably due to relatively thicker and softer gel compared to that reported in other literature29. Despite this limitation, our 3D BBB model could still be a convenient platform for various assays utilizing various surfaces: top and bottom surfaces for evaluating pathology-related assays destructing the BBB tightness, side surfaces for monitoring real-time penetration across the BBB upon the tightness destruction, and a single-layered platform for providing simple integration with other components.


Three-Dimensional Blood-Brain Barrier Model for in vitro Studies of Neurovascular Pathology.

Cho H, Seo JH, Wong KH, Terasaki Y, Park J, Bong K, Arai K, Lo EH, Irimia D - Sci Rep (2015)

BBB model response to neuroinflammation stimulus.(a) The changes in the levels of twenty-nine cytokines were measured after stimulations of the BBB model with tumor necrosis factor alpha (TNF-α, upper row) compared to unstimulated baseline (second row). (b) Seven inflammation involving cytokines were noticeably further secreted by the BBB model with TNF-α treatment [100 ng.mL−1] for 24 hours. (c) The levels of ZO-1 decreased with increasing treatment duration and concentration of TNF-α. The levels of ZO-1 decrease to 60% of control after 6 hours of exposure to TNF-α [100 ng.mL−1]. The dotted line represents the value of ‘Control’ group (no treatment). ncytokine = 4, ncell = 20 for each condition. Data represented as mean ± s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4622078&req=5

f3: BBB model response to neuroinflammation stimulus.(a) The changes in the levels of twenty-nine cytokines were measured after stimulations of the BBB model with tumor necrosis factor alpha (TNF-α, upper row) compared to unstimulated baseline (second row). (b) Seven inflammation involving cytokines were noticeably further secreted by the BBB model with TNF-α treatment [100 ng.mL−1] for 24 hours. (c) The levels of ZO-1 decreased with increasing treatment duration and concentration of TNF-α. The levels of ZO-1 decrease to 60% of control after 6 hours of exposure to TNF-α [100 ng.mL−1]. The dotted line represents the value of ‘Control’ group (no treatment). ncytokine = 4, ncell = 20 for each condition. Data represented as mean ± s.e.m.
Mentions: We constructed our 3D BBB model in the form of a tube inside a single layered microfluidic platform. The tube of endothelial cells inside the platform is geometrically similar to small brain blood vessels. The single-layered platform is convenient for integrating with other functional components and assays, i.e. chemotactic transmigration (Fig. 1). Our current model is static, with no fluid flow and no shear stress on the constructed model during culturing. To achieve faster maturation of barrier function, cells were seeded at high density on both the top and bottom surfaces and were confined along the compartments through the use of surface tension at the ends (Figs 1b, 2, 3, Supplementary Fig. S1). The seeded cells formed strong attachment to the thin gel layer on microstructured PDMS substrate (Supplementary Fig. S2) and formed tight monolayers two or three days after plating. We validated the formation of endothelial monolayers on 3D surfaces and the tightness of the junctions between adjacent cells by imaging each surface (Figs 1b, 4, supplementary Fig. S3). The confocal images showed a 3D membrane (Fig. 1c, 1) and monolayers of endothelial cells on all surfaces (Figs 1c, 2). The tightness of the BBB was confirmed by immunostaining of proteins: ZO-1 and VE-Cadherin, which are known to be responsible for the integrity of the BBB2728. The tightness of the BBB was confirmed by immunostaining of proteins representative for the BBB tightness: ZO-1 and VE-Cadherin. We found ZO-1 and VE-cadherin expressed selectively along the cellular boundaries, in representative patterns (Fig. 1d). The localization of expressed ZO-1 and VE-Cadherin on our model was not as clear as other endothelial monolayers on thin gel-coated or flat substrates, probably due to relatively thicker and softer gel compared to that reported in other literature29. Despite this limitation, our 3D BBB model could still be a convenient platform for various assays utilizing various surfaces: top and bottom surfaces for evaluating pathology-related assays destructing the BBB tightness, side surfaces for monitoring real-time penetration across the BBB upon the tightness destruction, and a single-layered platform for providing simple integration with other components.

Bottom Line: We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants.To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic conditions and measured the protective function of antioxidant and ROCK-inhibitor treatments.Overall, our 3D BBB model provides a robust platform, adequate for detailed functional studies of BBB and for the screening of BBB-targeting drugs in neurological diseases.

View Article: PubMed Central - PubMed

Affiliation: BioMEMS Resource Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02129, United States.

ABSTRACT
Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple. In vitro models of BBB are simpler, however they have limited functionality and relevance to disease processes. To address these limitations, we developed a 3-dimensional (3D) model of BBB on a microfluidic platform. We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants. Moreover, we verified the localization at endothelial cell boundaries of ZO-1 and VE-Cadherin, two components of tight and adherens junctions. To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic conditions and measured the protective function of antioxidant and ROCK-inhibitor treatments. Overall, our 3D BBB model provides a robust platform, adequate for detailed functional studies of BBB and for the screening of BBB-targeting drugs in neurological diseases.

No MeSH data available.


Related in: MedlinePlus