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A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Deosarkar SP, Prabhakarpandian B, Wang B, Sheffield JB, Krynska B, Kiani MF - PLoS ONE (2015)

Bottom Line: The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10-6 cm/s to 2.9±1.0 x 10-6 cm/s or 1.1±0.4 x 10-6 cm/s, respectively).Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC.In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, Temple University, Philadelphia, Pennsylvania, 19122, United States of America.

ABSTRACT
Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10-6 cm/s to 2.9±1.0 x 10-6 cm/s or 1.1±0.4 x 10-6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

No MeSH data available.


Related in: MedlinePlus

Passage of fluorescent dextran from the vascular channel to tissue compartment of B3C under shear flow.Permeability of Texas Red 40 kDa dextran from vascular channel to the tissue compartment in a cell-free B3C after 5 min (A), 15 min (B), 30 min (C), 60 min (D) and 120 min (E) from the initiation of flow in vascular channel. Normalized tissue intensity in a cell-free B3C increases linearly with time in the tissue compartment (F).
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pone.0142725.g002: Passage of fluorescent dextran from the vascular channel to tissue compartment of B3C under shear flow.Permeability of Texas Red 40 kDa dextran from vascular channel to the tissue compartment in a cell-free B3C after 5 min (A), 15 min (B), 30 min (C), 60 min (D) and 120 min (E) from the initiation of flow in vascular channel. Normalized tissue intensity in a cell-free B3C increases linearly with time in the tissue compartment (F).

Mentions: Since the B3C developed here is a dynamic in vitro model of neonatal BBB suitable for real-time monitoring and direct measurement of permeability across BBB using microscopic methods, we first optimized the techniques for quantifying permeability in B3C. Permeation of the fluorescent 40 kDa dextran from the vascular channel to the tissue compartment of cell-free B3C was characterized by imaging over time as dextran was injected into the vascular channel at a flow rate of 0.2 μl/min (i.e. shear stress of 7.6x10-2 dynes/cm2). As shown in Fig 2A, 2B, 2C, 2D and 2E, fluorescent dextran accumulates in the tissue compartment in a time-dependent manner over a 120 min period. Quantification of permeability was performed by calculating the average intensity of fluorescent dextran in the entire tissue compartment and normalizing it to the maximum intensity of fluorescent dextran in the vascular channel. The results of a typical cell-free experiment shown in Fig 2F indicate that the normalized intensity increases linearly with time in the tissue compartment. The slope of the line (dIt/dt) in Fig 2F is used to calculate the permeability of dextran from the vascular channel to the tissue compartment using Eq 1, which gives (P)Cell-Free (in this case 10 x 10−6 cm/s) used in Eq 2.


A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Deosarkar SP, Prabhakarpandian B, Wang B, Sheffield JB, Krynska B, Kiani MF - PLoS ONE (2015)

Passage of fluorescent dextran from the vascular channel to tissue compartment of B3C under shear flow.Permeability of Texas Red 40 kDa dextran from vascular channel to the tissue compartment in a cell-free B3C after 5 min (A), 15 min (B), 30 min (C), 60 min (D) and 120 min (E) from the initiation of flow in vascular channel. Normalized tissue intensity in a cell-free B3C increases linearly with time in the tissue compartment (F).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4640840&req=5

pone.0142725.g002: Passage of fluorescent dextran from the vascular channel to tissue compartment of B3C under shear flow.Permeability of Texas Red 40 kDa dextran from vascular channel to the tissue compartment in a cell-free B3C after 5 min (A), 15 min (B), 30 min (C), 60 min (D) and 120 min (E) from the initiation of flow in vascular channel. Normalized tissue intensity in a cell-free B3C increases linearly with time in the tissue compartment (F).
Mentions: Since the B3C developed here is a dynamic in vitro model of neonatal BBB suitable for real-time monitoring and direct measurement of permeability across BBB using microscopic methods, we first optimized the techniques for quantifying permeability in B3C. Permeation of the fluorescent 40 kDa dextran from the vascular channel to the tissue compartment of cell-free B3C was characterized by imaging over time as dextran was injected into the vascular channel at a flow rate of 0.2 μl/min (i.e. shear stress of 7.6x10-2 dynes/cm2). As shown in Fig 2A, 2B, 2C, 2D and 2E, fluorescent dextran accumulates in the tissue compartment in a time-dependent manner over a 120 min period. Quantification of permeability was performed by calculating the average intensity of fluorescent dextran in the entire tissue compartment and normalizing it to the maximum intensity of fluorescent dextran in the vascular channel. The results of a typical cell-free experiment shown in Fig 2F indicate that the normalized intensity increases linearly with time in the tissue compartment. The slope of the line (dIt/dt) in Fig 2F is used to calculate the permeability of dextran from the vascular channel to the tissue compartment using Eq 1, which gives (P)Cell-Free (in this case 10 x 10−6 cm/s) used in Eq 2.

Bottom Line: The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10-6 cm/s to 2.9±1.0 x 10-6 cm/s or 1.1±0.4 x 10-6 cm/s, respectively).Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC.In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, Temple University, Philadelphia, Pennsylvania, 19122, United States of America.

ABSTRACT
Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10-6 cm/s to 2.9±1.0 x 10-6 cm/s or 1.1±0.4 x 10-6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

No MeSH data available.


Related in: MedlinePlus