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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

Schematic illustration and images of neonatal blood-brain barrier on a chip (B3C).Schematic illustration of B3C showing the tissue compartment in the center of the device surrounded by two independent vascular channels with flow access openings. The dimensions of vascular channels are 200 μm x 100 μm x 2762 μm (width x height x length) and the dimensions of tissue compartment are 1575 μm x 100 μm (diameter x height). Vascular channels are in communication with the tissue compartment through a series of 3μm porous interface (pore dimensions are: 3μm x 3μm x 100 μm, width x height x length, spaced every 50 μm) along the length of the vascular channels (A). Schematic illustration of cell culture in B3C device showing one of two vascular channels (blue) with endothelial cells lining the channel walls, the tissue compartment (red) containing astrocytes, and the porous interface (white) separating the vascular channel and tissue compartment (B). The B3C device is assembled on a microscope glass slide with plastic tubes (dark blue) allowing access to individual vascular channels and the tissue compartment (C).
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pone.0142725.g001: Schematic illustration and images of neonatal blood-brain barrier on a chip (B3C).Schematic illustration of B3C showing the tissue compartment in the center of the device surrounded by two independent vascular channels with flow access openings. The dimensions of vascular channels are 200 μm x 100 μm x 2762 μm (width x height x length) and the dimensions of tissue compartment are 1575 μm x 100 μm (diameter x height). Vascular channels are in communication with the tissue compartment through a series of 3μm porous interface (pore dimensions are: 3μm x 3μm x 100 μm, width x height x length, spaced every 50 μm) along the length of the vascular channels (A). Schematic illustration of cell culture in B3C device showing one of two vascular channels (blue) with endothelial cells lining the channel walls, the tissue compartment (red) containing astrocytes, and the porous interface (white) separating the vascular channel and tissue compartment (B). The B3C device is assembled on a microscope glass slide with plastic tubes (dark blue) allowing access to individual vascular channels and the tissue compartment (C).

Mentions: To fabricate the microfluidic neonatal BBB on a chip (B3C), a photomask of the design shown in Fig 1A was created and soft photolithography was used to fabricate the final B3C model shown in Fig 1C on a microscope slide as described previously [32]. Briefly, Sylgard 184 Polydimethylsiloxane (PDMS) was prepared according to manufacturer’s (Dow Corning, Midland, MI) instructions and was poured over the developed master in a 150 mm Petri dish which was degassed for 15 min. The polymer was then allowed to cure overnight in an oven at 65°C. Inlet and outlet holes were punched using a 1.5 mm punch. The bonding surfaces of the PDMS and glass slide were plasma treated in a plasma generator (Harrick Scientific, Ithaca, NY). The assembly was heated at 75°C for 10 min to achieve a seal between the PDMS and glass yielding the complete device. The resulting B3C comprises of a disposable optically clear polydimethylsiloxane (PDMS) microfluidic chip containing a tissue and vascular channels [32, 33]. B3C is designed to allow culturing of brain cells in a tissue compartment and endothelial cells in two independent vascular channels with dimensions of 200 μm x 100 μm x 2762 μm (width x height x length) encompassing the tissue compartment. The tissue compartment and vascular channels are separated by an interface with a series of 3 μm pores along the length of the vascular channels, replacing the use of membranes in conventional models. Vascular channels and the tissue compartment in B3C are fabricated from optically clear PDMS and their side-by-side placement permits simultaneous real-time visualization of both compartments. The porous interface allows for biochemical and cellular communication between the two compartments. The size of the vascular channel is in the range of diameters observed in neonatal rats evaluated using a cranial window model and fluorescence microscopy [11].


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)

Schematic illustration and images of neonatal blood-brain barrier on a chip (B3C).Schematic illustration of B3C showing the tissue compartment in the center of the device surrounded by two independent vascular channels with flow access openings. The dimensions of vascular channels are 200 μm x 100 μm x 2762 μm (width x height x length) and the dimensions of tissue compartment are 1575 μm x 100 μm (diameter x height). Vascular channels are in communication with the tissue compartment through a series of 3μm porous interface (pore dimensions are: 3μm x 3μm x 100 μm, width x height x length, spaced every 50 μm) along the length of the vascular channels (A). Schematic illustration of cell culture in B3C device showing one of two vascular channels (blue) with endothelial cells lining the channel walls, the tissue compartment (red) containing astrocytes, and the porous interface (white) separating the vascular channel and tissue compartment (B). The B3C device is assembled on a microscope glass slide with plastic tubes (dark blue) allowing access to individual vascular channels and the tissue compartment (C).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142725.g001: Schematic illustration and images of neonatal blood-brain barrier on a chip (B3C).Schematic illustration of B3C showing the tissue compartment in the center of the device surrounded by two independent vascular channels with flow access openings. The dimensions of vascular channels are 200 μm x 100 μm x 2762 μm (width x height x length) and the dimensions of tissue compartment are 1575 μm x 100 μm (diameter x height). Vascular channels are in communication with the tissue compartment through a series of 3μm porous interface (pore dimensions are: 3μm x 3μm x 100 μm, width x height x length, spaced every 50 μm) along the length of the vascular channels (A). Schematic illustration of cell culture in B3C device showing one of two vascular channels (blue) with endothelial cells lining the channel walls, the tissue compartment (red) containing astrocytes, and the porous interface (white) separating the vascular channel and tissue compartment (B). The B3C device is assembled on a microscope glass slide with plastic tubes (dark blue) allowing access to individual vascular channels and the tissue compartment (C).
Mentions: To fabricate the microfluidic neonatal BBB on a chip (B3C), a photomask of the design shown in Fig 1A was created and soft photolithography was used to fabricate the final B3C model shown in Fig 1C on a microscope slide as described previously [32]. Briefly, Sylgard 184 Polydimethylsiloxane (PDMS) was prepared according to manufacturer’s (Dow Corning, Midland, MI) instructions and was poured over the developed master in a 150 mm Petri dish which was degassed for 15 min. The polymer was then allowed to cure overnight in an oven at 65°C. Inlet and outlet holes were punched using a 1.5 mm punch. The bonding surfaces of the PDMS and glass slide were plasma treated in a plasma generator (Harrick Scientific, Ithaca, NY). The assembly was heated at 75°C for 10 min to achieve a seal between the PDMS and glass yielding the complete device. The resulting B3C comprises of a disposable optically clear polydimethylsiloxane (PDMS) microfluidic chip containing a tissue and vascular channels [32, 33]. B3C is designed to allow culturing of brain cells in a tissue compartment and endothelial cells in two independent vascular channels with dimensions of 200 μm x 100 μm x 2762 μm (width x height x length) encompassing the tissue compartment. The tissue compartment and vascular channels are separated by an interface with a series of 3 μm pores along the length of the vascular channels, replacing the use of membranes in conventional models. Vascular channels and the tissue compartment in B3C are fabricated from optically clear PDMS and their side-by-side placement permits simultaneous real-time visualization of both compartments. The porous interface allows for biochemical and cellular communication between the two compartments. The size of the vascular channel is in the range of diameters observed in neonatal rats evaluated using a cranial window model and fluorescence microscopy [11].

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