Engineered human blood–brain barrier microfluidic model for vascular permeability analyses

Cynthia Hajal, Giovanni S. Offeddu, Yoojin Shin, Shun Zhang, Olga Morozova, Dean Hickman, Charles G. Knutson, Roger D. Kamm

Published: 2022-01-06 DOI: 10.1038/s41596-021-00635-w

stem-cell-derived endothelial cells

Extended

Extended Data Fig. 1 Morphological properties of the BBB MVNs over time starting at day 2 of culture after device seeding.

Parameters considered are ( a ) average vessel branch length (µm), ( b ) average vessel diameter (µm), and ( c ) number of branches per ROI 12 , 69 , 73 .

Source data

Extended Data Fig. 2 Steps for vascular permeability measurements via confocal microscopy.

( a ) Representative images of perfused plasma IgG (green) at times 0 and 12 min, and increasing matrix intensity over time; the single data points represent the same single pixels in the matrix imaged over time. Scale bar, 100 μm. ( b ) Permeability of plasma IgG in the BBB MVNs over time. At short times (<12 min), variability in the measurement derives from low signal-to-noise ratio. At long times (>20 min), higher permeability values derive from progressive perfusate diffusion from the side channels. ( c ) Comparison in 40 kDa dextran permeability measured in the micro and macro devices; n = 3 device repeats, each the average of 3 regions of interest (ROIs). The higher permeability in the micro device derives from increased perfusate diffusion from the side channels over the same time.

Source data

Extended Data Fig. 3 Steps for vascular permeability measurements via fluid sampling.

( a ) Example vascular and matrix intensities measured for IgG as a function of depth imaged within the device. Both intensities are normalized to the vascular intensity near the bottom glass. They decrease with depth as a result of light scattering, falling within the background intensity range (shaded area) in the case of the matrix intensity. ( b ) Example microscope z -drift measured during a typical experiment. ( c ) Permeability of plasma IgG as a function of imaged region of interest (ROI) size. Average and standard deviation between 3 devices are reported. For ROIs smaller than 600 µm by side, the varying permeability values are an artifact due to incomplete capture of the BBB MVN average morphology.

Source data

Extended Data Fig. 4 Effective permeability of FITC (assumed σ = 0) across the BBB MVNs as a function of intravascular pressure applied.

The slope of the linear fit represents the hydraulic conductivity L p 14 .

Source data

Extended Data Fig. 5 Immunofluorescence staining for various proteins of interest in the BBB MVNs with iPS-ECs (steps 52-59 in the protocol).

( a ) Staining of water channel protein (aquaporin 4) in ACs in the BBB MVNs. ( b ) Staining of glycocalyx protein (hyaluronic acid) in the BBB MVNs. ( c ) Staining of tight junction protein (claudin-5) in the BBB MVNs. Scale bars are 100 µm for (a-b) and 50 µm for (c).

Extended Data Fig. 6 Immunofluorescence staining for various proteins of interest in the BBB MVNs with HBMECs (steps 52-59 in the protocol).

( a ) Staining of PC marker (PDGFR-β) in the MVNs, along with F-actin to visualize interactions between HBMECs and PCs. ( b ) Staining of AC marker (GFAP) in the MVNs, along with F-actin to visualize interactions between HBMECs and ACs. ( c ) Staining of tight junction protein (ZO-1) in the BBB MVNs with HBMECs. ( d ) Staining of basement membrane proteins (collagen IV and laminin) in the BBB MVNs with HBMECs. Scale bars are 100 µm for (a, d) and 50 µm for (b-c).

[ Extended Data Fig. 7 Gene levels measured via qRT-PCR in the different conditions described in Fig. 12a for tight junctions. ](https://www.nature.com/articles/s41596-021-00635-w/figures/22)

( a ) Claudin 1, ( b ) claudin 3, ( c ) claudin 5, ( d ) occludin, and ( e ) ZO-1.

[ Extended Data Fig. 8 Gene levels measured via qRT-PCR in the different conditions described in Fig. 12a for adherens junctions. ](https://www.nature.com/articles/s41596-021-00635-w/figures/23)

( a ) VE-cadherin, ( b ) JAM-A, and EC adhesion markers ( c ) PDGF-B and ( d ) VCAM1.

[ Extended Data Fig. 9 Gene levels measured via qRT-PCR in the different conditions described in Fig. 12a for transporter receptors. ](https://www.nature.com/articles/s41596-021-00635-w/figures/24)

( a ) LRP1, ( b ) LAT1, ( c ) CAT1, ( d ) GLUT1, ( e ) TfR, ( f ) BCRP, ( g ) MOT1, ( h ) CERP, ( i ) MRP1, ( j ) MRP2, ( k ) RAGE, ( l ) MFSD2A, and ( m ) P-GP.

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References

The blood–nobreakbrain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant invitro human BBB models are n
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
- Reporting Summary
- References
References
ACKNOWLEDGMENTS

Figure 1
Fig. 1: Summary of protocol steps for the formation of BBB MVNs in the micro- and macrodevices, along with downstream applications following 7 d of culture in the devices.
Figure 2
Fig. 2: Summary of protocol steps for the measurement of permeability in the BBB MVNs by confocal microscopy of fluorophore-labeled molecules.
Figure 3
Fig. 3: Summary of protocol steps for the measurement of permeability in the BBB MVNs by collection and analysis of interstitial fluid after intravascular pressurization.
Figure 4
Fig. 4: Summary of protocol steps for the fabrication of the microdevice with an SU-8 wafer and the macrodevice with a 3D printed mold (Steps 1–2 in the protocol).
Figure 5
Fig. 5: Summary of protocol steps for the addition of EC monolayers in the side medium reservoirs on day 4 of culture (Steps 15–19 in the protocol).
Figure 6
Fig. 6: Schematic visualization of molecular perfusion through the BBB MVNs (Steps 21–22 in the protocol).
Figure 7
Fig. 7: Fabrication of flow stoppers and pressure line to prepare the BBB MVN device for pressurization (Steps 38–43 in the protocol).
Figure 8
Fig. 8: Access and collection of the BBB MVN gel within PDMS microfluidic devices (Steps 62–64 in the protocol).
Figure 9
Fig. 9: Immunofluorescence staining for various proteins of interest in the BBB MVNs with iPS-ECs (Steps 52–59 in the protocol).
Figure 10
Fig. 10: Immunofluorescence staining for various proteins of interest in the BBB MVNs with iPS-ECs (Steps 52–59 in the protocol).
Figure 11
Fig. 11: Scanning electron microscopy images of histological sections collected from BBB MVNs fixed in 2% PFA, 2% glutaraldehyde in 0.15 M cacodylate buffer and sectioned using the methodology described in Fang et al.70.
Figure 12
Fig. 12: EC culture conditions and isolation for gene analysis.
Figure 13
Fig. 13: Assessing transport across the BBB MVNs.
Figure 14
Fig. 14: Functional permeability assay.
Figure 15
Fig. 15: Evaluating secreted cytokines in the BBB MVNs.

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Engineered human blood–brain barrier microfluidic model for vascular permeability analyses

Extended

[ Extended Data Fig. 7 Gene levels measured via qRT-PCR in the different conditions described in Fig. 12a for tight junctions. ](https://www.nature.com/articles/s41596-021-00635-w/figures/22)

[ Extended Data Fig. 8 Gene levels measured via qRT-PCR in the different conditions described in Fig. 12a for adherens junctions. ](https://www.nature.com/articles/s41596-021-00635-w/figures/23)

[ Extended Data Fig. 9 Gene levels measured via qRT-PCR in the different conditions described in Fig. 12a for transporter receptors. ](https://www.nature.com/articles/s41596-021-00635-w/figures/24)

Supplementary information

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