This work is supported by NIH 3-UG3-NS115064-01, R01NS14239, Cure Alzheimer&#39;s Fund, NASA 80ARCO22CA004, Chan-Zuckerberg Initiative, MJFF/ASAP Foundation, and Brain Injury Association of America. C.G. is supported by NIH F31NS130909. Figure 1A was created with BioRender.com.

1. Differentiating iPSCs into iBBB cells
NOTE: These differentiation protocols have been previously described in Mesentier-Louro et al.34.
<ol>
	<li>Coating cell culture plates
	<ol>
		<li>Thaw reduced growth factor (GF) membrane matrix overnight at 4 &#176;C. Dilute 500 &#181;L of basement membrane matrix in 49.5 mL of DMEM. Keep this solution cold to prevent premature polymerization of the coating solution.</li>
		<li>Add 1-2 mL per well of a 6-well tissue culture-treated plate. Leave the coating solution on for at least 20 min at 37 &#176;C before replacing it with the appropriate warmed culture medium.</li>
	</ol>
	</li>
	<li>Differentiation of Human iPSCs into ETV2-inducible endothelial cells 
	Timing: 8 days 
	NOTE: This protocol is adapted from Blanchard et al.24 and Qian et al.35. We use the doxycycline-inducible expression of the transcription factor ETV2, as described by Wang et al.36 to improve the yield. ETV2 was introduced via PiggyBac plasmid and the cells were then transfected.
	<ol>
		<li>Culture iPSCs on reduced GF basement membrane matrix-coated plates in feeder-free pluripotent stem cell medium until the cells reach ~70% confluency. 
		NOTE: We recommend the inducible iPSCs be maintained with selection for the PiggyBac plasmid (i.e., puromycin)</li>
		<li>Day 0: Dissociate the cells with a protease-collagenase mixture for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to stem cell medium. Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet with stem cell medium and plate the cells at 20,800 cells/cm2 on reduced GF basement membrane matrix-coated plates in stem cell medium supplemented with 10 &#181;M Y27632.</li>
		<li>Day 1: Replace the medium with DeSR1 [DMEM/F12 + glutamine supplement, 1x Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA), 1% penicillin-streptomycin (P/S)], supplemented with 10 ng/mL BMP4, 6 &#181;M CHIR99021, and 5 &#181;g/mL doxycycline.</li>
		<li>Day 3: Replace the medium with DeSR2 [DMEM/F12 + glutamine supplement, 1x MEM-NEAA, 1x N-2, 1x B-27, 1% P/S] supplemented with 5 &#181;g/mL doxycycline.</li>
		<li>Day 5: Replace medium with hECSR [Human Endothelial Serum-Free Medium, 1x MEM-NEAA, 1x B-27, 1% P/S] supplemented with 50 ng/mL VEGF-A, 2 &#181;M forskolin, and 5 &#181;g/mL doxycycline.</li>
		<li>Day 7: Replace medium with hECSR supplemented with 50 ng/mL VEGF-A, 2 &#181;M forskolin, and 5 &#181;g/mL doxycycline.</li>
		<li>Day 8: Split ECs 1:3 with a protease-collagenase mixture and re-seed onto fresh reduced GF basement membrane matrix-coated plates in hECSR supplemented with 50 ng/mL VEGF-A and 5 &#181;g/mL doxycycline. 
		NOTE: This is the best time point to encapsulate ECs into iBBBs for uniform vasculature formation.</li>
		<li>Day 10+: Feed the cells every 2-3 days with hECSR supplemented with 50 ng/mL VEGF-A and 5 &#181;g/mL doxycycline. Continue to split the cells 1:3 with a protease-collagenase mixture when the plates are confluent. 
		NOTE: iPSC-derived ECs can be frozen at any point starting Day 8. Lift the cells, as if to passage. Resuspend the pellet in EC freezing medium [60% knockout serum replacement (KSR), 30% hECSR, 10% DMSO,&#160;50 ng/mL VEGF-A,&#160;and 10 &#181;M Y27632], splitting the cells 1:3, and freeze using a freezing container at -80 &#176;C. Thaw onto reduced GF basement membrane matrix-coated plates in hECSR supplemented with&#160;50 ng/mL VEGF-A, 5 &#181;g/mL doxycycline.</li>
		<li>Confirm brain microvascular endothelial cell differentiation by immunocytochemistry using antibodies against CD31/PECAM1 or VE-cadherin (Figure 2A).</li>
	</ol>
	</li>
	<li>Differentiation of Human iPSCs into pericytes 
	Timing: 6 days 
	NOTE: This protocol was adapted from Patsch et al.36.
	<ol>
		<li>Culture iPSCs on reduced GF basement membrane matrix-coated plates in feeder-free conditions in stem cell medium until the cells reach ~70% confluency.</li>
		<li>Day 0: Dissociate the cells with a protease-collagenase mixture for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to stem cell medium. Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet with stem cell medium and plate the cells at 37,000-40,000 cells/cm2 (depending on the cell line) on reduced GF basement membrane matrix-coated plates in stem cell medium supplemented with 10 &#181;M Y27632.</li>
		<li>Day 1: Replace the media with N2B27 [50% DMEM/F12 + glutamine supplement, 50% Neurobasal, 1x MEM-NEAA, 0.5x, glutamine supplement, 1x N-2, 1x- B-27, 1% P/S] supplemented with 25 ng/mL BMP4 and 8 uM CHIR99021.</li>
		<li>Days 3-4: Replace the media with N2B27 supplemented with 2 ng/mL Activin A and 10 ng/mL PDGF-BB, and feed daily.</li>
		<li>Day 5: Split PCs with a protease-collagenase mixture and seed onto 0.1% gelatin-coated plates at 35,000 cells/cm2 in N2B27 supplemented with 10 ng/mL PDGF-BB. 
		NOTE: Only split cells once confluent. Some lines might require a few more days before they are ready to split; continue to change the media daily until cells are confluent.</li>
		<li>Expand PCs in N2B27 supplemented with 10 ng/mL PDGF-BB, changing media every 2-3 days. Remove PDGF-BB from the media after the cells once again reach confluency and undergo a second passage. 
		NOTE: Pericytes are ready to be used in iBBBs after the second passage. Once expanded, PCs can be frozen. Lift the cells as if to passage, resuspend the cell pellet in freezing media [90% KSR and 10% DMSO] and freeze using a freezing container at -80 &#176;C. Thaw onto 0.1% gelatin-coated plates at 35,000 cells/cm2 in N2B27.</li>
		<li>Confirm pericyte differentiation by immunocytochemistry using antibodies against PDGFRB and NG2 (Figure 2B).</li>
	</ol>
	</li>
	<li>Differentiation of Human iPSCs into neural progenitor cells (NPCs) and astrocytes 
	Timing: 45 days total (15 days for NPCs followed by 30 days for astrocytes) 
	&#8203;NOTE: The NPC differentiation protocol was adapted from Chambers et al.38, and the astrocyte differentiation is as described in TCW et al.39.
	<ol>
		<li>Culture iPSCs on reduced GF basement membrane matrix-coated plates in feeder-free conditions in stem cell medium until the cells reach ~70% confluency.</li>
		<li>Dissociate the cells with a protease-collagenase mixture for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to stem cell medium. Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet with stem cell medium and plate the cells at 100,000 cells/cm2 on reduced GF basement membrane matrix-coated plates in stem cell medium supplemented with 10 &#181;M Y27632.</li>
		<li>Replace the stem cell medium the following day. Continue to feed the cells every other day until they reach &#62;95% confluency (3-4 days depending on the cell line).</li>
		<li>NPC Day 0: Replace the media with N2B27 [50% DMEM/F12 + glutamine supplement, 50% Neurobasal, 1x MEM-NEAA, 0.5x, glutamine supplement, 1x N-2, 1X- B-27, 1% P/S] supplemented with 10 &#181;M SB43152 and 100 nM LDN193189.</li>
		<li>NPC Days 1-9: Feed the cells daily with N2B27 supplemented with 10 &#181;M SB43152 and 100 nM LDN193189.</li>
		<li>NPC Day 10: Split NPCs 1:3 with the protease-collagenase mixture and seed onto fresh reduced GF basement membrane matrix-coated plates in N2B27 supplemented with 20 ng/mL FGF-basic and 10 &#181;M Y27632.</li>
		<li>NPC Days 11 - 13: Feed NPCs daily with N2B27 supplemented with 20 ng/mL FGF-basic.</li>
		<li>NPC Day 14: Split NPCs 1:3 with the protease-collagenase mixture and reseed onto fresh reduced GF basement membrane matrix-coated plates in N2B27 supplemented with 20 ng/mL FGF-basic and 10 &#181;M Y27632.</li>
		<li>NPC Day 15/Astrocyte Day 0: This is Day 0 of the astrocyte differentiation. Feed the cells with complete Astrocyte medium (AM). 
		NOTE: NPCs can be maintained in N2B27 supplemented with 20 ng/mL FGF-basic and passaged when confluent. To freeze NPCs, resuspend the cell pellet in freezing media [90% KSR and 10% DMSO], split the cells 1:3, and freeze using a freezing container at -80 &#176;C. Thaw onto reduced GF basement membrane matrix-coated plates in N2B27 supplemented with 20 ng/mL bFGF and 10 &#181;M Y27632.</li>
		<li>Astrocyte Days 1-30: Feed the cells every 2-3 days with AM. When cells are &#62;90% confluent, passage using the protease-collagenase mixture and plate at 15,000 cells/cm2 on fresh reduced GF basement membrane matrix-coated plates in AM. 
		NOTE: Astrocytes should be fully differentiated in 30 days. Astrocytes can be frozen in freezing media [90% KSR and 10% DMSO] in a freezing container at -80 &#176;C. Thaw onto reduced GF basement membrane matrix-coated plates in AM at 25,000-35,000 cells/cm2.</li>
		<li>Confirm NPC differentiation by immunocytochemistry using antibodies against Nestin and SOX2. Confirm astrocyte differentiation by immunocytochemistry using antibodies against S100B and CD44 (Figure 2C).</li>
	</ol>
	</li>
</ol>
2. Assembling the iBBB
<ol>
	<li>Thaw reduced growth factor (GF) basement membrane matrix overnight at 4 &#176;C. Do not dilute this matrix in DMEM as the iBBBs are encapsulated in 100% reduced GF basement membrane matrix. Each iBBB requires 50 &#181;L of the matrix.</li>
	<li>Dissociate the differentiated ECs with a protease-collagenase mixture at 37 &#176;C for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to hECSR (Human Endothelial Serum-Free Medium, 1x MEM-NEAA, 1x B-27, 1% P/S). Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet in hECSR. 
	NOTE: Use a conical tube, either 15 mL or 50 mL, that is large enough to accommodate the volume of dissociated cells plus media. Cells can also be divided among multiple tubes and then recombined upon resuspension.</li>
	<li>Dissociate the differentiated PCs with the protease-collagenase mixture at 37 &#176;C for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to N2B27 (50% DMEM/F12 + glutamine supplement, 50% Neurobasal, 1x MEM-NEAA, 0.5x glutamine supplement, 1x N-2, 1x B-27, 1% penicillin-streptomycin [P/S]). Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet in N2B27.</li>
	<li>Dissociate the differentiated astrocytes with the protease-collagenase mixture at 37 &#176;C for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to complete Astrocyte Medium (AM). Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet in AM.</li>
	<li>Using a hemocytometer, count each cell type. Dilute each type of cell suspension to a final concentration of 1 &#215; 106 cells/mL in the appropriate medium.</li>
	<li>Combine the calculated number of cells for each cell type in a conical tube: 50 &#181;L of iBBB requires 2.5 &#215; 105 ECs, 5 &#215; 104 PCs, and 5 &#215; 104 astrocytes. Include 10% more than the calculated number of cells to account for pipetting errors. Spin down the cell mixture at 300 &#215; g for 3 min. 
	NOTE: It is highly recommended to plate some leftover cells of each cell type in 2D monocultures to fix and stain for quality control of the input cells. Plate each cell type at 35,000 cells/cm2 on previously prepared plates coated with reduced GF basement membrane matrix and fix after 3-4 days. See Table 1 for cell-type specific antibodies.</li>
	<li>Aspirate medium from the cell pellet leaving approximately 50 &#181;L of the supernatant above the cell pellet. Using a P200, gently resuspend the cell pellet in residual medium to create a single-cell slurry.</li>
	<li>Mix the cell slurry in enough reduced GF basement membrane matrix supplemented with 10 ng/mL VEGF-A for the desired number of iBBBs at 50 &#181;L of reduced GF basement membrane matrix per iBBB, taking care to mix the cells homogeneously while not introducing any air bubbles. 
	NOTE: It is critical to keep the cells and reduced GF basement membrane matrix on ice at this stage to prevent premature polymerization.</li>
	<li>Pipette 50 &#181;L of reduced GF basement membrane matrix mixture into the well of a 48-well glass-bottom culture dish. Distribute the volume evenly throughout the bottom of the dish (Figure 1C).</li>
	<li>Incubate the iBBBs at 37 &#176;C for 30-40 min to allow the reduced GF basement membrane matrix to polymerize, encapsulating the cells in the matrix.</li>
	<li>Add 500 &#181;L of AM supplemented with 10 ng/mL VEGF-A, 5 &#181;g/mL doxycycline, and 10 &#181;M Y27632.</li>
	<li>Change the medium the following day to AM supplemented with 10 ng/mL VEGF-A. Continue to change the medium every 2-3 days. After 2 weeks, stop supplementing with VEGF-A; iBBBs are ready for downstream assays after 2 weeks.</li>
</ol>
3. Inducing cerebral amyloid angiopathy (CAA) with A&#946; fibrils
<ol>
	<li>Prepare stock solutions of amyloid-&#946;1-40 and amyloid-&#946;1-42 at 200 &#181;M in PBS.</li>
	<li>Add A&#946; to iBBB medium on 2-week iBBBs to a final concentration of 20 nM. Incubate the cells in A&#946;-supplemented medium for 96 h (4 days).</li>
	<li>After 96 h, remove the medium and continue with fixing (section 4).</li>
</ol>
4. Fixing and staining the iBBB
<ol>
	<li>To fix the iBBB, remove the medium, wash in 1 mL of PBS, and incubate in 500 &#181;L of 4% paraformaldehyde overnight at 4 &#176;C.</li>
	<li>Remove the 4% paraformaldehyde solution, and rinse for 4 x (at least) 15 min in 1 mL of PBS.</li>
	<li>Permeabilize the samples in 0.3% PBST (PBS with 0.3% Triton X-100) for 1 h at room temperature with gentle shaking.</li>
	<li>Incubate the cultures in blocking buffer (5% Normal Serum in 0.3% PBST) at room temperature for at least 1 h with gentle shaking. 
	NOTE: The type of serum used is dependent on the host species for the secondary antibodies used. For example, if the host species for the secondary antibodies is&#160;goat, use normal goat serum; if the host species is&#160;donkey, use normal donkey serum. This can also be done overnight at 4 &#176;C with gentle shaking.</li>
	<li>Dilute primary antibodies to the recommended or determined concentration in blocking buffer.</li>
	<li>Replace the blocking solution with the primary antibody dilution. Make sure that the iBBBs are fully submerged in antibody solution for even incubation; 100-150 &#181;L is enough to cover a 50 &#181;L iBBB in a 48-well glass-bottom culture dish. Incubate overnight at 4 &#176;C with gentle shaking.</li>
	<li>Remove the primary antibody. Wash iBBBs for 3 x 10-20 min in 0.3%&#160;PBS.</li>
	<li>Dilute the secondary antibody to the recommended or determined concentration in blocking buffer. Additionally, add a nuclear stain, such as Hoechst, to the secondary antibody solution at the recommended or determined concentration.</li>
	<li>Replace the last PBS wash with the secondary antibody dilution. Make sure that the iBBBs are fully submerged in antibody solution for even incubation; 100-150 &#181;L is enough to cover a 50 &#181;L iBBB in a 48-well glass-bottom culture dish. Incubate at room temperature for 2 h with gentle shaking. 
	NOTE: This can also be done overnight at 4 &#176;C with gentle shaking.</li>
	<li>Wash iBBBs 4-5x for at least 1 h per wash in&#160;PBS. Store in PBS or anti-fading mounting media at 4 &#176;C until the samples are imaged.</li>
	<li>To image on an inverted microscope, keep iBBBs in glass-bottom plates or dishes. Place a glass coverslip over the glass well to keep the iBBBs in place (Figure 1D).</li>
</ol>

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

The authors report no conflicts of interest.

BBB dysfunction is a co-morbidity, and potentially, a cause or exacerbating factor in numerous neurological diseases7,40,41. However, it is nearly impossible to study the molecular and cell biology underlying the dysfunction and breakdown of the BBB in humans with neurovascular disease. The inducible-BBB (iBBB) presented in this protocol provides an in vitro system that recapitulates important cell interactions of the B...

The blood-brain barrier (BBB) is a key microvascular network separating the central nervous system (CNS) from the periphery to maintain an ideal environment for proper neuronal function. It has a critical role in regulating the influx and efflux of substances into the CNS by maintaining metabolic homeostasis1,2,3,4, clearing waste4,5,6, and protecting the brain from pathogens and toxins7,8.
The primary cell type of the BBB is the endothelial cell (EC). Endothelial cells, derived from the mesoderm lineage, form the walls of the vasculature1,9. Microvascular ECs form tight junctions with each other to greatly decrease the permeability of their membrane10,11,12,13,14 while expressing transporters to facilitate the movement of nutrients into and out of the CNS1,4,12,14. Microvascular ECs are encircled by pericytes (PCs)-mural cells that regulate microvascular function and homeostasis and are critical for regulating the permeability of the BBB to molecules and immune cells15,16,17. The astrocyte, a major glial cell type, is the final cell type comprising the BBB. Astrocyte end-feet wrap around the EC-PC vascular tubes while the cell bodies extend into the brain parenchyma, forming a connection between neurons and vasculature1. Distinct solute and substrate transporters are localized on astrocyte&#160;end-feet (e.g., aquaporin 4 [AQP-4]) that have a critical role in BBB function18,19,20,21.
The BBB is critical in maintaining proper brain health function, and dysfunction of the BBB has been reported in many neurological diseases, including Alzheimer&#39;s disease (AD)22,23,24,25, multiple sclerosis7,26,27,28, epilepsy29,30, and stroke31,32. It is increasingly recognized that cerebrovascular abnormalities play a central role in neurodegeneration, contributing to increased susceptibility to ischemic and hemorrhagic events. For example, more than 90% of AD patients have cerebral amyloid angiopathy (CAA), a condition characterized by the deposition of amyloid &#946; (A&#946;) along the cerebral vasculature. CAA increases BBB permeability and decreases BBB function, leaving the CNS vulnerable to ischemia, hemorrhagic events, and accelerated cognitive decline33.
We recently developed an in vitro model of the human BBB, derived from&#160;patient-induced pluripotent stem cells, which incorporates ECs, PCs, and astrocytes encapsulated in a 3D matrix (Figure 1A). The iBBB recapitulates physiologically relevant interactions, including vascular tube formation and localization of astrocyte end-feet with vasculature24. We applied the iBBB to model the susceptibility of CAA mediated by APOE4 (Figure 1B). This enabled us to identify the causal cellular and molecular mechanisms by which APOE4 promotes CAA, and leverage these insights to develop therapeutic strategies that reduce CAA pathology and improve learning and memory in vivo in APOE4 mice24. Here, we provide a detailed protocol and video tutorial for reconstructing the BBB from human iPSCs and modeling CAA in vitro.

<table><tbody><tr><td>6e10 amyloid-&amp;beta; antibody</td><td>Biolegend</td><td>SIG-39320</td><td>Used at 1:1000</td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT104</td><td></td></tr><tr><td>Activin A</td><td>Peprotech</td><td>20-14E</td><td></td></tr><tr><td>Alexa Fluor 488, 555, 647 secondary antibodies</td><td>Invitrogen</td><td>Various</td><td>Used at 1:1000</td></tr><tr><td>Amyloid-beta 40 fibril</td><td>AnaSpec</td><td>AS-24235</td><td></td></tr><tr><td>Amyloid-beta 42 fibril</td><td>AnaSpec</td><td>AS-20276</td><td></td></tr><tr><td>Aquaporin-4 antibody</td><td>Invitrogen</td><td>PA5-53234</td><td>Used at 1:300</td></tr><tr><td>Astrocyte basal media and supplements</td><td>ScienCell</td><td>1801</td><td></td></tr><tr><td>B-27 serum-free supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>BMP4</td><td>Peprotech</td><td>120-05ET</td><td></td></tr><tr><td>CHIR99021</td><td>Cyamn Chemical</td><td>13112</td><td></td></tr><tr><td>DMEM/F12 with GlutaMAX medium</td><td>Gibco</td><td>10565018</td><td></td></tr><tr><td>Doxycycline</td><td>Millipore-Sigma</td><td>D3072-1ML</td><td></td></tr><tr><td>FGF-basic</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Fluoromount-G slide mounting medium</td><td>VWR</td><td>100502-406</td><td></td></tr><tr><td>Forskolin</td><td>R&amp;amp;D Systems</td><td>1099/10</td><td></td></tr><tr><td>GeltrexTM LDEV-Free hESC-qualified Reduced Growth Factor Basement&amp;nbsp;</td><td>Gibco</td><td>A1413302</td><td></td></tr><tr><td>Glass Bottom 48-well Culture Dishes</td><td>Mattek Corporation</td><td>P48G-1.5-6-F</td><td></td></tr><tr><td>GlutaMAX supplement</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>Human Endothelial Serum-free medium</td><td>Gibco</td><td>11111044</td><td></td></tr><tr><td>LDN193189</td><td>Tocris</td><td>6053</td><td></td></tr><tr><td>Minimum Essential Medium Non-essential Amino Acid Solution (MEM-NEAA)&amp;nbsp;</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>N-2 supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Millipore-Sigma</td><td>S30-100mL</td><td>Use serum to match secondary antibody host</td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>ThermoFisher</td><td>28908</td><td></td></tr><tr><td>PDGF-BB</td><td>Peprotech</td><td>100-14B</td><td></td></tr><tr><td>PDGFRB (Platelet-derived growth factor receptor beta) antibody</td><td>R&amp;amp;D Systems</td><td>AF385</td><td>Used at 1:500</td></tr><tr><td>Phosphate Buffered Saline (PBS), pH 7.4</td><td>Gibco</td><td>10010031</td><td></td></tr><tr><td>Pecam1 (Platelet endothelial cell adhesion molecule 1) antibody</td><td>R&amp;amp;D Systems</td><td>AF806</td><td>Used at 1:500</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>PiggyBac plasmid (PB_iETV2_P2A_GFP_Puro)</td><td>AddGene</td><td>&amp;nbsp;Catalog #168805</td><td></td></tr><tr><td>S100B antibody</td><td>Sigma-Aldrich</td><td>S2532-100uL</td><td>Used at 1:500</td></tr><tr><td>SB43152</td><td>Reprocell</td><td>04-0010</td><td></td></tr><tr><td>Thioflavin T</td><td>Chem Impex</td><td>22870</td><td>Used at 25uM</td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787-250mL</td><td></td></tr><tr><td>VE-cadherin (CD144) antibody</td><td>R&amp;amp;D systems</td><td>AF938</td><td>Used at 1:500</td></tr><tr><td>VEGF-A</td><td>Peprotech</td><td>100-20</td><td></td></tr><tr><td>Y27632</td><td>R&amp;amp;D Systems</td><td>1254/10</td><td></td></tr><tr><td>ZO-1</td><td>Invitrogen</td><td>MA3-39100-A488</td><td>Dilution = 1:500</td></tr></tbody></table>

reconstruction of the blood-brain barrier in vitro to model and therapeutically target neurological disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

A properly formed iBBB solidifies into a single translucent disc (Figure 3A). It is normal for the iBBB to detach from the surface onto which it was first pipetted after a few days. This cannot be avoided, but is not a major concern to the proper formation of the iBBB if care is taken when changing media to not accidentally aspirate the iBBB. After 24 h, evenly distributed, single cells can be identified under a brightfield microscope (Figure 3B). After 2 weeks,...

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Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

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Video: Auteur in de schijnwerpers: een gepersonaliseerde benadering voor het onderzoeken van de ziekte van Alzheimer met behulp van een in vitro bloed-hersenbarrièremodel 

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

Cancer Research

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Bioengineering

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Medicine

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

Biochemistry

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Immunology and Infection

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

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