J.L.M., B.E., T.R.G., M.C.M., P.K., M.T., K.C., and L.W. were funded by NIH grant R33 HL154249. J.L.M. was funded by R44 GM137651. M.M. was funded by the Schmidt Program Award Del Monte Institute for Neuroscience, University of Rochester. M.T. was funded by RF1 AG079138. K.C. was funded by International Foundation for Ethical Research.

Analysis of Blood-Brain Barrier Models via Immunofluorescence Staining

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoporous+silicon+nitride+membranes+fabricated+from+porous+nanocrystalline+silicon+templates.">Nanoporous silicon nitride membranes fabricated from porous nanocrystalline silicon templates.</a> Nanoscale. 6 (18), 10798-10805 (2014).</li><li>Hill, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+generation+nanoporous+silicon+nitride+membranes+for+high+toxin+clearance+and+small+format+hemodialysis.">Second generation nanoporous silicon nitride membranes for high toxin clearance and small format hemodialysis.</a> Advanced Healthcare Materials. 9 (4), e1900750 (2020).</li><li>Salminen, A. 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J.L.M. is a co-founder of SiMPore and holds an equity interest in the company. SiMPore is commercializing ultrathin silicon-based technologies including the membranes used in this study.

While membrane chips have been designed for stability, they can crack or break if handled incorrectly during assembly. Thus, it is critical to grip the chip in the chip tweezer notches and gently place it in the fixture. When handling the devices in general, extra precaution must be taken not to bump or drop the devices. During the bonding of the membrane chip to fixture A1, the chip must be laid flat and centered on the pillar of fixture A1 to avoid membrane cracking during the bonding to component 1. Further, any contact with the exposed PSA layers must be avoided after removing protective masks. When handling component 2 following the removal of the protective masks, it is advisable to hold it along the component&#39;s edge and use tweezers to grab the triangle corner that is PSA-free.
While cell culture protocols were described for possible BBB models, the BMEC/pericyte co-culture model of the BBB described here may be sufficient or insufficient depending on the physiological context and questions of interest. For example, immune cell trafficking largely occurs in the postcapillary venules of the brain41,42. In these regions, a perivascular space separates the BMEC/pericyte barrier from the glial limitans established by astrocytes. Thus, in postcapillary venules, the neurovascular unit (NVU) comprises two physically separated barriers in series, and astrocytic end-feet do not directly contact the BMEC/pericyte blood barrier, which is well represented by the current model. If the goal is an NVU model that accounts for the impact of astrocyte-secreted factors on the blood barrier, an astrocyte compartment could be added to the device that allows soluble factor exchange through the perivascular space. This example was illustrated previously34 and could be extended to include other cells such as microglia and neurons in a 3D &#39;brain&#39; compartment. The modular architecture of the platform enables simple assembly strategies to be used to achieve these reconfigurations so that the platform&#160;is as simple or complex as needed to address the hypotheses at hand. Each cell culture setup;&#160;however, must be optimized for new cell lines and multi-cultures. For example, due to the properties of the silicon-nitride membranes, coating solutions may need to be adjusted compared to tissue culture plates. The inclusion of fibronectin typically aids in cell attachment and survival. Further, users could culture endothelial cells and pericytes in opposite directions. In this case, it may be necessary to modify, for example, the way the permeability measurements are made and interpreted. Providing steps for this type of culture, however, is beyond the scope of this paper.
Another challenge one may encounter during cell culture is the quick evaporation of media since the devices can be more sensitive to alterations in the environment compared to standard tissue culture plates and flasks. If excess evaporation is seen or cell growth is slowed, all critical incubator parameters must be measured to ensure accurate settings. More water can be added to the tissue cap or&#160;small Petri dish placed inside the cell culture chamber, or media must be exchanged more frequently. Further, bubbles can enter the bottom channel and get stuck in the trench for trench-down devices. While they can be removed, it is easiest to avoid adding bubbles in the first place. To do this, it is important to check that there are no bubbles in the pipette tip or air at the end of the pipette tip prior to pipetting media into the bottom chamber. Further, media evaporation in the channel can lead to a gap between the media surface and the port top. A small volume of media can be pipetted into one port until the media reaches the surface of the opposite port, after which the media can be exchanged in that opposite port. While hECSR and E6 + 10% FBS media should not be warmed in the water bath, other media can be prewarmed to reduce bubble formation. If a bubble does get into the bottom chamber, it can be removed by quickly pipetting 100 &#181;L through the channel. However, this method can lead to contamination between chambers or media spilled across the device&#39;s surface. It may also disrupt cell layers. Alternatively, the media can be removed from the channel first and then reintroduced with a 50 &#181;L volume. Removing the media first, however, can result in more bubble formation in the channel. If a bubble is not directly under the membrane area, it can be left in the channel with no effects on cell culture.
Pericyte attachment and growth, as demonstrated in Figure 2, can be challenging. Using an optimized seeding density is essential for the formation of a layer with a physiologically relevant pericyte-to-endothelial cell ratio. Further, as the pericytes are sensitive to shear, all media exchanges in the channel should be done very slowly to protect the cells. For BPLC culture, improved attachment can be achieved by coating the bottom chamber with 800 &#181;g/mL collagen type IV or 100 &#181;g/mL fibronectin.
Immunocytochemistry in devices described here enables qualitative analysis of cell health and function. Methods for staining in tissue culture plates or other platforms should be directly translatable into the platform. For cell culture in only the top chamber, following fixation, PBS can be added into the bottom chamber and left for the remaining steps, with blocking and staining done in the top chamber only. This minimizes the risks of breaking the membranes or getting bubbles into the bottom chamber. For co-culture staining, we recommend using both chambers in all steps. It is important to note that the viscosity of the fixative and PBS is different from that of media. Thus, it can be easier to add bubbles into the bottom chamber, and extra care should be taken to check pipette tips for air at the end of the tip prior to pipetting into the bottom chamber.
The protocol described for the small molecule permeability assay allows functional and quantitative assessment of the barrier function of the endothelial cells cultured in the &#181;SiM device. One issue that may be encountered during this assay is drawing air bubbles into the pipette during sample collection from the bottom channel at protocol step 4.1.7.4. To avoid this issue, it is important to make sure that the tip is sealed into the port before starting sample collection and the sample should not be drawn too fast. If that does not solve the problem, the size of the tips used might be too small or too large to fit into the ports; use the tips listed in the Table of Materials. If unexpectedly high permeability values are measured in spite of a healthy-looking confluent monolayer, the integrity of the monolayer must be checked for disruption during sample collection. We recommend always checking the monolayer under the microscope immediately after sample collection. If the monolayer still seems intact and healthy, the sample can be fixed and biological markers of barrier function can be assessed, for instance, via immunostaining of the junctional proteins. Conversely, if unexpectedly low permeability values are measured, it is important to ensure that 50 &#181;L of media is sampled from the bottom channel without any air bubbles. If medium comes out from the sampling port as soon as the reservoir tip is placed, that media must be collected before fitting the pipette into the sampling port as most of the fluorescent small molecule will be present in the initial 10 &#181;L volume sampled from the bottom channel. Drawing circles around the ports using a hydrophobic pen or placing a hydrophobic tape with a hole around the port prevents any&#160;passively pumped&#160;media from spreading. If bubbles are withdrawn during sampling or the full 50 &#181;L sample is not removed, the sample must not be used. Alternatively, the exact volume can be determined and used in the permeability calculation; however, this should only be done if the volume removed is &#8805;40 &#181;L, which corresponds to ~98-99% dye recovery34.

Living tissues are compartmentalized by specialized cells that create and maintain barriers and regulate which cells and molecules are transported from one compartment to another. The improper regulation of barrier functions can be the source of both acute illnesses and chronic disease. The blood-brain barrier (BBB) is the most restrictive tissue barrier in the human body1. Dysfunction of the BBB underlies a wide range of diseases of the central nervous system (CNS), including Alzheimer&#39;s disease2, Parkinson&#39;s disease3,4, and multiple sclerosis5,6. Injury to the BBB is also linked to long-term cognitive impairment as an outcome of acute disorders, such as sepsis7, COVID-198, and postoperative delirium9. The development of drugs with the potential to treat brain disorders has been frustratingly difficult because of the challenge of intentionally breaching the BBB to deliver bioactive molecules to targets in the brain10. For these reasons, methods to study BBB function in vitro are of paramount importance to the understanding and treatment of diseases of the CNS.
The basic methods for measuring barrier function in vitro involve the establishment of a monolayer or co-culture on a semi-permeable membrane and measuring the resistance imparted by cells to either small molecule diffusion or small electrical currents11,12,13,14. While the emergence of microphysiological systems (MPS) has produced an abundance of choices for modeling the BBB in 3D15,16, the diversity of system geometries makes it difficult to compare permeability measurements between MPS or to established literature values. The establishment of trusted baseline values is particularly important in BBB research where, because of the extensive barrier regulation by brain vascular endothelial cells, in vitro permeability values are highly scrutinized12,17,18. For these reasons, permeability measurements across monolayers established on 2D membranes will remain a staple in BBB studies for years to come. This holds true for other tissue barriers, including epithelial barriers, where absolute values of baseline permeability are used to validate and compare in vitro models19,20,21.
With the goal of establishing a valuable new tool for the BBB research community, we have introduced22 and advanced23,24,25,26 the microdevice featuring a silicon membrane (&#181;SiM) platform for use in barrier tissue modeling over the last 5 years. The enabling feature of the platform is an ultrathin (&#60;100 nm thick) membrane with hundreds of millions of nanopores27,28 or a mix of nanopores and micropores29. The free-standing membrane chips are produced on a 300 &#181;m silicon &#39;chip&#39; that stabilizes the ultrathin structures30 and allows them to be handled by tweezers for device assembly. Because of their ultrathin nature, the membranes have a permeability that is two orders of magnitude higher than that of conventional track-etched membranes used in commercial membrane culture devices31,32. In practice, this means that the membrane&#39;s hindrance to the diffusion of molecules smaller than the nanopores (&#60;60 nm) is negligible33. Thus, for cellular barriers, only the cells and the matrices they deposit will determine the rate of small molecule transport from the apical to basal compartments that are separated by the membrane34. The device design and the ultrathin nature of the membranes also provide many advantages for optical microscopy. These include 1) the ability to follow live cultures using phase contrast or bright field imaging, 2) the ability to fluorescently stain and image in situ without the need to extract and transfer the membrane to a cover glass, and 3) the fact that the membranes are thinner than confocal &#39;slices&#39; so that direct co-cultures have a more natural spacing between cell types than can be achieved with 6-10 &#181;m-thick track-etched membranes.
Most recently, we advanced the platform to a modular format to facilitate rapid assembly34 and customization35,36. We leveraged the modular format to distribute device components between our bioengineering and collaborating brain barrier laboratories. We then jointly developed protocols for device assembly, monoculture and co-culture, immunofluorescent staining, and small molecule permeability and showed that these methods were reproducible between labs. Using these protocols, we also showed that the modular platform supports a validated BBB developed using the extended endothelial culture method (EECM) for creating brain microvascular-like endothelial cells (BMEC) from human induced pluripotent stem cells (hiPSCs)37. The purpose of the current report is to review these methods in greater detail and, with the aid of the accompanying video, facilitate&#160;broader adoption of the platform in the BBB community.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD31 Polyclonal antibody</td><td>Thermo Fisher Scientific</td><td>PA5-32321</td><td></td></tr><tr><td>Claudin-5 mouse IgG antibody, 4C3C2</td><td>Thermo Fisher Scientific</td><td>35-2500</td><td></td></tr><tr><td>Goat &amp;alpha;-Mouse IgG Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A11001</td><td></td></tr><tr><td>Goat &amp;alpha;-Rabbit IgG Alexa Fluor 568</td><td>Thermo Fisher Scientific</td><td>A11011</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>NG2, mouse IgG2a, 9.2.27</td><td>Millipore</td><td>&amp;nbsp;MAB2029</td><td></td></tr><tr><td>Occludin mouse IgG antibody, OC-3F10</td><td>Thermo Fisher Scientific</td><td>33-1500</td><td></td></tr><tr><td>PDGFR&amp;beta;, rabbit IgG antibody, 28E1</td><td>Cell Signaling Technology</td><td>3169</td><td></td></tr><tr><td>VE-cadherin mouse IgG2B Clone # 123413</td><td>R&amp;amp;D Systems</td><td>MAB9381</td><td></td></tr><tr><td>ZO-1 rabbit IgG antibody, polyclonal</td><td>Thermo Fisher Scientific</td><td>40-2200</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals, peptides, and recombinant proteins&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100% Methanol</td><td>VWR</td><td>101443-718</td><td>Can be substituted with similar products</td></tr><tr><td>16% Paraformaldehyde</td><td>Thermo Fisher Scientific</td><td>28908</td><td>Can be substituted with similar products</td></tr><tr><td>Accutase</td><td>Thermo Fisher Scientific</td><td>A1110501</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td></td></tr><tr><td>B27 supplement</td><td>Thermo Fischer Scientific</td><td>17504044</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>Collagen IV from human placenta</td><td>Sigma-Aldrich</td><td>C5533</td><td></td></tr><tr><td>Collagen, Type I solution from rat tail</td><td>Sigma-Aldrich</td><td>C3867-1VL</td><td>Can be substituted with similar products</td></tr><tr><td>DMEM/Ham&amp;rsquo;s F12</td><td>Thermo Fisher Scientific</td><td>11320033</td><td></td></tr><tr><td>EGM-2 Endothelial Cell Growth Medium-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Essential 6 medium</td><td>Thermo Fisher Scientific</td><td>A1516401</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Peak Serum</td><td>PS-FB1</td><td></td></tr><tr><td>Fibronectin from bovine plasma</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Fibronectin human protein, plasma</td><td>ThermoFisher Scientific</td><td>33016015</td><td>Can be substituted with similar products</td></tr><tr><td>hESFM</td><td>Thermo Fisher Scientific</td><td>11111044</td><td></td></tr><tr><td>Lucifer Yellow CH dilithium salt</td><td>Sigma-Aldrich</td><td>861502</td><td></td></tr><tr><td>Normal goat serum</td><td>Thermo Fisher Scientific</td><td>500627</td><td>Can be substituted with similar products</td></tr><tr><td>PBS without calcium, magnesium</td><td>Cytiva</td><td>SH30256.01</td><td>Can be substituted with similar products</td></tr><tr><td>Pericyte Medium</td><td>ScienCell</td><td>1201</td><td>Use complete kit (includes supplements)</td></tr><tr><td>Poly-L-Lysine, 10 mg/mL</td><td>ScienCell</td><td>0413</td><td></td></tr><tr><td>Triton X-100</td><td>JT Baker</td><td>X198-05</td><td>Can be substituted with similar products</td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>Invitrogen&amp;trade;</td><td>10977015</td><td>Can be substituted with similar products</td></tr><tr><td>&lt;strong&gt;Experimental models: Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Blood-brain barrier hCMEC/D3 cell line</td><td>Sigma-Aldrich</td><td>SCC066</td><td></td></tr><tr><td>Human brain vascular pericytes</td><td>ScienCell</td><td>1200</td><td></td></tr><tr><td>iPS(IMR90)-4 human induced pluripotent stem cells</td><td>WiCell</td><td>RRID:CVCL_C437</td><td></td></tr><tr><td>&lt;strong&gt;Glassware and Plasticware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>150 mm TC-treated Cell Culture Dish with 20 mm Grid</td><td>Falcon</td><td>353025</td><td></td></tr><tr><td>Clamps</td><td>VWR</td><td>MFLX06832-10</td><td></td></tr><tr><td>Corning tissue culture plates (6-well)</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Flat 96-well non-binding microplates</td><td>Greiner Bio-One</td><td>655900</td><td></td></tr><tr><td>Microscope Slide Device Holder</td><td>&amp;nbsp;SiMPore</td><td>USIM-SB</td><td>Dimensions compatible with micropscope slide holders</td></tr><tr><td>Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fischer Scientific</td><td>339651</td><td></td></tr><tr><td>Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fischer Scientific</td><td>339653</td><td>Tube caps can be filled with water for cell culture</td></tr><tr><td>P20-200 pipette tips</td><td>VWR</td><td>76322-516</td><td>Alternate brands may not seal as effectively into ports</td></tr><tr><td>Transwell filters (12 well, 12 mm, 0.4 &amp;mu;m pore size, PC)</td><td>CoStar</td><td>3401</td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X Objective (For Andor)</td><td>Nikon Instruments Inc.</td><td>MRD00105</td><td>Air; WD: 4 mm; NA 0.45</td></tr><tr><td>10X Objective (For Nikon)</td><td>Nikon Instruments Inc.</td><td>MRP40102</td><td>Air; WD: 6.2 mm; NA 0.25</td></tr><tr><td>40X Objective (LWD) (For Andor)</td><td>Nikon Instruments Inc.</td><td>MRD77410</td><td>Water immersion; WD: 590-610 mm; NA 1.15</td></tr><tr><td>Andor Spinning Disc Confocal microscope</td><td>Oxford Instruments, Andor</td><td></td><td></td></tr><tr><td>Nikon Eclipse Ts2 Phase Contrast Microscope</td><td>Nikon Instruments Inc.</td><td></td><td>Can be substituted with similar products</td></tr><tr><td>TECAN Infinite M200</td><td>TECAN</td><td></td><td>Can be substituted with similar products</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advanced PAP Pen</td><td>Millipore Sigma</td><td>Z672548</td><td>2 mm tip is recommended</td></tr><tr><td>Assembly kit with fixtures</td><td>SiMPore</td><td>USIM-JIGSET</td><td>Including fixure A1, A2, B1, and B2</td></tr><tr><td>Camera Adapter for Microscopes</td><td>AmScope</td><td>CA-CAN-SLR Canon SLR / D-SLR</td><td>&amp;Phi;23.2 - &amp;Phi;30 adapter fits Nikon Eclipse Ts2</td></tr><tr><td>Canon EOS Rebel SL3 DSLR Camera</td><td>Canon</td><td>EOS 250D, BH #CAEDRSL3B, MFR #3453C001</td><td>Can be substituted with similar products</td></tr><tr><td>Cell strainer, 40 &amp;micro;m</td><td>Millipore Sigma (Corning)</td><td>CLS431750</td><td></td></tr><tr><td>Component 1</td><td>SiMPore</td><td>USIM-C1</td><td></td></tr><tr><td>Component 2</td><td>SiMPore</td><td>USIM-C2</td><td></td></tr><tr><td>Membrane chips</td><td>SiMPore</td><td>NPSN100-1L</td><td>Other chips formats are available</td></tr><tr><td>SMD handling tweezer double angle</td><td>Techni-Tool</td><td>758TW003</td><td>&quot;Chip Tweezers&quot;</td></tr><tr><td>Stainless steel precision type GG tweezer</td><td>Techni-Tool</td><td>758TW534</td><td>&quot;Straight Tweezers&quot;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fiji</td><td>ImageJ</td><td></td><td>For image processing</td></tr><tr><td>Fusion</td><td>Oxford Instruments, Andor</td><td></td><td>Instructions for version 2.3.0.44</td></tr><tr><td>i-control</td><td>TECAN</td><td></td><td>Instructions for version 2.0</td></tr><tr><td>Imaris</td><td>Oxford Instruments</td><td></td><td>For image processing. Instructions for version 9.9.0</td></tr></tbody></table>

use of the microsim (&#181;sim) barrier tissue platform for modeling the blood-brain barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

The assembly of a trench-down device is shown in Figure 1. The fixtures guide the assembly of the components and the membrane chip. Component 1 is primarily acrylic with a PSA surface for bonding to the chip and an opening to the bottom chamber and ports for pipet access to the bottom chamber. Component 2 is the channel layer and contains a non-adhesive, PSA-free &#34;triangle&#34; in the top right for gripping. Trench-down devices provide a flat cell culture growth area in the top chamber, whereas trench-up devices have a flat surface for cell culture in the bottom chamber.
We performed endothelial monocultures and co-cultures of hCMEC/D3 and HBVPs and of hiPSC IMR90-4-derived EECM-BMEC-like cells and BPLCs and acquired phase images using a Nikon Eclipse Ts2 phase contrast microscope and 10x objective (Figure 2). Optimal seeding densities for primary cell culture are shown (images taken 1 day after seeding), as well as under-seeded HBVPs (Figure 2A). Final coculture and monoculture phase images (6 days endothelial cell culture) can be difficult to distinguish (Figure 2C), and confirmation of successful primary cell co-culture may require immunofluorescence staining (see protocol section 3). Low, high, and optimal hiPSC-derived BPLC seeding densities are also illustrated (Figure 2B). hiPSC-derived co-culture is clearer to distinguish in phase contrast imaging compared to primary co-cultures (Figure 2D). Low BPLC seeding results in poor pericyte coverage and pericyte clumping, whereas overseeding results in the pericyte layer peeling off the membrane. Further, exchanging the medium in the bottom chamber too quickly may result in pericyte loss, as these cells are very sensitive to shear. Optimal coverage by pericytes is ~90% for a BBB model, with no gaps in the endothelial layer.
Representative images of immunostained hiPSC-derived co-culture are illustrated in Figure 3 (6 days endothelial cell culture). IMR90-4-derived BPLCs were stained for the pericyte marker PDGFR&#946;, and IMR90-4-derived EECM-BMEC-like cells were stained for the adherens junction marker VE-cadherin. Hoechst was used to stain the nuclei. Images were acquired on a Spinning Disc confocal microscope using a 40x LWD objective with 0.2 &#181;m slices and processed with Imaris. Both cell layers can be visualized even though the thin nanomembrane is not seen.
We performed the sampling-based small molecule permeability assay using the same experimental conditions in two physically distant laboratories at the University of Bern, Switzerland, and the University of Rochester, NY, USA to demonstrate interlaboratory reproducibility of results (Figure 4)34. hiPSC IMR90-4-derived EECM-BMEC-like cells were cultured in the &#181;SiM device for 2, 4, or 6 days and on transwell filters for 6 days. The assay was performed using 150 &#181;g/mL Lucifer Yellow (457 Da) in both laboratories. High variability in the permeability of the endothelial cells cultured for 2 days in the device indicates that 2 days of culture was insufficient for barrier maturation. There were no significant differences in permeability between the labs upon barrier maturation-from 4 days on. We also showed that the permeability of the endothelial cells cultured in &#181;SiM and transwell filters for 6 days matched those previously published40.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65258/65258fig01.jpg" /> 
Figure 1: Steps of &#181;SiM assembly. (A) Prepare the chip by placing it on fixture A1. Place the chip trench up for a final trench-down device. Place the chip trench down for a final trench-up device. (B) Bond component 1 to the chip by removing the protective masks from component 1 and placing it face-down into FA1. Bond by applying pressure with fixture A2. (C) Bond component 2 and component 1 by removing component 2 from its sheet and peeling off the top protective layers. Place the channel up into fixture B1 and place component 1 on top of component 2 face-up. Bond by applying pressure with fixture B2. Abbreviations: FAn = fixture An; FBn = fixture Bn. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65258/65258fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65258/65258fig02.jpg" /> 
Figure 2: Schematic of coculture and representative phase contrast images of cell culture in devices. (A) Location of pericyte and endothelial cell seeding. (B) Side view schematic of cell locations across the trench of the membrane chip. (C) Representative images of low and optimal seeding densities for primary&#160;HBVP and hCMEC/D3 brain endothelial cell line. Images were acquired 1 day after seeding (HBVP) and 2 h after seeding (hCMEC/D3). (D) Representative images of low, high, and optimal seeding densities for hiPSC-derived brain pericyte-like cells. Images were acquired 1 day after seeding. (E) Representative images of final HBVP and hCMEC/D3 co-culture and hCMEC/D3 monoculture. Images were acquired 8 days following HBVP seeding and 7 days following hCMEC/D3 seeding. (F) Representative images of failed and successful BPLC and EECM-BMEC-like cell co-culture and EECM-BMEC-like cell monoculture. Images were acquired 7 days following BPLC seeding and 6 days following EECM-BMEC seeding. Underseeded BPLC cultures fail to have sufficient coverage, whereas overseeding BPLC cultures will grow overconfluent and start clumping/receding. Scale bars = 100 &#181;m (C-F). Abbreviations: HBVPs = human brain vascular pericytes; hiPSC = human induced pluripotent stem cell; BPLCs = hiPSC-derived brain pericyte-like cells. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65258/65258fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65258/65258fig03.jpg" /> 
Figure 3: Representative images of immuno-stained hiPSC-derived cell co-culture in devices. Cells were stained for endothelial cell marker VE-cadherin (green), pericyte marker PDGFR&#946; (red), and nuclear stain (blue). Two layers of cells can be seen in close proximity, separated only by a thin silicon-nitride nanomembrane (white arrow marks membrane location on left image). Scale bar = 50 &#181;m. Abbreviations: hiPSC = human induced pluripotent stem cell; PDGFR&#946; = platelet-derived growth factor receptor beta. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65258/65258fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65258/65258fig04.jpg" /> 
Figure 4: Sampling-based small molecule permeability assay. (A) Schematic of the experimental workflow. (B) Demonstration of the interlaboratory reproducibility between two physically distant laboratories at the University of Bern (UniBe), Switzerland, and the University of Rochester (UR), NY, USA: hiPSC-derived endothelial cells were cultured in the &#181;SiM device for 2, 4, or 6 days and in transwell filters for 6 days. Permeability assay was performed using 150 &#181;g/mL Lucifer Yellow (457 Da). The red bar indicates the previously published sodium fluorescein (376 Da) permeability data of the same hiPSC-derived endothelial cells cultured for 6 days in transwell filters40. N = 4-16 per group. Two-way ANOVA with Tukey&#39;s post hoc test was used, and comparisons were only displayed for relevant p &#60; 0.05. (C) Demonstration of cytokine response using hiPSC-derived EECM-BMEC-like cells cultured in the &#181;SiM for 2 days; 0.1 ng/mL TNF&#945; + 2 IU/mL IFN&#947;) or media control (non-stimulated, NS) was added to the top chamber for 20 hr prior to permeability assay using 150 &#181;g/mL Lucifer Yellow. N = 3 per group. Student&#8217;s t-test, p &#60; 0.05. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65258/65258fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Top Chamber Seeding Surface Area</td>
			<td>Top Well Volume</td>
			<td>Bottom Chamber Seeding Surface Area</td>
			<td>Bottom Channel Volume</td>
		</tr>
		<tr>
			<td>~37 mm2</td>
			<td>100 &#181;L (can hold &#8805;115 &#181;L)</td>
			<td>~42 mm2</td>
			<td>10 &#181;L (pipet 20 &#181;L to avoid bubbles)</td>
		</tr>
	</tbody>
</table>
Table 1: Critical &#181;SiM surface area and volumes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Target</td>
			<td>Fixative</td>
			<td>Blocking Solution</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>VE-cadherin</td>
			<td>4% PFA or 100% MeOH</td>
			<td>5% GS + 0.4% Tx-100 or 10% GS + 0.3% Triton X-100</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>4% PFA or 100% MeOH</td>
			<td>5% GS + 0.3-0.4% Triton X-100</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Claudin-5</td>
			<td>100% MeOH</td>
			<td>5-10% GS + 0.3% Triton X-100</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>ZO-1</td>
			<td>100% MeOH</td>
			<td>5-10% GS + 0.3% Triton X-100</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Occludin</td>
			<td>100% MeOH</td>
			<td>5-10% GS + 0.3% Triton X-100</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>PDGFR&#946;</td>
			<td>4% PFA</td>
			<td>5% GS + 0.4% Triton X-100</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>NG2</td>
			<td>4% PFA</td>
			<td>5% GS + 0.4% Triton X-100</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Goat &#945;-Mouse IgG Alexa Fluor 488</td>
			<td></td>
			<td></td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Goat &#945;-Mouse IgG Alexa Fluor 568</td>
			<td></td>
			<td></td>
			<td>1:200</td>
		</tr>
	</tbody>
</table>
Table 2: Antibodies and staining methods validated for co-culture immunocytochemistry in &#181;SiM devices. Abbreviations: PFA = paraformaldehyde; MeOH = methanol; GS = goat serum.
Supplemental File 1: Template for calculation of permeability value. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65258/65258_SupplementalFile_SamplingCalcTemplate_Updated.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing the Use of Tissue Chip Technology for Studying Human Tissues at JoVE.com

Author Spotlight: Advancing the Use of Tissue Chip Technology for Studying Human Tissues 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

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2.6K Views.  University of Rochester. This report provides protocols for assembly, cell culture, and assays on the µSiM platform for the construction of blood-brain barrier models.

Video: Author Spotlight: Advancing the Use of Tissue Chip Technology for Studying Human Tissues 

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 ...

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Neuroscience

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 ...

Medicine

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

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

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

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 ...

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

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.

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.

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

Use of the MicroSiM (µSiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

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

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

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

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

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

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

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

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

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

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