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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</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 &#945;-Mouse IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat &#945;-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>&#160;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&#946;, 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&#38;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>Chemicals, peptides, and recombinant proteins</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&#38;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&#8217;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&#8482;</td>
			<td>10977015</td>
			<td>Can be substituted with similar products</td>
		</tr>
		<tr>
			<td>Experimental models: Cell lines</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>Glassware and Plasticware</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>&#160;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 &#956;m pore size, PC)</td>
			<td>CoStar</td>
			<td>3401</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</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>Other</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>&#934;23.2 - &#934;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 &#181;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>&#34;Chip Tweezers&#34;</td>
		</tr>
		<tr>
			<td>Stainless steel precision type GG tweezer</td>
			<td>Techni-Tool</td>
			<td>758TW534</td>
			<td>&#34;Straight Tweezers&#34;</td>
		</tr>
		<tr>
			<td>Software</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.

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

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

We are developing a tissue GI model of the blood-brain barrier to understand the impact of sepsis on the brain and the contribution of blood-borne factors. Using human-induced pluripotent stem cells. We plan on evaluating the contribution of genetic factors to the susceptibility of certain patient populations.Current experimental challenges within the tissue chip community are the reproducibility of results and the adoption of techniques by outside labs. Unique tissue chip designs, small volumes within tissue chips, and lack of standard functional assays all contribute to these challenges and often limit users to fluorescent based assays as their primary output. Micro simm devices offer modularity for versatile in vitro modeling, a glass light surface ideal for high resolution lifestyle imaging and ultra syn 100 nanometer, highly produced nano membrane, ensuring seamless soluble factor crosstalk and precise permeability assays.With the micro simm, we have seen that specific cell types tend to produce more of the matrix proteins that provide a supporting scaffold around brain blood vessels. Now we can ask whether inflammation causes these same cells to strengthen or break down their matrices, thereby altering barrier integrity. In the future, we will add another compartment to the model with green cells such as astrocytes and microglia to study how those cells interact with the blood-brain barrier in cases of systemic inflammation like sepsis.We also plan to investigate additional causes of systemic inflammation like postoperative delirium, superimposed on dementia.

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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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

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

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...

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