We are grateful to Annika Engbers and Frank Kurth for their excellent technical support and Dr. Markus Sch&#228;fer (nanoAnalytics GmbH) for helpful discussions regarding TEER measurements. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), SFB1009 project A03 to HW and LK, CRC TR128, projects A08; Z1 and B01 to LK and HW, and the Interdisciplinary Center for Clinical Research (Medical Faculty of M&#252;nster) grant number Kl2/2015/14 to LK.

For all experiments, mice were bred and maintained under specific pathogen-free conditions in the central animal facility at the University of M&#252;nster, according to German guidelines for animal care. All experiments were performed according to the guidelines of the animal experimental ethics committee and approved by the local authorities of North Rhine-Westphalia, Germany (LANUV, AZ 84-02.05.20.12.217).
1. MBMEC Isolation and Culture
NOTE: Isolate MBMECs as previously described in detail14 with the following modifications:
<ol>
	<li>Sacrifice 20 adult C57BL/6 mice (6-8 weeks old) by CO2 inhalation and confirm their death by ascertaining cardiac and respiratory arrest.</li>
	<li>Rinse the mouse with 70% ethanol and decapitate it using scissors; remove the scalp using forceps and scissors. Make incisions on the left and right side of the skull, starting from the foramen magnum. Lift the skull from its caudal side and take out the brain with forceps.</li>
	<li>Under a laminar flow hood, use sterile forceps to place the brain in a Petri dish and remove the brain stem, cerebellum, and thalamus, keeping only the cortex. 
	NOTE: Use of a microscope is not necessary for any of these steps.</li>
	<li>Place the cortex onto a piece of sterile Western blotting paper and roll it gently with forceps until meninges are no longer visible.</li>
	<li>After mechanical and enzymatic digestion (following the protocol14), collect endothelial cells from the density gradient by using a long, sterile needle and a 5 ml plunger. Endothelial cells appear as a murky layer above the red ring with erythrocytes. Transfer 20-25 ml of this layer into a new 50 ml centrifugation tube; top up the tube with DMEM.</li>
	<li>After two rounds of washing14, resuspend the cells in 6 ml of MBMEC medium containing Puromycin (4 &#181;g/ml) and seed them onto six coated wells of a 24-well plate; leave them in a tissue culture incubator at 37 &#176;C and 5% CO2 (referred to as &#39;incubator&#39; from here on) for three days. 
	NOTE: For details of MBMEC coating solution, see Materials Table.</li>
	<li>Change the medium by adding 1 ml to each well of fresh MBMEC medium without Puromycin; put the plate back at 37 &#176;C and 5% CO2 for two more days.</li>
</ol>
2. Harvesting MBMECs
<ol>
	<li>On day five after MBMEC isolation, pre-coat permeable inserts with MBMEC coating solution: add 80 &#181;l of solution per insert and leave them at 37 &#176;C and 5% CO2 for 3 hr.</li>
	<li>Carefully pipette out the coating solution and let the inserts air-dry for 30 min. Add 2 ml of room temperature (RT) PBS to each well of the plate, using MBMECs to wash the medium; repeat this step. Add 0.05% Trypsin-EDTA (300 &#181;l per well) and leave the plate in the incubator for 5-10 min.</li>
	<li>Add 2 ml of MBMEC medium to each well to stop trypsinization. Transfer the collected cells to a 15 ml centrifugation tube and centrifuge them at 700 x g for 8 min at 4 &#176;C. Discard the supernatant and resuspend the cells in 1 ml of MBMEC medium without Puromycin.</li>
	<li>Count the cells; mix 10 &#181;l of cell suspension with 90 &#181;l of 0.04% Trypan Blue (10x dilution of cells). Pipette 10 &#181;l of this mix between the glass cover and the cell counting chamber (hemocytometer). Count Trypan Blue-free cells in all four quadrants of the chamber (N) and determine the average number of counted cells (N&#39;) as follows: N&#39; = N/4.</li>
	<li>Calculate the cell concentration (in 106 cells/ml) using the following formula: C = N&#39; x 104 x 10, where 104 is given by the dimensions of the hemocytometer and 10 is the dilution factor from step 2.4.</li>
	<li>Seed 2 x 104 cells in a volume of 260 &#181;l per insert. Add 810 &#181;l of MBMEC medium to the lower compartment of each well. Place the plate with inserts in the incubator until ready to proceed with section 4. 
	NOTE: The volumes for the upper and lower compartment are insert-specific and do not work for all 24-well formats.</li>
</ol>
3. CD4+ T Cell Isolation and Stimulation
<ol>
	<li>CD4+ T Cell Isolation
	<ol>
		<li>Sacrifice one adult mouse (6-8 weeks old) by CO2 inhalation and confirm its death by ascertaining cardiac and respiratory arrest.</li>
		<li>Place the mouse on its back on a clean dissection board and rinse it with 70% ethanol; use sterile scissors and forceps to open the peritoneum and remove the spleen and lymph nodes (inguinal, axillary, brachial and cervical); finally, transfer the tissue into a 15 ml centrifugation tube containing 5 ml of PBS on ice.</li>
		<li>Under the laminar flow hood, transfer the tissue by decanting PBS with the tissue into a 50 ml centrifugation tube with a 70 &#181;m cell strainer on top; homogenize the tissue by pressing it with a 1 ml plunger through the strainer.</li>
		<li>Add 30 ml of FACS buffer and centrifuge it at 500 x g for 5 min at 4 &#176;C. Discard supernatant and resuspend the cells in 10 ml of FACS buffer. Filter the suspension through a 40 &#181;m cell strainer, add another 20 ml of FACS buffer to the tube.</li>
		<li>Centrifuge it at 500 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cells in 500 &#181;l of FACS buffer.</li>
		<li>Add 20 &#181;l of mouse CD4 magnetic microbeads, mix well and incubate for 15 min at 4 &#176;C. Add 25 ml of FACS buffer and centrifuge it at 500 x g for 5 min at 4 &#176;C.</li>
		<li>In the meantime, place an LS separation column into the magnet and rinse it with 3 ml of FACS buffer. Discard the supernatant and resuspend the cells in 3 ml of FACS buffer.</li>
		<li>Add cells to the column. Wash the column with 3 ml of FACS buffer three times. Remove the column from the magnet and place it onto a new 15 ml centrifugation tube. Add 5 ml of FACS buffer, flush out the labelled cells with the column plunger and centrifuge it at 500 x g for 5 min 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the cells in 3 ml of T cell medium. Count the cells as described in 2.4 and seed 1 x 105 cells in 100 &#181;l of medium per well.</li>
	</ol>
	</li>
	<li>CD4+ T Cell Stimulation
	<ol>
		<li>Polyclonal CD4+ T cell stimulation
		<ol>
			<li>Pre-coat a round-bottom 96-well plate with purified anti-mouse CD3 antibody (clone 145-2C11) in PBS, at a desired final concentration. For example, to pre-coat the full plate with &#945;-CD3 at 1 &#181;g/ ml, mix 10 &#181;l of the antibody (stock concentration = 0.5 mg/ml) with 5 ml of PBS, vortex and add 50 &#181;l of the mix to each well with a multichannel pipette.</li>
			<li>Leave the plate in the incubator for 3 hr. After isolating the T cells, wash the pre-coated plate twice with PBS. Add purified anti-CD28 antibody (clone 37.51) to isolated T cells at a desired final concentration (e.g., at 1 &#181;g/ml); mix well. Seed the T cells and leave them in the incubator for two to three days.</li>
		</ol>
		</li>
		<li>Antigen-specific CD4+ T cell stimulation with dendritic cells (DCs) 
		NOTE: If DCs are used as antigen-presenting cells (APCs), follow the Protocol for T cell isolation, with these exceptions:
		<ol>
			<li>Before homogenizing the spleen, inject it with 1 ml of Collagenase type IA in PBS at 0.5 mg/ml and transfer it to a 15 ml centrifugation tube.</li>
			<li>Incubate in the water bath at 37 &#176;C for 15 min. After washing with PBS, resuspend the pellet in FACS buffer and add 20 &#181;l of mouse CD11c magnetic microbeads, instead of CD4 microbeads.</li>
			<li>Use an MS separation column and the appropriate volumes: rinse the column with 1 ml of FACS buffer; resuspend cells in 1 ml of FACS buffer and wash the column with 1 ml of FACS buffer three times.</li>
			<li>Add antigen of choice to DCs (e.g., myelin oligodendrocyte glycoprotein (MOG) a.a. 35-55 at 20 &#181;g/ml), mix well and seed 5 x 104 cells in 100 &#181;l of T cell culture medium per 96-round-bottom-well.</li>
			<li>Add 1 x 105 T cells in 100 &#181;l of T cell culture medium per 96-round-bottom-well at the end, as described in the Protocol for T cell isolation.</li>
		</ol>
		</li>
		<li>Antigen-specific CD4+ T cell stimulation with B cells 
		NOTE: If B cells are used as APCs, follow the Protocol for T cell isolation, with these exceptions:
		<ol>
			<li>Use spleens from transgenic mice whose B cells are antigen-specific (e.g., IgHMOG (Th) mice, whose B cells specifically recognize MOG35-55).</li>
			<li>Use antigen-specific T cells (e.g., from TCRMOG (2D2) mice). Add 20 &#181;l of mouse CD19 magnetic microbeads instead of CD4 microbeads.</li>
			<li>Add the antigen of choice to the B cells (e.g., MOG35-55 at 20 &#181;g/ml), mix well and seed 5 x 104 cells in 100 &#181;l of B cell culture medium per 96-round-bottom-well.</li>
			<li>Add 1 x 105 T cells in 100 &#181;l of T cell culture medium per 96-round-bottom-well, as described in the Protocol for T cell isolation.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Setting Up and Performing TEER Measurement
<ol>
	<li>Place the 24-well module of the TEER instrument under the laminar flow hood. Remove the lids and place inserts with MBMECs in the instrument using forceps.</li>
	<li>Pipette 810 &#181;l of fresh medium to the lower compartment of the module wells: add it carefully between the insert and the wall of the module well.</li>
	<li>Close the lids and place the instrument in the incubator. Connect the instrument to its computer; turn on the instrument controller and open the software.</li>
	<li>Select &#39;new measurement&#39; in the pop-up window. Check &#39;show TEER&#39; and &#39;show Ccl&#39; boxes; then press &#39;start&#39;. After completing the first measurement, select &#39;check all wells&#39; in the &#39;results&#39; tab to see all TEER and Ccl values.</li>
	<li>Save the file: File&#62;Save as&#62;&#39;the name of your file&#39;. 
	NOTE: As TEER and Ccl are continuously measured in an automated fashion, monitor their values over a period of three to five days. Changing the medium is not necessary, unless cell viability is suboptimal, as visualized by a lack of increase in TEER.</li>
	<li>Choose the time point to co-culture MBMECs with T cells when Ccl is stable and lower than 1 &#181;F/cm2 and TEER has reached its maximum level.</li>
	<li>Carefully inspect absolute TEER and Ccl values and exclude the wells in which MBMECs have not developed confluent enough monolayers by unchecking such wells.</li>
	<li>Group the rest of the wells: right-click on a well and select &#39;add well to new average well&#39;. Name it in the pop-up window; do the same for all individual wells to be grouped.</li>
	<li>Check all average wells to confirm that all of them have the same initial conditions before the co-culture. If some have significantly different absolute TEER values or TEER slopes, or the standard errors are too large, redo the grouping. 
	NOTE: The optimal grouping of wells provides minimal variation in TEER values, both within and between experimental groups.</li>
	<li>In the &#39;experiment&#39; tab, press &#39;pause&#39;, disconnect the instrument and take it out of the incubator. Remove the lids under the laminar flow hood.</li>
	<li>Prepare pre-activated and/or na&#239;ve T cells, with or without specific cytokines, antibodies or other substances, as desired: For example: mix purified NA/LE rat anti-mouse IFN-&#947; antibody (clone XGM1.2) with prepared T cells, at 20 &#181;g/ml per well of the TEER instrument. 
	NOTE: If substances such as granzyme B inhibitor are used, they are added to T cell culture at the beginning of T cell stimulation: e.g., Granzyme B Inhibitor II (Calbiochem) is mixed with isolated T cells at a final concentration of 10 &#181;M in DMSO. See Materials and Equipment for more details.</li>
	<li>Remove some of the medium from the insert (the upper well compartment): e.g., carefully pipette out 150 &#181;l (removing all the medium should be avoided as it could disturb the MBMEC monolayer).</li>
	<li>Add T cells to MBMECs by carefully pipetting 150 &#181;l of medium containing 2 x 105 cells/insert. 
	NOTE: When harvesting pre-activated T cells, count only blasting, Trypan Blue-free cells.</li>
	<li>Close the lids and place the instrument back to the incubator. Reconnect the instrument and press &#39;resume measurement&#39;. After 24 hr, press &#39;stop&#39; in the &#39;experiment&#39; tab and save the file (File&#62;Save).</li>
</ol>
5. Data Export and Statistical Analysis
<ol>
	<li>Export the results by choosing File&#62;Export.</li>
	<li>In the &#34;settings&#34; tab of the pop-up window, select &#34;.dot&#34; in the &#34;decimal separator&#34; option and &#34;tabulator&#34; in the &#34;field delimiter&#34; option.</li>
	<li>In the &#34;export results&#34; tab, name the file, check all the wells to be exported, and check the &#34;TEER&#34; and &#34;Ccl&#34; boxes in the &#34;choose data&#34; option.</li>
	<li>Press &#34;export data,&#34; open the exported file, and copy the data to a spreadsheet.</li>
	<li>Normalize the exported data (sorted by each replicate, with TEER and Ccl values given for each run (measurement)): Set TEER and Ccl values of the last time point before the co-culture to 100% and change accordingly the values for all other runs, relative to the &#39;100%&#39; run.</li>
	<li>Copy normalized data to a software of choice to generate a graph, using individual wells and displaying the standard error of the mean for each treatment group.</li>
	<li>Perform Two-way ANOVA statistical test with a Bonferroni correction for multiple comparisons, using the statistical software of choice.</li>
</ol>

I. Kuzmanov, A. M. Herrmann, S.G. Meuth, H. Wiendl and L. Klotz have nothing to disclose. H.-J. Galla is a scientific advisor of nanoAnalytics GmbH, the manufacturer of the automated cell monitor.

Several steps of the described protocol are essential for a successful experiment. During the initial MBMEC isolation and culture, it is crucial that work is performed under sterile conditions as much as possible, to prevent the contamination of the cell culture with fungal spores or bacteria. In order to obtain a pure culture of ECs, it is recommended to use a medium containing Puromycin for the first three days, which enables survival of ECs, but not other cells types (especially pericytes)41,42. Another cri...

The blood-brain barrier separates the systemic circulation from the central nervous system (CNS)1-3. It provides a physical barrier that inhibits the free movement of cells and the diffusion of water-soluble molecules and protects the brain from pathogens and potentially harmful substances. In addition to its barrier function, the BBB enables the delivery of oxygen and nutrients to the brain parenchyma, which ensures proper functioning of the neuronal tissue. Functional properties of the BBB are highly regulated by its cellular and acellular components, with highly specialized ECs being its main structural element. ECs of the BBB are characterized by the presence of tight junction (TJ) complexes, the lack of fenestrations, extremely low pinocytic activity, and permanently active transport mechanisms. Other components of the BBB the EC basement membrane, pericytes embedding the endothelium, astrocytic end feet and = associated parenchymal basement membrane also contribute to the development, maintenance and function of the BBB2,4-6 and, together with neurons and microglia, form the neurovascular unit (NVU), which enables proper functioning of the CNS7-9.
In a variety of neurological diseases, such as neurodegenerative, inflammatory or infectious diseases, the function of the BBB is compromised2,5,10. Dysregulation of TJ complexes and molecular transport mechanisms leads to increased BBB permeability, leukocyte extravasation, inflammation and neuronal damage. In order to study BBB properties under such pathophysiological conditions, various in vitro BBB models have been established9,11,12. Together they have provided valuable insights into the changes of barrier integrity, permeability as well as transport mechanisms. These models employ endothelial cells of human, mouse, rat, porcine or bovine origin13-18; primary endothelial cells or cell lines are cultured either as a monoculture or together with pericytes and/or astrocytes in order to mimic more closely the BBB in vivo19-25. In recent years, measurement of transendothelial electrical resistance (TEER) has become a widely accepted tool to assess endothelial barrier properties26,27.
TEER reflects the impedance to the ion flux across the cell monolayer and its decrease provides a sensitive measure of compromised endothelial barrier integrity and hence increased permeability. Various TEER measurement systems have been developed, including Epithelial Voltohmmeter (EVOM), Electric Cell-substrate Impedance Sensing (ECIS), and real-time cell analysis15,28-30. TEER reflects the resistance to the ion flux between adjacent ECs (paracellular route) and is directly proportional to the barrier integrity. In impedance spectroscopy27,31, complex total impedance (Z) is measured, which provides additional information about the barrier integrity by measuring Ccl. Ccl relates to the capacitive current through the cell membrane (transcellular route): the cell layer acts like a capacitor in the equivalent electric circuit, separating the charges on both sides of the membrane and is inversely proportional to the barrier integrity. When grown on permeable inserts, ECs adhere, proliferate and spread over the microporous membrane. This resists the background capacitive current of the insert (which itself acts like a capacitor)&#160;and leads to a decrease in the capacitance until it reaches its minimal level. This is followed by the establishment of TJ complexes that seal off the space between adjacent ECs. This restricts the ion flux through the paracellular route, and TEER increases until it reaches its plateau. Under inflammatory conditions, however, the endothelial barrier is compromised: TEER decreases as TJ complexes get disrupted and Ccl increases as the capacitive component of the insert rises again.
Our TEER measurement uses the automated cell monitoring32 system: it follows the principle of impedance spectroscopy and extends its previous applications. Here, we describe an in vitro BBB model that enables the study of the barrier properties, including the interaction of brain endothelium with immune cells; in particular activated T cells. Such pathophysiological conditions are observed in autoimmune diseases of the CNS, such as multiple sclerosis and its animal model experimental autoimmune encephalomyelitis33-37. Here, a crucial step is the transmigration of encephalitogenic, myelin-specific T cells across the BBB. This is followed by their reactivation in the perivascular space and entry into the brain parenchyma, where they recruit other immune cells and mediate inflammation and subsequent demyelination1,35,38. However, molecular mechanisms of the interaction between such T cells and endothelial cells, the main constituents of the BBB, are not well understood. Our protocol aims to fill this gap and give new insights into the consequences on endothelial cells (i.e., barrier integrity and permeability) upon their direct contact and complex interplay with activated T cells.
The protocol described here makes use of primary mouse brain microvascular endothelial cells, grown as a monolayer on permeable inserts with microporous membranes. Endothelial cells are co-cultured with CD4+ T cells, which can be pre-activated either polyclonally or in an antigen-specific fashion. Co-culture of MBMECs with pre-activated, but not na&#239;ve T cells induces a decrease in TEER and an increase in Ccl, which provides a quantitative measure of the MBMEC dysfunction and barrier disruption. The technique is non-invasive: it uses built-in instead of chopstick electrodes, which prevent major disturbance of the EC monolayer; it can be used to monitor barrier function without the use of cell markers. It makes continuous measurements in an automated fashion and enables an independent assessment of the two barrier parameters (TEER and Ccl) simultaneously over time. The method is also sensitive enough to distinguish between different levels of T cell activation and effects of such T cells on ECs.
It can be used in a wide range of functional assays: different cytokines and/or chemokines implicated in inflammatory processes can be added to the co-culture of MBMECs and T cells; blocking antibodies against cell adhesion molecules on either the EC or T-cell side can be used; and inhibitors of T cell activation markers or of their cytolytic properties can be added during the T-cell priming or their co-culture with ECs. The assay is also useful for T-cell transmigration assays, as it can serve as a quality control of the MBMEC monolayer integrity prior to the addition of T cells. All this makes this method a versatile and reliable tool to study the BBB in vitro, with a focus on the effect of activated T cells on EC monolayer integrity. This is of particular importance for understanding the mechanisms of the BBB disruption in the pathogenesis of autoimmune diseases, such as MS and its animal model EAE, where self-reactive, encephalitogenic T cells cross the BBB and cause inflammation and neuronal damage.

<table><tbody><tr><td>cellZscope</td><td>nanoAnalytics GmbH</td><td>www.nanoanalytics.com</td><td>including: 24-well Cell Module, Controller, PC with cellZscope software v2.2.2&amp;nbsp;</td></tr><tr><td>Ultracentrifuge</td><td>Thermo Scientific</td><td>www.thermoscientific.com</td><td>SORVALL RC 6+; rotor F21S-8x50y; for MBMEC isolation</td></tr><tr><td>flow cytometer</td><td>Beckman Coulter</td><td>www.beckmancoulter.com</td><td>for analysis of T cell transmigration</td></tr><tr><td>FlowJo7.6.5 software</td><td>Tree Star</td><td>www.flowjo.com</td><td>for analysis of T cell transmigration</td></tr><tr><td>Oak Ridge centrifuge tubes, PC</td><td>Thermo Fisher Scientific</td><td>3118-0050</td><td>50 ml; for MBMEC isolation</td></tr><tr><td>Transwell membrane inserts - pore size 0.4 &amp;micro;m</td><td>Corning</td><td>3470</td><td>for TEER measurement as the main readout</td></tr><tr><td>Transwell membrane inserts - pore size 3 &amp;micro;m</td><td>Corning</td><td>3472</td><td>for TEER measurement as the quality control prior to T-cell transmigration assay</td></tr><tr><td>24-well cell culture plate</td><td>Greiner</td><td>650 180</td><td>flat-bottom; for MBMEC culture</td></tr><tr><td>96-well cell culture plate</td><td>Costar</td><td>3526</td><td>round-bottom; for immune cell culture</td></tr><tr><td>QuadroMACS Separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td>for T cell and B cell isolation; supports MACS LS columns</td></tr><tr><td>OctoMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-109</td><td>for dendritic cell isolation; supports MACS MS columns</td></tr><tr><td>Neubauer counting chamber</td><td>Marienfeld</td><td>MF-0640010</td><td>for cell counting</td></tr><tr><td>Cell strainer, 70 &amp;micro;m</td><td>Corning</td><td>352350</td><td>for immune cell isolation</td></tr><tr><td>Cell strainer, 40 &amp;micro;m</td><td>Corning</td><td>352340</td><td>for immune cell isolation</td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td>for immune cell isolation</td></tr><tr><td>MACS LS separation columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>for T cell and B cell isolation</td></tr><tr><td>MACS MS separation columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td>for dendritic cell isolation</td></tr><tr><td>Mouse CD4 MicroBeads</td><td>Miltenyi Biotec</td><td>130-049-201</td><td>for CD4&lt;sup&gt;+&lt;/sup&gt; T cell isolation</td></tr><tr><td>Mouse CD19 MicroBeads</td><td>Miltenyi Biotec</td><td>130-052-201</td><td>for B cell isolation</td></tr><tr><td>Mouse CD11c MicroBeads</td><td>Miltenyi Biotec</td><td>130-052-001</td><td>for dendritic cell isolation</td></tr><tr><td>Collagen type IV from human placenta</td><td>Sigma</td><td>C5533</td><td>for MBMEC coating solution</td></tr><tr><td>Fibronectin from bovine plasma</td><td>Sigma</td><td>F1141-5MG</td><td>for MBMEC coating solution</td></tr><tr><td>Collagenase 2 (CSL2)</td><td>Worthington</td><td>LS004176</td><td>for MBMEC isolation</td></tr><tr><td>Collagenase/Dispase (C/D)</td><td>Roche</td><td>11097113001</td><td>for MBMEC isolation</td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25</td><td>for MBMEC isolation</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F7524</td><td>for MBMEC isolation</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Amresco</td><td>0332-100G</td><td>for MBMEC isolation</td></tr><tr><td>Percoll</td><td>Sigma</td><td>P1644-1L</td><td>for MBMEC isolation</td></tr><tr><td>DMEM (+ GlutaMAX)</td><td>Gibco</td><td>31966-021</td><td>for MBMEC isolation and MBMEC culture medium</td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma</td><td>P4333</td><td>for MBMEC isolation and MBMEC culture medium</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Sigma</td><td>D8537</td><td>for MBMEC and immune cell isolation</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td>for MBMEC culture medium</td></tr><tr><td>Human Basic Fibroblast Growth Factor (bFGF)</td><td>PeproTech</td><td>100-18B</td><td>for MBMEC culture medium</td></tr><tr><td>Puromycin</td><td>Sigma</td><td>P8833</td><td>for MBMEC culture medium; only for the first three days</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>for harvesting MBMECs</td></tr><tr><td>Collagenase Type IA</td><td>Sigma</td><td>C9891</td><td>for dendritic cell isolation</td></tr><tr><td>Trypan Blue solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250061</td><td>for cell counting</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td>for immune cell isolation</td></tr><tr><td>IMDM + 1% L-Glutamin</td><td>Gibco</td><td>21980-032</td><td>for T cell culture medium</td></tr><tr><td>X-VIVO 15</td><td>Lonza</td><td>BE04-418Q</td><td>protect from light; for B cell culture medium</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>31350-010</td><td>for B cell culture medium</td></tr><tr><td>L-Glutamine (100x Glutamax)</td><td>Gibco</td><td>35050-061</td><td>for B cell culture medium</td></tr><tr><td>mouse MOG&lt;sub&gt;35&amp;mdash;55 &lt;/sub&gt;peptide</td><td>Biotrend</td><td>BP0328</td><td>for antigen-specific T cell activation</td></tr><tr><td>purified anti-mouse CD3 Ab</td><td>BioLegend</td><td>100302</td><td>clone 145-2C11; for polyclonal T cell activation</td></tr><tr><td>purified NA/LE anti-mouse CD28 Ab</td><td>BD Pharmingen</td><td>553294</td><td>clone 37.51; for polyclonal T cell activation</td></tr><tr><td>Recombinant Murine IFN-&amp;gamma;</td><td>PeproTech</td><td>315-05</td><td>for T-cell transmigration assays</td></tr><tr><td>Recombinant Murine TNF-&amp;alpha;</td><td>PeproTech</td><td>315-01A</td><td>for T-cell transmigration assays</td></tr><tr><td>NA/LE purified anti-mouse IFN-&amp;gamma; antibody</td><td>BD Biosciences</td><td>554408</td><td>clone XMG1.2; recommended final concentration: 20 &amp;micro;g/ml</td></tr><tr><td>Granzyme B Inhibitor II</td><td>Calbiochem</td><td>368055</td><td>recommended final concentration: 10 &amp;micro;M</td></tr><tr><td>PE anti-mouse CD4 antibody</td><td>Biolegend</td><td>116005</td><td>clone RM4-4; for analysis of T cell transmigration</td></tr></tbody></table>

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.

Figure 1 provides a general overview of the in vitro BBB model used to study the interaction between T cells and endothelial cells. The experiment consists of three major steps. The first step is the isolation of primary MBMECs from brain cortices, and their culture for five days. When they reach confluence in the cell culture plate, MBMECs are trypsinized and reseeded onto permeable inserts, which are then placed in the TEER instrument. The TEER and Ccl of MBMEC...

Watch this Scientific Journal Video about An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction at JoVE.com

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.

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 ...

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Research

JoVE Journal

Immunology and Infection

11.2K Views.  University Hospital Münster. 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.

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

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

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

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 ...

Neuroscience

Analysis of Blood-Brain Barrier Models via Immunofluorescence Staining

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 

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, ...

Bioengineering

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

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 ...

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

Biology

Impedance-Based Biosensors in Investigating Brain Endothelial Barrier Strength

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising pericytes, astrocytic foot processes, and tightly adhered endothelial cells. Here, the brain endothelial cells form the first line of barrier against blood-borne pathogens. In conditions like cancer and neuroinflammation, circulating factors in the blood can disrupt this barrier. Disease progression significantly worsens post barrier disruption, which permits access to or impairment of regions of the brain. This significantly worsens the prognoses, particularly due to limited treatment options available at the level of the brain. Hence, emerging studies aim to investigate potential therapeutics that can prevent these detrimental factors in the blood from interacting with the brain endothelial cells.
The commercially available Electric Cell-Substrate Impedance Sensing (ECIS) and cellZscope instruments measure the impedance across cellular monolayers, such as the BBB endothelium, to determine their barrier strength. Here we detail the use of both biosensors in assessing brain endothelial barrier integrity upon the addition of various stimuli. Crucially, we highlight the importance of their high-throughput capability for concurrent investigation of multiple variables and biological treatments.

Author Spotlight: Studying Brain Endothelial Barrier in Metastatic Cancer Using Impedance-Based Biosensors

Here we demonstrate the technique of using impedance-based biosensors: ECIS and cellZscope, for measuring brain endothelial barrier strength. We detail the preparation and technique of adding various stimuli to an in vitro model of the brain endothelium. We measure, record, and give a representative analysis of the findings.

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising ...

An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection

Source: da Costa, A., et al. <a href="https://app.jove.com/v/59220">A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain.</a> J. Vis. Exp. (2019).
This video demonstrates an in vitro model to study pathogens crossing the blood-brain barrier (BBB). Microvascular endothelial cells, which express tight junctions, form an endothelial layer in the upper chamber, while neurons, astrocytes, and microglia represent the brain parenchyma in the lower chamber, separated by a porous membrane. Virus infection in the BBB model mimics the pathogen crossing from blood to the brain. Upon introducing a neurotropic virus, it crosses the BBB via transcytosis, infects neural cells, replicates, and releases viral progeny, demonstrating successful neuroinvasion.

Source: da Costa, A., et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the ...

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

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Chapters in this video

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

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

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

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

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

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

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines

An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection