This study has been supported by the Collaborative Research Centre CRC TR128 "Initiating/Effector versus Regulatory Mechanisms in Multiple Sclerosis-Progress towards Tackling the Disease" (Project A9 to H.W. and C.C.G., project B1 to N.S.).

1. Cell Culture of Human Brain Microvascular Endothelial Cells (HBMEC)
<ol>
	<li>Coating of cell culture flasks
	<ol>
		<li>To prepare the fibronectin solution, add 10 mL PBS to a 15 mL centrifuge tube. Add 150 &#181;L fibronectin and mix well.</li>
		<li>To cover the bottom a T-25 cell culture flask add 2 mL of the fibronectin solution. Incubate the cell culture flask for at least 3 h at 37 &#176;C in the incubator. Fibronectin coated flasks can be stored for 2 weeks at 37 &#176;C / 5% CO2.</li>
	</ol>
	</li>
	<li>Seeding and cell culture of HBMEC
	<ol>
		<li>Aspirate fibronectin solution from the bottom of the cell culture flask. Add 7.2 x 104 HBMEC/cm&#178; suspended in 6 mL ECM-b medium (= ECM-b supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement). Incubate at 37 &#176;C / 5% CO2. Check cell growth daily using a microscope.</li>
		<li>Change the medium every 3 days. Harvest or split cells, when HBMEC reach approximately 80% confluence. HBMEC should be used between passage 1 and 15 to avoid loss of physiological properties.</li>
	</ol>
	</li>
	<li>Harvest HBMEC.
	<ol>
		<li>Prepare accutase solution by mixing accutase (1x) with PBS at a ratio of 1:1. Keep accutase solution at 37 &#176;C in a water bath until further use.</li>
		<li>Transfer ECM-b medium from the cell culture flask to a 15 mL centrifuge tube. Wash HBMEC by adding 5 mL PBS to the bottom of the cell culture flask. Aspirate PBS and repeat the washing step two more times.</li>
		<li>Add 2 mL pre-warmed accutase solution. Incubate at 37 &#176;C for 2 min. Afterwards, HBMEC are re-suspended by firmly tapping the cell culture flask several times. Cell detachment is controlled using a microscope</li>
		<li>The ECM-b-medium previously stored in a 15 mL tube is added back to the cell culture flask as soon as HBMEC start to detach. Rinse the bottom of the flask repeatedly until most HBMEC are re-suspended.</li>
		<li>Transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 300 x g for 10 min at room temperature. Discard supernatant and re-suspend cells in 1 mL ECM-b medium. Count cells and dilute the cell suspension to achieve a final concentration of 3 x 105 HBMEC per mL ECM-b medium.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Cell Culture Inserts
<ol>
	<li>Coating of cell culture inserts 
	Important note: Avoid touching the membrane of the cell culture inserts.
	<ol>
		<li>Add 100 &#181;L fibronectin solution (see 1.1.1) to each cell culture insert (Figure 1A) and one well of a 96-well flat bottom plate (optical control well). Incubate for at least 3 h at 37 &#176;C. After incubation aspirate fibronectin solution.</li>
		<li>Add 100 &#181;L HBMEC suspension to the cell culture inserts and the optical control well. Add 600 &#181;l ECM-b medium to the lower compartment of the cell culture inserts. Incubate for 3 - 4 days at 37 &#176;C / 5% CO2 until barrier integrity (Figure 1B) is reached, check cell growth by microscopic evaluation of the HBMEC in the optical control well. Note: Cell growth beyond four days is not recommended.</li>
		<li>Optional: To mimic inflammatory conditions aspirate the medium from the lower compartment and replace it with ECM-b medium supplemented with 500 U/mL IFN-&#947;/TNF-&#945; 24 h prior the migration assay.</li>
	</ol>
	</li>
</ol>
3. Quality Control with Evans Blue on the Day of the Transmigration Assay
<ol>
	<li>Preparation of evans blue solution
	<ol>
		<li>To prepare PBS/B27 solution mix 10 mL PBS with 200 &#181;l B27 supplement using a 15 mL centrifuge tube. Dilute evans blue stock solution (20 mg/mL PBS) 1:1,000 with PBS/B27.</li>
	</ol>
	</li>
	<li>Evans blue permeability assay
	<ol>
		<li>Aspirate the medium from the lower compartment followed by the upper compartment of one cell culture insert containing a confluent HBMEC monolayer. Add 100 &#181;L evans blue solution to the cell culture insert.</li>
		<li>Add 600 &#181;L PBS/B27 to the lower compartment and incubate for 60 min at 37 &#176;C / 5% CO2. Carefully remove the cell culture insert using forceps.</li>
	</ol>
	</li>
	<li>Evans blue measurement
	<ol>
		<li>Remove PBS/B27 from the lower compartment and transfer 100 &#181;L each to two wells of a black polystyrol 96-well flat bottom plate. Insert plate in a Tecan Infinite M200 Pro plate reader and determine optimal z-position.</li>
		<li>Measure excitation of evans blue using respective settings (for example: excitation: 620 nm, emission: 680 nm, excitation bandwidth: 9 nm, emission bandwidth: 20 nm, 175x enhancement, 25 flashes, time of integration: 20 &#181;s).</li>
		<li>To determine HBMEC barrier functions compare acquired data to a standard curve depicting evans blue permeation across HBMEC at different time points after seeding cells (Figure 1B, right).</li>
	</ol>
	</li>
</ol>
4. Migration Assay
<ol>
	<li>Preparation of peripheral blood mononuclear cells (PBMC).
	<ol>
		<li>Add 10 mL RPMI into a 15 mL centrifuge tube and add 200 &#181;L B27 supplement. Count PBMC and centrifuge cells at 300 x g for 5 min. Re-suspend PBMC to a final concentration of 5 x 106 cells/mL RPMI/B27.</li>
	</ol>
	</li>
	<li>Set-up of the migration assay
	<ol>
		<li>Aspirate medium from the lower compartment followed by the upper compartment of cell culture inserts containing confluent HBMEC monolayers (Figure 1A). Per donor add 100 &#181;L PBMC suspension each to the cell culture inserts and also to one well of a 24-well plate per (in vitro control).</li>
		<li>Add 600 &#181;L RPMI/B27 to the lower compartment of the cell culture inserts and 500 &#181;L to the PBMC of the in vitro control and incubate 6 h at 37 &#176;C / 5% CO2.</li>
	</ol>
	</li>
	<li>Harvesting of migrated PBMC
	<ol>
		<li>Take out the cell culture insert using forceps and carefully rinse the bottom with 400 &#181;L PBS without touching the membrane. Discard the cell culture insert.</li>
		<li>Add 20 &#181;L flow count fluorospheres (approximately 1,000 beads/&#181;L) to the lower compartment of the cell culture insert as well as to the in vitro control and mix well. Transfer 1 mL of resulting PBMC suspension to flow cytometry tubes.</li>
	</ol>
	</li>
</ol>
5. Flow Cytometry
<ol>
	<li>Sample preparation
	<ol>
		<li>Centrifuge PBMC at 300 x g for 5 min at room temperature.</li>
		<li>Prepare antibody solution by adding fluorochrome-conjugated antibodies to 100 &#181;L flow cytometry buffer (PBS/1% BSA/2 mM EDTA) per sample. For the results presented below 1 &#181;L CD4-FITC, 1 &#181;LCD3-PerCP/Cy5.5, 1 &#181;L CD56-PC7, 1 &#181;L CD8-A700, and 1 &#181;L CD16-A750 were used per sample.</li>
		<li>Re-suspend PBMC in 100 &#181;L of the antibody solution and incubate for 30 min at 4 &#176;C.</li>
		<li>Add 250 &#181;L flow cytometry buffer and centrifuge at 300 x g for 5 min.</li>
	</ol>
	</li>
	<li>Sample acquisition
	<ol>
		<li>Re-suspend PBMC in the required amount (varies depending on the flow cytometer used) of flow cytometry buffer.</li>
		<li>Acquire stained PBMC using a flow cytometer with an active detector between 525 and 700 nm wavelength to detect flow count fluorospheres (excitation 488 nm, emission 525 - 700 nm). 
		(The following steps are an example if a Gallios flow cytometer operated with Kaluza G software is used: (1) Start the computer. (2) When the operating system is fully loaded, start the flow cytometer by pressing the &#34;cytometer on&#34; button. (3) Load the respective acquisition protocol by pressing &#34;open protocol&#34; button. (4) Choose the required protocol and select &#34;open&#34;. (5) Duplicate the protocol for every sample by clicking with the right mouse button on the protocol visible in the virtual carousel and a left click on the field &#34;duplicate&#34;. (6) Label each sample in the sample list. (7) Transfer the samples to the indicated positions of the carousel and start the acquisition.)</li>
	</ol>
	</li>
	<li>Sample analysis
	<ol>
		<li>Open resulting flow cytometry data using the respective software. Determine number of subpopulations of interest for transmigrated PBMC as well as cells from in vitro control wells and flow count fluorospheres using the respective analysis software. 
		(An example of the gating strategy is given in the results part (Figure 1 C: To analyze the transmigration of NK-cell subsets, first select lymphocytes in a sideward scatter channel (SSC) versus forward scatter channel (FSC) plot. Lymphocytes are then displayed in a CD3 versus CD56 plot and CD56+CD3- NK cells are selected. To distinguish between NK-cell subsets, NK cells are displayed in a CD56 versus CD16 plot and CD56brightCD16dim/- as well as CD56dimCD16+ NK cells are selected. In addition, flow count fluorospheres are selected from a FSC versus SSC plot and subsequently displayed in a plot of a channel with an emission between 525 and 700 nm versus time to determine their number.)</li>
		<li>To calculate the total cell number of each sample, normalize the detected number of cells using flow count fluorospheres: 
		<img alt="Equation" src="/files/ftp_upload/55390/55390eq1.jpg" /></li>
		<li>Determine percentage of migrated cells as ratio between total migrated cells and total cells in the in vitro control.</li>
	</ol>
	</li>
</ol>

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.S.-M. and U.B. have no financial disclosures. T. S.-H. received travel and conference expenses from Biogen. N.S. received speaker and advisory board honoraria from Biogen and Novartis Pharma, as well as travel expenses from Biogen. H.W. received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. He also received speaker honoraria and travel support from Bayer Vital GmbH, Bayer Schering AG, Biogen, CSL Behring, Fresenius Medical Care, Glaxo Smith Kline, GW Pharmaceuticals, Lundbeck, Merck Serono, Omniamed, Novartis, and Sanofi-Genzyme. He received compensation as a consultant from Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. H.W. received research support from Bayer Vital, Biogen, Genzyme, Merck Serono, Novartis, Sanofi-Aventis Germany, and Sanofi US. C.C.G. received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis Pharma GmbH, and Bayer Health Care.

Here we present a technique to investigate the transmigration of lymphocytes across the human blood-brain barrier. In vitro analysis of lymphocyte migration to the CNS is important to study basic processes of lymphocyte extravasation, potential disease-related alterations, and new therapeutic approaches.
Several modifications of the blood-brain barrier model are possible. For example, cells from the upper compartment could be analyzed to investigate the composition of the non-migrated cell population. Furthermore, treatment of the HBMEC monolayer with IFN-&#947; and TNF-&#945; 24 hours prior to the assay can be used to mimic an inflamed blood-brain barrier in order to study the effect of inflammatory CNS disorders21,25. Similarly, treatment of HBMEC or lymphocytes with other substances allows investigating their effects on lymphocyte extravasation (e.g. their effect on adhesion molecules)30,31. The involvement of certain adhesion molecules can be studied using blocking antibodies for integrins or their ligands32. Furthermore, the experimental setup presented here allows analysis of chemotactic effects from chemokines or supernatants derived from astrocytes or other cells33,34. Replacement of HBMEC with primary human brain derived epithelial cells widens the spectrum of this experimental setup for investigation of the blood-cerebrospinal fluid barrier15. HBMEC should not be replaced with immortalized endothelial cells or cells derived from other organs to maintain CNS-specificity of the model. However, brain-derived endothelial cells from other species might be used to analyze transmigration in the respective animals35. In addition, retinoic acid or hydrocortisone have been described to increase barrier functions and might thus be employed36,37. In case of limited cell numbers the amount of cells subjected to the assay might be reduced, because our model provides linear recovery rates between 2 x 105 and 1 x 106 PBMC (data not shown). To analyze rare cell populations following transmigration it might be necessary to pool cells from several wells in order to obtain sufficient cells required for flow cytometry. Finally, our experimental setup could also be used to study the drug delivery into the CNS38. A pore size of 3 &#181;m is typically used to allow lymphocyte transmigration, whereas a pore size of 0.4 &#181;m prevents lymphocyte transmigration, but allows to study drug delivery39,40,41,42.
Despite the various applications of the experimental setup described here, several points have to be kept in mind to ensure meaningful results. The integrity of the porous membrane as well as the HBMEC monolayer is critical. Thus, it is mandatory to avoid physical damage of the membrane, for example with pipet tips. Addition of fluids or cell suspension to the upper part of the cell culture insert should be performed without direct contact. Droplets might be wiped off at the border of the cell culture insert. Horizontal placement of the pipet tip ensures protection of the membrane, when the abluminal membrane is rinsed at the end of the migration assay. The quality of the HBMEC monolayer can be analyzed by microscopic evaluation of confluence as well as evans blue permeation as demonstrated for the assessment of blood-brain barrier integrity in rodent experiments43. Centrifugation of HBMEC at low g forces and usage of early passages (i.e. 1 - 15) ensures quality and uniformity of the monolayer. To prevent dissolution from the porous membrane, it is important to adhere to the described order of aspiration and addition of medium, when working with the HBMEC monolayer.
Although adherence to the protocol described here ensures meaningful and reproducible results, this technique has some limitations. First of all, in vivo the blood-brain barrier is formed by a number of cells, which interact in various ways and strengthen the barrier function by affecting the formation of tight junctions1. Therefore, although this blood-brain barrier model is a good approximation of the in vivo situation, important aspects are missing. In addition, lymphocyte extravasation into the CNS across the blood-brain barrier is a multistep process. While each single step can be investigated separately, the technique presented here does not provide information on the whole process of extravasation under the influence of shear forces2,44,45,46. Finally, analysis of migrated cells from PBMC might be challenging depending on the frequency of the cells of interest, because usually only single-digit frequencies of cells transmigrate. Therefore, separation of the populations of interest prior to the assay or pooling of cells migrated across multiple cell culture inserts might be necessary. Other models for the analysis of leukocyte migration into the CNS cover some of the aspects missing in our model. Co-culture with astrocytes, pericytes, and/or neurons are used to mimic the complexity of the blood-brain barrier better47. Models including shear forces such as DIV-BBB reflect more the physiological conditions, thus, enabling a more sophisticated analysis of lymphocyte diapedesis48.
In summary, we present an easily accessible technique suitable for the investigation of qualitative and quantitative lymphocyte diapedesis across the human blood-brain barrier.

Lymphocyte migration from the blood into tissues is crucial for immune surveillance. A sequence of specific molecular interactions ensures site specific extravasation into small intestine, skin, lymph nodes, the central nervous system (CNS), and other tissues1. Alterations in lymphocyte migration are involved in the pathophysiology of a number of wide spread diseases2. Migration into the immune-privileged CNS is tightly regulated and accordingly alterations of this process are involved in CNS-related diseases like encephalomyelitis3, neuromyelitis optica, stroke, and multiple sclerosis (MS)2,4,5,6,7. Therefore, it is important to study lymphocyte extravasation to better understand disease pathophysiology and to develop tools for amelioration of disease burden8,9,10,11,12.
Lymphocytes migrate into the CNS via distinct routes. Extravasation through postcapillary venules into the subarachnoid space via the blood-cerebrospinal fluid barrier within the choroid plexus and across the blood-brain barrier have been described1,13,14,15. Migration across the blood-brain barrier is conducted by the interaction of lymphocytes with endothelial cells14. In contrast to endothelial cells in the periphery, endothelial cells of the CNS express high amounts of tight junction molecules, thereby strictly limiting the amount of cells and proteins capable of crossing the blood-brain barrier16. Inflammation results in loosening of tight junctions and induces the expression of adhesion molecules; thus, enhancing lymphocyte migration into the CNS1,17,18.
Extravasation via the blood-brain barrier is a multistep process. Lymphocytes tether to the endothelial cells and then roll along the endothelium in a process mainly mediated by selectins1,15. Subsequently, interactions between chemokines secreted by the endothelium and the respective chemokine receptors expressed on lymphocytes induce conformational changes of integrins, thereby promoting firm adhesion to the endothelial cells1. Finally, lymphocytes either crawl along the endothelial barrier against the blood flow before transmigrating into the perivascular space, or stall immediately and directly transmigrate at the site of firm adhesion1,19,20. All these steps of lymphocyte extravasation can be analyzed in vitro using distinct techniques21. Time-lapse video microscopy is used to study the initial tethering and rolling15. Adhesion assays provide detailed information about firm arrest to endothelial barriers22. Transmigration assays as demonstrated here allow analysis of immune-cell transmigration21,23,24,25,26,27,28,29.
Using the human in vitro blood brain barrier model, we could recently show that a higher migratory capacity of CD56brightCD16dim/- NK cells compared to their CD56dimCD16+ counterparts was reflected by a predominance of this NK cell subset in the intrathecal compartment21. Thus, our experimental setup seems to be suitable to mimic the in vivo situation.

<table><tbody><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td>without CaCl&lt;sub&gt;2&lt;/sub&gt; or MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Fibronectin 1 mg/mL</td><td>Sigma</td><td>F1141-5MG</td><td>from bovine plasma</td></tr><tr><td>T-25 cell culture flask</td><td>Greiner BioOne</td><td>690160</td><td></td></tr><tr><td>HBMEC</td><td>ScienCell</td><td>1000</td><td></td></tr><tr><td></td><td>Pelobiotech</td><td>PB-H-6023</td><td></td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964-100ML</td><td></td></tr><tr><td>ECM-b</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>FBS</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Endothelial cell growth supplement</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Transwell</td><td>Corning</td><td>3472</td><td>clear, 6.5 mm diameter, 3.0 &amp;micro;m pore size</td></tr><tr><td>96-well flat bottom plate</td><td>Corning</td><td>3596</td><td></td></tr><tr><td>Evans blue</td><td>Sigma</td><td>E2129-10G</td><td>stock solution: 1 g/50 mL PBS</td></tr><tr><td>B27</td><td>Gibco</td><td>17504-044</td><td>50x concentrated</td></tr><tr><td>Infinite M200Pro</td><td>Tecan</td><td></td><td></td></tr><tr><td>96-well black flat bottom plate</td><td>Greiner BioOne</td><td>675086</td><td></td></tr><tr><td>48-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>61870-010</td><td></td></tr><tr><td>Flow Count Fluorospheres</td><td>Beckman Coulter</td><td>7547053</td><td></td></tr><tr><td>Na-EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>Gallios 10-color flow cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Kaluza 1.5a</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>TNF-&amp;alpha;</td><td>Peprotech</td><td>300-01A</td><td></td></tr><tr><td>IFN-&amp;gamma;</td><td>Peprotech</td><td>300-02</td><td></td></tr><tr><td>CD3-PerCP/Cy5.5</td><td>Biolegend</td><td>300430</td><td>clone UCHT1</td></tr><tr><td>CD56-PC7</td><td>Beckman Coulter</td><td>A21692</td><td>clone N901</td></tr><tr><td>CD16-A750</td><td>Beckman Coulter</td><td>A66330</td><td>clone 3G8</td></tr><tr><td>CD4-FITC</td><td>Biolegend</td><td>300506</td><td>clone RPA-T4</td></tr><tr><td>CD8-A700</td><td>Beckman Coulter</td><td>A66332</td><td>clone B9.11</td></tr></tbody></table>

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.

Representative results showing transmigration of NK-cell and T-cell subsets using the human blood-brain barrier model (Figure 1A) are shown. The integrity of the HBMEC monolayer was validated by staining of the tight junction molecule ZO-1, transendothelial electrical resistance (TEER) measurements, and evans blue permeation (Figure 1B). Following 3 - 4 days culture HBMEC expressed the tight junction molecule ZO-1 (Figure 1B, left). Furthermore, HBMEC grew in monolayers exhibiting transendothelial electrical resistance (Figure 1B middle) as well as reduced permeation for evans blue (Figure 1B, right). HBMEC monolayers were used to study the transmigration of NK cells including CD56brightCD16dim/- and CD56dimCD16+ NK-cell subsets and T cells including CD4+ and CD8+ T cells as two examples (Figure 1D+E, respectively). The percentage of migrated cells was calculated based on cell counts obtained by flow cytometry and normalized using flow count fluorospheres as an internal control (Figure 1C). The HBMEC monolayer was stimulated with IFN-&#947; and TNF-&#945; 24h prior the assay to mimic inflammatory conditions. Cytokine stimulation resulted in increased migration of all analyzed lymphocyte populations. This might be due to an increased expression of adhesion molecules including ICAM-1 (data not shown). CD56brightCD16dim/- NK cells exhibited a higher migratory capacity when compared to their CD56dimCD16+ NK across both unstimulated (10.88% vs. 0.86%) and IFN-&#947;&#947;/TNF-&#945; stimulated (18.22% vs. 2.94%) HBMEC (Figure 1D). However, the relative increase of transmigration as a result of inflammation was higher for the CD56dimCD16+ NK-cell subset (+342% vs. +167% for CD56brightCD16dim/-). These results mimic the in vivo observations that CD56brightCD16dim/- NK cells are enriched in the intrathecal compartment. Thus, the blood-brain barrier model seems to be suitable to analyze basic principles of immune-cell diapedesis of rare lymphocyte populations into the CNS21. Finally, the transmigratory capacity of CD4 and CD8 T-cell subsets is shown (Figure 1E).
<img alt="Figure 1" src="/files/ftp_upload/55390/55390fig1.jpg" /> 
Figure 1: Differential Migration of NK-cell Subsets across Uninflamed and Inflamed HBMEC Monolayers. A. A picture of transwell inserts (left) and illustration of the experimental setup (right). B. Validation of HBMEC barrier functions. Left: immunohistochemical staining of the tight junction molecule ZO-1 using rabbit anti-human ZO-1 (abcam, 1:200) and goat anti-rabbit IgG-Cy3 (1:300) on HBMEC cultivated for 3 days. Center: transendothelial electrical resistance (TEER) of HBMEC between day 2 and day 4 of cultivation. Right: standard curve for evans blue permeation for HBMEC with (&#34;inflamed&#34;, red) or without (&#34;uninflamed&#34;, black) stimulation with 500 U/mL IFN-&#947; and TNF-&#945; for 24 hours. Transmigration assays are performed 72 - 96 hours after seeding of the HBMEC (black arrow). C-E. PBMC derived from 16 healthy individuals were subjected to migration assays as described in the protocol section. 24 h prior to the assay, half of the cell culture inserts were stimulated with 500 IU/mL IFN-&#947; and TNF-&#945;. Transmigrated cells were harvested and analyzed by flow cytometry. C. Representative results for PBMC derived from the in vitro control well (top) and after migration across uninflamed HBMEC (bottom). NK cells were gated from total lymphocytes as CD3-CD56+ cells and further distinguished into CD56brightCD16dim/- (&#34;CD56bright&#34;) and CD56dimCD16+ (&#34;CD56dim&#34;) NK-cell subsets. Flow count fluorospheres (&#34;beads&#34;) were gated based on FSC/SSC characteristics and their number was determined in a FL3 versus time plot. Exemplary calculations to determine the percentage of migrated CD56bright and CD56dim NK cells are shown on the right. D. Percentage of migrated NK cells as well as CD56bright and CD56dim NK-cell subsets, and E. percentage of migrated T cells including CD4+ and CD8+ T-cell subsets following transmigration across uninflamed (black) or inflamed (red) HBMEC depicted as mean &#177; SEM. P-values were calculated by paired student t-test; **p&#60;0.01, ***p&#60;0.001. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55390/55390fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier at JoVE.com

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.

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 ...

analysis-lymphocyte-extravasation-using-an-vitro-model-human-blood

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.3K Views.  University Hospital Münster. Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro. 

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

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

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

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

Bioengineering

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

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 ...

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.

We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

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, ...

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

Neuroscience

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Source: Fattakhov, N., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/64469">A triple primary cell culture model of the human blood-brain barrier for studying ischemic stroke in vitro.</a> J. Vis. Exp.&#160;(2022)
This video demonstrates the co-culturing of human brain pericyte, astrocyte, and endothelial cells. Each cell layer is formed separately and then cultured together, creating a three-layered system that mimics the cellular arrangement of the blood-brain barrier

1. Triple cell culture BBB model setting

	Seeding pericytes
	
		Cultivate HBVP (Human brain vascular pericytes) in T75 culture flasks with an activated ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

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.bdigitaluss.remotexs.co/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 ...

Neuropathology

Infectious Diseases

Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer

Source: Schulte-Mecklenbeck, A., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55390">Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier.</a> J. Vis. Exp. (2017)
This video demonstrates an experimental investigation of lymphocyte transmigration across a blood-brain barrier (BBB) endothelium model. A monolayer of human brain microvascular endothelial cells (HBMECs) is generated in vitro, and peripheral blood mononuclear cells (PBMCs) containing lymphocytes are added to the monolayer. The cells are then incubated to allow lymphocyte transmigration, and the migrated cells are quantified using flow cytometry.

Source: Schulte-Mecklenbeck, A., et al., Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier. J. Vis. Exp. ...

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

Summary

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Chapters in this video

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

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

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

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

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

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

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

Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer