Akshata Anchan was funded by the Neurological Foundation of New Zealand for the Gillespie Scholarship (grant reference: 1628-GS) and First Fellowship (grant reference: 2021 FFE). The research cost was also partially funded by the Neurological Foundation Fellowship-2021 FFE and the University of Auckland Faculty Research Development Fund. James Hucklesby was funded by a scholarship from the Auckland Medical Research Foundation. Thanks to the Baguley team and Auckland Cancer Society Research Centre for the patient-derived New Zealand Melanoma NZM cell lines.

1. Using ECIS to monitor changes in brain endothelial barrier integrity in response to various treatments
<ol>
	<li>Setting up the software, array station, and machine
	<ol>
		<li>Connect the 96-well array station to the machine. Place the array station in a plastic bag and place it in the incubator at least 1 h before the assay to allow it to warm up. Remove the plastic bag immediately before the experiment.</li>
		<li>Once ready, turn on the machine and open the corresponding software on the computer. Press the Setup button under the Collect Data tab. The software will complete a connection check, which will detect the type of adapter attached (e.g., 96-well array station) and display red wells in the depicted plate map. This states that the software detects the adapter but cannot sense a plate attached. The system is now ready for the 96-well plate.</li>
	</ol>
	</li>
	<li>Preparing the 96-well biosensor plate 
	NOTE: The 96-well plate arrangement is used. The base of each well is patterned with interdigitating fingers of gold-plated electrodes. The plates come in sterile packs that should only be opened in a sterile hood/biological safety cabinet. The gold electrodes must first be chemically stabilized by adding 10 mM cysteine to maintain the electrode capacitance.
	<ol>
		<li>Prepare the 10 mM cysteine in a sterile trough and using a multi-channel, carefully pipette 100 &#181;L into each well. Incubate the cysteine for 15 min at room temperature and then carefully aspirate, pipetting at the edges of the well only, to avoid scratching the electrode. Wash the cysteine off at least 2x with sterile deionized water (not PBS) using the same pipetting technique. 
		NOTE: The plate electrodes can also be stabilized electrically using warm media on the ECIS apparatus, instead of using cysteine coating, as detailed in the manufacturer&#39;s protocol.</li>
		<li>Prepare ECM substrate to act as a basement membrane on the bottom of the well. 
		NOTE: ECM substrates like collagen, or laminin may be chosen depending on the requirement of the brain endothelial cell line. Here we use collagen for a human cerebral microvascular endothelial cell line (hCMVEC).</li>
		<li>Prepare 1 &#181;g/cm2 rat-tail collagen I dissolved in 0.02 M acetic acid in a fresh, sterile trough (100 &#181;L per well). Leave the collagen for 1 h at low light in the sterile hood, then wash 2x carefully with deionized water and careful pipetting. 
		NOTE: Ensure that as much of the deionized water is removed as carefully as possible after the last wash and let the plate dry in the hood. It is recommended to use the plate soon after coating; however, it can be stored in water at 4 &#176;C temporarily (under a week). The plate is now ready for use.</li>
	</ol>
	</li>
	<li>Preparing the brain endothelial cells for seeding onto the 96-well biosensor plate 
	NOTE: It is recommended to first optimize the growth phase and seeding density of the endothelial cell line to be used. The hCMVEC line was used in this protocol, which takes 48 h to reach confluency after seeding at 20,000 cells per well in a 96-well plate.
	<ol>
		<li>Harvest the brain endothelial cells in a sterile hood using a gentle dissociation reagent and conduct an accurate cell count. Use 20,000 hCMVECs in 100 &#181;L of media per well for a steady growth phase for 48 h. Prepare excess brain endothelial cells to ensure optimal pipetting of all 96 wells. 
		NOTE: Hence, for a 96-well plate, approximately 2,000,000 cells in 10 mL of prewarmed media are needed.</li>
		<li>Transfer the brain endothelial cells to a fresh sterile trough and using a multi-channel pipette, carefully add 100 &#181;L (20,000 cells) of the cell suspension per well. Resuspend the cells gently in the trough regularly to avoid the endothelial cells settling over time. Ensure 1 well has media only and no cells to allow mathematical modeling (cell-free well) and that the entire plate is seeded within 30 s.</li>
	</ol>
	</li>
	<li>Starting the experiment
	<ol>
		<li>Carefully attach the plate to the adapter in the incubator by moving the red clips on either side of the plate adapter outwards. Align the A1 well of the plate with the A1 region of the adaptor. Gently hold the plate in place with one hand on top and click the red clips back in place with the other hand. 
		NOTE: Ensure that the plate is stable in the adaptor.</li>
		<li>Close the incubator and press Setup again. Look for green wells in the plate map indicating correctly detected wells. Any wells showing red have a connection error. To fix this, re-attach the plate quickly, 
		NOTE: Excessive time spent trying to fix wells with poor connections may compromise cell health.</li>
		<li>Press Check to re-check the attachments as absolute impedance readouts. This will take several minutes.</li>
		<li>Press the arrow under the plate map to ensure the correct plate catalog is selected. In this case, it is 96w20idf.</li>
		<li>Press the multifrequency button to ensure impedance is measured at multiple AC frequencies to allow future mathematical modeling.</li>
		<li>Press Start to begin the experiment. Allow the cells to proliferate for ~48 h and form their typical monolayer of high resistance, as measured by an increase in ohms.</li>
	</ol>
	</li>
	<li>Treating the brain endothelial cells with cancer cells or cytokines on the 96-well plate 
	NOTE: As the 96-well plate allows concurrent testing of multiple stimuli, prepare a clear plate map with desired treatment options (Supplemental Figure S1-bottom). Each treatment can and must be conducted at least in triplicate (three replicate wells) to allow statistical analyses; however, four replicates are shown in Supplemental Figure S1.
	<ol>
		<li>At 48 h, check the growth of the brain endothelial cells. Ensure a plateau is reached before preparing treatment solutions. 
		NOTE: Different endothelial cell lines may take different lengths of time to reach a plateau. Hence, their growth phase and seeding density must be optimized prior to conducting this experiment. Wells should typically plateau within the same hour. If not, they might be compromised or pipetted inaccurately and must not be included.</li>
		<li>For treatment with cytokines, prepare appropriate concentrations of the cytokines in prewarmed complete media along with their relevant vehicle and media controls.
		<ol>
			<li>Prepare the cytokines at 2x the final desired concentration as they will be diluted 1:1 when added to each well containing brain endothelial cells.</li>
			<li>To allow each treatment to be conducted in triplicate, prepare all cytokines in over 300 &#181;L of total volume (i.e., at least 350 &#181;L total), for treatment addition of 100 &#181;L per well.</li>
		</ol>
		</li>
		<li>For treatment with cells, harvest the cells (three different patient-derived melanoma cell lines used in this protocol) with a gentle dissociation reagent and conduct an accurate cell count. Perform cell-based treatments at top effector:target ratios (E:T ratios) of 1:1 where the treatment cells are the effector cells (e.g., melanoma cells) and the brain endothelial cells are the target cells i.e. add 20,000 melanoma cells to 20,000 brain endothelial cells per well.
		<ol>
			<li>Serially dilute for smaller E:T ratios of 1:10 or 1:100 where required. To allow treatment in triplicate wells, count the cells and prepare them in over 300 &#181;L of total volume (i.e., at least 350 &#181;L total) for treatment addition of 100 &#181;L per well.</li>
		</ol>
		</li>
		<li>Label 1.1 mL polypropylene cluster tubes, which come in 8-tube strip format (i.e., 1 mL strip tubes), according to the 96-well plate map (Supplemental Figure S1-Top). Insert the strip tubes in the strip tube plates and autoclave them before the experiment. Add the total volume of treatment cytokines or cells (i.e.&#160;full 350 &#181;L of treatment) to the sterile strip tubes and leave them in the incubator to keep warm.</li>
		<li>On the software, press Pause to pause the experiment. This may take a few seconds to complete. Once paused, open the incubator and carefully unclip both red clips with one hand while stabilizing the plate with the other hand. Keep the experiment paused and bring the plate to the sterile hood.</li>
		<li>Remove the treatment strip tubes from the incubator. Using a multichannel pipette, carefully resuspend the contents of the treatment strip tubes and then add 100 &#181;L of treatment from the strip tubes to the correct wells on the 96-well plate without touching the bottom of the well. Carefully add treatment to all wells and media only to the cell-free well and complete this step in under 5 min for a full 96-well plate, as excessive cooling will affect the resistance of the endothelial monolayer.</li>
		<li>Return the treated plate to the machine, attach it as done previously, and then check the electrode connections to the adaptor again by pressing Setup | Check.</li>
		<li>Ensure that the correct plate catalog and multifrequency acquisition settings are selected, and press Resume to resume the experiment. Allow the experiment to progress and run it until the desired endpoint.</li>
		<li>Press Finish to complete the experiment, which automatically saves the recorded abp. file. Navigate to File | Export Data to retrieve this file as a xls. or csv. file for further analyses.</li>
	</ol>
	</li>
</ol>
2. Using cellZscope to monitor changes in brain endothelial barrier integrity in response to various treatments
<ol>
	<li>Preparing the apparatus&#39; metal components, adaptor, and porous membrane insert
	<ol>
		<li>Clean the metal components sequentially with deionized water, then 70% ethanol, and then deionized water again.</li>
		<li>Autoclave the bottom electrode or &#34;pots&#34; and top dipping electrodes and sterilize all other components of the Cell Module with 70% ethanol.</li>
		<li>In a sterile hood, install all of the 24 bottom electrodes/&#34;pots&#34; by screwing them into the Cell Module, taking care to maintain sterility throughout.</li>
		<li>Add 900 &#181;L of base media to the wells. 
		NOTE: Base media is added to the bottom well, which acts as the basal compartment of the endothelium. It is recommended to not use serum in this compartment to more reliably replicate the basal environment of the brain endothelium.</li>
		<li>Connect the dipping electrodes magnetically to the Cell Module lid and close the lid over the wells so the dipping electrodes slot into the bottom electrode pots.</li>
		<li>Attach the Cell Module to the adaptor in an incubator and leave it for at least 1 h to equilibrate. At this stage, open the software to load the experimental plate map under the Layout tab, thereby embedding the treatment plate map into the files, which will ultimately contain the data.</li>
		<li>Where coating is required, coat the membrane inserts in a sterile hood by using a sterile 24-well culture plate to hold the inserts and adding the ECM substrate (e.g., collagen) to the inner section of the well, as detailed in section 1.2.3 Use a single-channel pipette to load the ECM substrate into each well carefully.</li>
	</ol>
	</li>
	<li>Preparing the brain endothelial cells for seeding onto the membrane insert and starting the experiment
	<ol>
		<li>Harvest and count the brain endothelial cells as per section 1.3, but prepare&#160;the final required cell titration in a 50 mL centrifuge tube. Ensure the harvested cells are in warm complete media, including all required growth factors and serum.</li>
		<li>Using a single-channel pipette, carefully seed the brain endothelial cells (80,000 in this protocol) into the apical chamber of a well insert in 350 &#181;L of complete media.</li>
		<li>Include a cell-free well by pipetting complete media only into one of the inserts.</li>
		<li>Remove the Cell Module from the incubator and place it in the sterile hood. Transfer the prepared inserts into the bottom electrode pots using a pair of tweezers to handle only the upper supports of the insert. Avoid bubbles forming underneath the membrane.</li>
		<li>Place the Cell Module back in the incubator over the adapter.</li>
		<li>In the software, in the experiment tab, under the Spectrum section, select Start at 1 Hz and Stop at 100 kHz to acquire data over the entire frequency range; select fine steps.</li>
		<li>Under Wait time, select 15 min to permit continuous measurements at the fastest rate conducted by the software.</li>
		<li>Press Start to begin the experiment and allow brain endothelial cells to proliferate and form a monolayer with high resistance. This step takes approximately 48 h.</li>
	</ol>
	</li>
	<li>Treating the brain endothelial cells with cancer cells and cytokines on the membrane inserts
	<ol>
		<li>At 48 h, prepare the treatment cytokines or cells (e.g., melanoma cells) as per section 1.5, including conducting cell counts where applicable, and collect in 1 mL strip tubes or equivalent.</li>
		<li>Prepare treatment at correct concentrations for adding 70 &#181;L to each well (insert). Place treatments in the incubator to keep warm.</li>
		<li>As the instrument herein conducts measurements every 15 min, choose a treatment time that begins immediately after a measurement is completed. At this time, remove the Cell Module from the incubator and gently wipe it with 70% ethanol using lint-free wipes.</li>
		<li>In a sterile hood, open the Cell Module and carefully pipette 70 &#181;L of the treatment into the apical chamber of the respective insert. Do this using a single channel pipette and add in a circular motion, avoiding bubbles. Complete this step in under 10 min to return the Cell Module to the adapter before the next measurement starts.</li>
		<li>Press Stop to end the experiment. Export the data directly to a xls. file under the File | Export Tab. Name and save this file appropriately after opening it.</li>
	</ol>
	</li>
</ol>

Impedance-Based Biosensors in Investigating Brain Endothelial Barrier Strength

The authors have no conflicts of interest to declare.

Therapeutic studies on diseases that affect the BBB must consider the importance of brain endothelial barrier integrity and regulation. For example, brain endothelial barrier disruption is critically investigated in the metastasis of cancer to the brain from other anatomical sites. This is because the brain endothelium forms the first barrier against circulating tumor cells. As mentioned earlier in the introduction, in vitro studies on endothelial barrier integrity need to be reproducible, non-invasive, label-fr...

This article discusses current trends in the assessment of microvascular cells. We specifically detail the use of two commercially available platforms for measuring the barrier properties of cerebral microvascular endothelial cells. Endothelial cells are blood-facing cells, which line the vessel wall. However, cerebral microvessels are unique as they help form the protective blood-brain barrier (BBB)1,2,3. The BBB functions to regulate the transport of molecules from the blood to the brain. Peripheral diseases that affect the central nervous system (CNS) are commonly linked to a functional failure of the BBB4,5. The anatomical structures that form the BBB are not present at the blood-tissue interface elsewhere in the body6. These anatomical structures comprise pericytes, which are located close to brain endothelial cells, and regulate their proliferation and permeability; astrocytic foot processes, which are involved in nutrient shuffling and anatomical support7,8; microglia, which are the resident macrophages in the brain, often implicated in neuroinflammation and ischaemia9,10,11,12, and the brain endothelium, which forms a monolayer of tightly adhered cells without fenestrations13,14. The brain endothelium is typically known as the &#39;brain endothelial barrier&#39; and forms a structural and functional barrier in five distinct ways. First, the paracellular barrier component is formed by adhesion at lateral cell-cell junctions. Second, the transcellular barrier component is sustained by regulating endocytosis. Third, a specialized basement membrane anchors and supports the endothelium via a rich extracellular matrix comprising largely of collagen15,16. The last two mechanisms are through enzymes and transporters that help regulate the metabolism of drugs and uptake of large molecules, respectively17.
The paracellular interactions form the major component of the brain endothelial barrier, facilitated by tight junctions (TJs), comprising membrane proteins claudins, occludin, and junctional adhesion molecules (JAMs)18. Strong homotypic binding of the membrane proteins forms the first structural barrier, though JAMs also link to accessory proteins zonula occludens, thus linking the TJs to the actin cytoskeleton19,20. The actin links place the TJs in the apical region of the endothelium21, which functionally polarizes the endothelial cells to form the structural barrier on this apical or &#34;blood-facing&#34; side. In the basolateral region of the endothelium, highly specialized adherens junctions (AJs) play a regulatory role in maintaining cell morphology. AJs comprise calcium-dependent cadherins, which link the cytoskeleton of neighboring endothelial cells through the catenin family complex20,22. Vascular endothelial cadherin (VE-Cadherin) is one such cadherin, which regulates the expression of TJ proteins and overall endothelial barrier function to maintain endothelial monolayer integrity23,24,25,26,27. Inflammatory modulators, such as tumor necrosis factor-alfa (TNF&#945;), signal VE-cadherin internalization away from cell junctions, leading to destabilization of the endothelial barrier28,29,30. Platelet endothelial cell adhesion molecule (PECAM)31 is another AJ cadherin that stabilizes and remodels endothelial junctions32,33. The density of these junctional proteins restricts the flow of electrons through the paracellular space between endothelial cells. This attribute is utilized to measure endothelial barrier strength or trans-endothelial electrical resistance (TER) across confluent cell monolayers like the brain endothelium.
Therapeutic studies focus on the brain endothelium due to its vital role at the blood-brain interface. Several diseases negatively affect the brain endothelium, including neuroinflammatory conditions like multiple sclerosis, stroke, neurodegenerative diseases, and cancer34,35,36,37. Once the brain endothelial barrier is disrupted, disease progression significantly worsens as the brain is effectively exposed to the harmful stimuli in the blood38. We have previously shown that inflammatory mediators and metastatic melanoma cells disrupt the brain endothelial barrier by using two technologies that measure endothelial barrier strength39,40,41.
Electric cell-substrate impedance sensing (ECIS) is an impedance-based biosensor that permits real-time and label-free assessment of endothelial cell barrier integrity. Herein, assay wells are lined with gold-plated electrodes, which introduces alternating current (AC) to the assay system. Brain endothelial cells are seeded into these wells, which means that AC can be applied through the cells. (Figure 1A-well; side view). This establishes the electrical circuit, which is used to calculate the impedance (Figure 1A-circuit diagram). The impedance increases when the brain endothelial cells adhere to the plate and begin forming their paracellular junctions. The impedance plateaus when the endothelial cells become confluent, forming a monolayer, and restricting current flow. Application of AC at different frequencies influences the route of flow of current through the endothelial cells. Current flows through the endothelial cell body when applied at a higher frequency (&#62;104 Hz). This provides information on the capacitance of the cell monolayer, used to assess cell attachment and spreading. At low frequencies (102-104 Hz) the membrane impedance is high, restricting current flow through the cells. In this case, the majority of the current navigates between the cells. At approximately 4,000 Hz, the resistance to current flow is attributed mostly to the endothelial cell-cell junctions, via the intercellular space.Therefore, any change in resistance at this frequency provides information regarding endothelial barrier integrity.
Whilst raw impedance measurements can provide insight into barrier properties, the ECIS software can then mathematically model the total resistance measured across multiple AC frequencies and more precisely, separate it into two key parameters of barrier integrity. These parameters are the paracellular resistance between the lateral membranes of neighboring cells (resistance beta-Rb; paracellular barrier; Figure 1A-green arrows), and the basolateral resistance between the basal cell layer and the electrode (resistance alpha-Alpha; basolateral barrier; Figure 1A-blue arrows). A third modeled parameter is also measured as the cell membrane capacitance (Cm; Figure 1A-red arrows). The Cm displays the capacitive flow of current through the cells, indicative of cell membrane composition. Herein, changes in the Rb or paracellular barrier indicate alterations in the TJs and AJs, crucial in maintaining endothelial barrier integrity. To reliably interpret the Rb, four key assumptions are made, as developed by Giaever and Keese42 and critically discussed by Stolwijk et al.43. Although these assumptions are important for ensuring the validity of ECIS modeling, they are readily met by a confluent endothelial monolayer.
Like ECIS, the cellZscope permits the measurement of changes in endothelial barrier resistance; however, the cells are cultured on a porous membrane insert. In this system, the electrical circuit is between two electrodes on either side of a membrane insert. The endothelial monolayer is cultured on top of this membrane insert, allowing for measurement of trans-endothelial electrical resistance (TER) (Figure 1B-well; side view). As with ECIS, in this system, the total impedance can be attributed to several barrier components, dependent on the frequency of current applied44. At low frequencies, electrode capacitance (CEl) dominates the total impedance of the system. Alternatively, at high frequencies, the resistance of the media (Rmedium) dominates the total impedance. Hence, the most useful measurements fall within the midfrequency range (102-104 Hz), which provides information regarding two key components of the endothelial barrier (Figure 1B-circuit diagram). First, at 103-104 Hz, the cell layer capacitance (CCl) dominates the overall impedance as the membrane resistance (Rmembrane) is high enough to be neglected, and current flows predominantly across the capacitor. Hence, the CCl indicates changes to resistance through the cell membrane. Alternatively, TER predominantly imparts the overall impedance at 102-103 Hz, where current flow is channeled through junctional spaces between neighboring cells, held together by junctional proteins. Hence, this provides information on the paracellular component of the endothelial barrier, as seen previously with Rb on ECIS.
Figure 1C details how specific regions of the brain endothelium are disrupted by&#160;treatment with&#160;melanoma cells. This disruption is detected by the biosensors by a change in the flow of current through the paracellular space (measured as Rb or TER); the basolateral space (measured as Alpha); and the cell membrane (measured as Cm or CCl). We used both biosensors detailed in this introduction to measure brain endothelial barrier change following treatment with various stimuli such as cytokines or invasive melanoma cells. The measured resistance decreases if a given stimulus disrupts the endothelial barrier, creating a path of least resistance to allow current flow. Hence, a decrease in &#34;barrier resistance&#34; suggests loss of barrier integrity or brain endothelial barrier disruption. In these assays, we have studied this disruption by interpreting resistance and modeled parameters in real time. The application of ECIS and cellZscope in addressing such research questions are detailed elsewhere39,41,45,46.
In vitro research allows the discovery of crucial disease mechanisms by revealing molecules and functional pathways, which progress the disease. However, this requires reliable replication of the disease in vitro, which substantially differs from a functioning body. In an ideal scenario, in vitro research should be reproducible, non-invasive, label-free, quantitative, and mimic structural influences found in vivo. In this article, we detail the methodology for using these two contending technologies to measure treatment-induced changes in brain endothelial barrier integrity. We discuss the advantages of combining their results to provide a more comprehensive picture of barrier disruption and share limitations that still need to be overcome.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>aMEM</td>
			<td>Gibco</td>
			<td>12561072</td>
			<td>Melanoma cell base media</td>
		</tr>
		<tr>
			<td>cellZscope array</td>
			<td>nanoAnalytics</td>
			<td>cellZscope2; software v4.3.1</td>
			<td>TER measuring biosensor array</td>
		</tr>
		<tr>
			<td>Collagen I&#8212;rat tail</td>
			<td>Gibco</td>
			<td>A1048301</td>
			<td>ECM substrate for coating</td>
		</tr>
		<tr>
			<td>dibutyryl-cAMP</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>ECIS array</td>
			<td>&#160;Applied Biophysics</td>
			<td>ECIS Z&#920;; software v1.2.163.0</td>
			<td>Rb/Alpha measuring biosensor array</td>
		</tr>
		<tr>
			<td>ECIS plate</td>
			<td>&#160;Applied Biophysics</td>
			<td>96W20idf&#160;</td>
			<td>96-well biosensor plate</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>12203C-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>305050-061</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>hCMVEC</td>
			<td>Applied Biological Materials</td>
			<td>T0259</td>
			<td>Brain endothelial cell line</td>
		</tr>
		<tr>
			<td>hEGF</td>
			<td>PeproTech</td>
			<td>PTAF10015100</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H-3393</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>hFGF</td>
			<td>PeproTech</td>
			<td>PTAF10018B50</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>Hydrocortison</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>PeproTech</td>
			<td>200-01B</td>
			<td>Cytokine</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>11074547001</td>
			<td>Melanoma cell media supplement</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>Gibco</td>
			<td>11150-067</td>
			<td>Brain endothelial cell base media</td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td></td>
			<td></td>
			<td>Deionized water</td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>PeproTech</td>
			<td>300-01A</td>
			<td>Cytokine</td>
		</tr>
		<tr>
			<td>Transwell insert</td>
			<td>Corning</td>
			<td>CLS3464</td>
			<td>Porous membrane insert</td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12604021</td>
			<td>Dissociation reagent</td>
		</tr>
	</tbody>
</table>

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

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

Interpreting ECIS impedance data
Understanding optimal experimental conditions 
Herein the data can be directly viewed using the software (Figure 2A) or exported for analysis and graph plotting (Figure 2B). Figure 2A shows an example of data displayed on the actual software interface. The left graph shows an example of a disrupted connection due to improper lo...

Watch this Scientific Journal Video about Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines at JoVE.com

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 ...

measuring-changes-brain-endothelial-barrier-integrity-with-two

Research

JoVE Journal

Biology

350 Views.  University of Auckland. 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.