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 c...

Impedance-Based Biosensors in Investigating Brain Endothelial Barrier Strength

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

Our research focuses on cerebral microvasculature and the blood-brain barrier. Brain endothelial cells regulate molecular flow into the brain. We use impedance-based biosensors to assess the barrier integrity in disease.With concurrent analysis and multiple treatment variables, we have already established that several cancer cell lines will disrupt the brain endothelium. Importantly, we have established that this is not predominantly mediated by proteases or cytokines but instead could be a function of cancer-related vesicular bodies. We are studying how metastatic cancers disrupt brain endothelial cells, enter the brain, and form secondary tumors.Using impedance-based biosensors, we explore this interaction to understand cerebral vasculature compromise and disease. Our goal is to identify and block targets to save our brain endothelial cells from disruption. In this protocol, we detail modifications made from five to seven years of experience that will help increase reproducibility and robustness of using both impedance-based technologies.Advantages and limitations of both systems compared to other technologies are discussed in the manuscript. Using this technology, we can investigate several properties of brain microvascular endothelial cells simultaneously and in real time. We have established that cancer cells and cytokines can disrupt the endothelial barrier.Next, we will assess if blocking these adhesion molecules or receptors helps to protect the brain endothelial barrier, as measured using these techniques.

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

Abstract