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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 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.
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 t....
1. Using ECIS to monitor changes in brain endothelial barrier integrity in response to various treatments
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
aMEM | Gibco | 12561072 | Melanoma cell base media |
cellZscope array | nanoAnalytics | cellZscope2; software v4.3.1 | TER measuring biosensor array |
Collagen I—rat tail | Gibco | A1048301 | ECM substrate for coating |
dibutyryl-cAMP | Sigma-Aldrich | D0627 | Brain endothelial media supplement |
ECIS array |  Applied Biophysics | ECIS ZΘ; software v1.2.163.0 | Rb/Alpha measuring biosensor array |
ECIS plate |  Applied Biophysics | 96W20idf | 96-well biosensor plate |
FBS | Sigma-Aldrich | 12203C-500ML | |
GlutaMAX | Gibco | 305050-061 | Brain endothelial media supplement |
hCMVEC | Applied Biological Materials | T0259 | Brain endothelial cell line |
hEGF | PeproTech | PTAF10015100 | Brain endothelial media supplement |
Heparin | Sigma-Aldrich | H-3393 | Brain endothelial media supplement |
hFGF | PeproTech | PTAF10018B50 | Brain endothelial media supplement |
Hydrocortison | Sigma-Aldrich | H0888 | Brain endothelial media supplement |
IL-1β | PeproTech | 200-01B | Cytokine |
Insulin-Transferrin-Sodium Selenite | Sigma-Aldrich | 11074547001 | Melanoma cell media supplement |
M199 | Gibco | 11150-067 | Brain endothelial cell base media |
MilliQ water | Deionized water | ||
PBS 1x | Gibco | 10010-023 | |
TNFα | PeproTech | 300-01A | Cytokine |
Transwell insert | Corning | CLS3464 | Porous membrane insert |
TrypLE Express Enzyme | Gibco | 12604021 | Dissociation reagent |
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