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 assist their barrier integrity in disease.

With concurrent analysis of 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 release of vesicular bodies. We're 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 safeguard 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'll assess if blocking these adhesion molecules or receptors helps to protect the brain endothelial barrier has measured using these techniques. To begin, use a multi-channel pipette to pipette 100 microliters of 10 millimolar cysteine into the wells of a 96 well plate, in a sterile hood. After incubating the plate, carefully aspirate the solution from the well and avoid scratching the electrode.

Rinse the wells two times with sterile deionized water. Next, add 100 microliters of freshly prepared Rat-Tail Collagen 1 solution into each well. Incubate the plate for one hour under low light before washing twice with deionized water.

Next, transfer the harvested brain endothelial cells into a fresh sterile trough. With a multi-channel pipette, add 100 microliters of the cell suspension into each well, completing seeding in 30 seconds. Keep one well media only for mathematical modeling purposes.

On a 96 well plate adapter, move the red clips on each side outward to allow the plate to be inserted. Precisely align the A1 well of the plate with the A1 region of the adapter and apply gentle pressure on top of the plate to hold it securely in place, then lock the red clips back. Press set to initiate the instrument after closing the incubator.

All correctly detected wells will be indicated by a green color on the plate map. Press the check button to authenticate the absolute impedance readouts. Now use the dropdown menu beneath the plate map to select the type of plate being used for the experiment.

Select multi frequency to measure the impedance at various alternating current frequencies. Finally, click on the start button to begin the experiment. Allow the cells to proliferate until a high resistance monolayer is formed, indicated by an increase in ohms.

Ensure the cell growth has reached a plateau before preparing treatment solutions. To prepare treatment solutions, place labeled 1.1 milliliter polypropylene cluster tubes into strip tube plates. Add three 50 microliters of the treatment cytokines or cells into the tube, following a pre-made plate map.

Then place them in the incubator for warmth. On the ECIS software, click on pause to temporarily halt the ongoing experiment. When prompted, open the incubator and carefully release both red clips while holding the plate steady.

With the experiment still in paused state, transfer the plate to the sterile hood, then remove the treatment strip tubes from the incubator. With a multi-channel pipette, carefully resuspend the contents of the treatment strip tubes. Now pipette out 100 microliters of treatment from the stripped tubes and transfer it to the appropriate wells on the 96 well plate.

Add only complete media to the cell-free wells. Try to finish pipetting in under five minutes as excessive cooling of the plate could affect the resistance of the endothelial cell monolayer. Reattach the treated plate to the machine, then check to assess the electrode well impedance.

Confirm the correct multi frequency acquisition settings and plate catalog have been selected, then press resume to continue the experiment. Once the experiment has progressed to the desired endpoint, press finish to automatically save the recorded file. Navigate to file and click on export data to export the data as an XLS or CSV file for further analysis.

Increase in resistance was observed over 30 hours, indicating the growth phase of the endothelial cells. During growth phase, the cells plateaued at different levels by 48 hours. Measurement normalization allowed a more reliable interpretation of resistance changes.

Incorrect cell count of the brain endothelial cells before treatment, resulted in a fluctuating growth phase, which gradually declined into a stable resistance plateau. Optimized cell seeding density and loading volume resulted in an optimal growth phase. Cytokines appeared to have a transient effect on the barrier.

The addition of melanoma cells drastically reduced the barrier resistance. The paracellular barrier and basal lateral component fluctuated with the addition of the cytokines. Addition of melanoma cells resulted in a larger magnitude of decrease of the paracellular barrier relative to the basolateral component.

To begin in the sterile hood, screw autoclave 24 well pots into the ethanol sterilized cell module. Add 900 microliters of base media into each pot. Now connect the autoclave dipping electrodes magnetically to the cell module lid.

Then close the lid over the wells so that the dipping electrodes fit into the bottom electrode pots. Next, attach the cell module to the adapter in an incubator. Then launch the software and click on the layout tab to load and annotate the experimental plate map.

Use a sterile 24 well culture plate to hold the membrane inserts. Where coating is required, add extracellular matrix solution to the intersection of the well. With a single channel pipette, carefully see the brain endothelial cells into the apical chamber of each well insert.

To create a cell-free well, pipette only complete media into one of the inserts. Then, transfer the cell module from the incubator into a sterile hood. Using tweezers, carefully transfer the prepared inserts into the electrode pots.

Place the cell module back in the incubator over the adapter. Then locate the spectrum section in the experiment control tab and select start at 1 Hz and stop at 100 KHz to acquire data over the entire frequency range. Next, under the wait time, select 15 minutes to permit continuous measurements at the fastest rate.

Press start to initiate data collection. After approximately 48 hours, once a plateau in the endothelial resistance is reached, place the prepared treatments in an incubator to maintain an optimal temperature. Remove the cell module at a selected treatment time that begins immediately after a 15 minute measurement.

In a sterile hood, open the cell module, then carefully pipette 70 microliters of the treatment into the apical chamber of the respective insert. Immediately return the cell module to the adapter before the next measurement begins. To end the experiment, press the stop button, then click on file, followed by export to export the data directly to an XLS file and name it appropriately.

While some variation was observed between replicate wells, data normalization, reduced this variation and allowed for a better comparison of the treatment with the control. A transient decrease in trans endothelial electrical resistance was observed when cytokines were added. The melanoma cell lines caused a substantial decrease in resistance within five hours.