This video demonstrates a method to monitor the invasion of an endothelial cell monolayer by cancer cells using a real-time electrical impedance based technique. First, human umbilical vein endothelial cells referred to as VE are seated in 16 well EPLs coated with gold electrodes at the well bottom. To generate a confluent monolayer cancer cells are added, which attach to the VE and invade the HVAC monolayer disrupting the endothelial cell junctions Impedance readings, which depend on the total area of the well bottom covered by cells are then acquired.
The extent of invasion can then be determined based on changes in electrical impedance. The main advantage of this technique or existing methods, such as void chamber and METROGEL assays, is that the endothelial cell tumor cell interactions more closely mimic the in vivo metastatic process, and the data is obtained in real time and is more easily quantifiable as opposed to endpoint analysis for other methods. Though this method can provide insight into cancer cell invasion.
It can also be applied to other systems such as investigating cell cell interactions in immune system. Furthermore, initial phase of experiment where we observe growth of UX cells can be used to evaluate cell proliferation of any cell line without further invasion steps demonstrating the procedure will be site Rahim graduate student from my laboratory. All steps of this protocol should be performed under sterile conditions in a tissue culture hood.
The Xcel system used for this experiment is kept permanently in a 37 degree Celsius 5%carbon dioxide incubator, solely dedicated for this instrument on its first use. The instrument should be left in the incubator for at least 16 hours to equilibrate to the new temperature and humidity environment. Next, prepare Thence EPL 16 by coating it with 0.1%gelatin for one hour at 37 degrees Celsius.
Once the plate has been coated, wash it once with PBS. Then add 100 microliters of reconstituted e gm two medium to perform a blank reading. To measure background impedance in the absence of cells.
Place the EPL in the Excel agent system on the computer. Open the Excel agent software by clicking on the RTCA software icon on the desktop in the software window. Click on the layout tab and select the wells containing samples.
To program the frequency of realtime data acquisition. Click on the schedule tab, then click on the add a step icon sweeps indicates the number of readings and intervals indicates the time interval between readings. These are automatically set to one and 1.00 minute respectively.
This programs the system to perform a background reading. Click on add a step and enter 300 in the sweeps box and 10 minutes in the intervals box. This will program the instrument to perform impedance measurements for up to 50 hours.
To begin the experiment, click on the start a step icon. The background impedance of each well will be measured after the background measurement has been performed. The window displays the message.
Ready for next step, please click. Next step to start. At this time, cells can be added and the formation of a monolayer can be monitored as described.
In the next section, the human umbilical vein, endothelial cells or VE used here are cultured in EGM two medium reconstituted with the EGM two bullet kit containing growth factors supplements, and 5%FBS cells should be maintained in a 37 degree Celsius incubator in the presence of 5%carbon dioxide. On the day of the experiment, check the co fluency of HUB E Under a microscope, the cells should be a low passage number, preferably no more than six and less than 75%Con fluent to generate a hue, monolayer harvest the cells by trypsin. Once the cells have detached from the flask, all traces of trypsin should be removed by centrifugation at 200 G.Following the spin, wash the cells once with PBS.
Then re suspend the cells and reconstituted e GM two media to a concentration of 2.5 times 10 to the fifth cells per milliliter. Once the cells are resuspended, remove the background calibrated EPL from the EXOGEN system and add 100 microliters of the HX suspension to the 100 microliters of E GM two media already in the plate. Immediately place the EPL in the Excel system cell analyzer.
Start acquiring impedance readings by clicking the start step button in the software window. Allow the endothelial cells to grow. The system will continue taking impedance readings every 10 minutes.
Uve show a characteristic transient flattening of cell index four to six hours after seeding, followed by another stabilization after 16 to 18 hours. Hence, it is important to let the cells form a confluent monolayer for at least 18 hours. Once a monolayer has formed, it is time to add the invading cells in preparation for the invasion assay.
Prepare the invading tumor cells. Here osteosarcoma cells are used. Begin by harvesting the cells by trypsin.
Remove all traces of trypsin by spinning the cells at 200 G and washing once with PPS. Following the wash reus, suspend the cells while final density of one times 10 to the fifth cells per milliliter in the medium used to grow the tumor cells such as RPMI or DMEA medium containing 10%FBS. Pause the experiment by clicking the pause step icon in the software window.
Remove the EPL and aspirate the EGM two medium from the vac monolayer. Add 100 microliters of tumor cell suspension containing one times 10 to the fourth cells. Generally, a ratio of one to 2.5 tumor cells to endothelial cells works best for the assay.
However, the ratio can be optimized for individual cell lines. Place the EPL back in the Excel agent system in the incubator. Click on the continue step to continue taking impedance readings at 10 minute intervals.
Monitor the invasion in real time over the next six to 12 hours. A drop in cell index will result from the retraction of endothelial junctions and penetration by invading tumor cells. When the experiment is finished, use the exergen software to normalize the results to the time of addition of invading tumor cells.
The exergen software permits normalization to any time point VEX were cultured on the exergen system as shown in this video, and either K seven M two cells or K 12 cells were added following the formation of the monolayer. As shown here, a steep decrease in cell index occurs within a few hours of the introduction of K seven M two cells. K seven M two is a highly metastatic osteosarcoma cell line.
These cells express high levels of the cytoskeletal linker protein ene, which accounts for the invasive properties of the cell line. K 12 cells, on the other hand, are less metastatic and are unable to penetrate the endothelial monolayer as efficiently as K seven M two cells. This is represented by a less deep decrease in cell index as shown here.
The control shown here represents impedance of the uve monolayer. In the absence of cancer cells, experiments were performed in triplicate. After watching this video, you should have a good understanding of how to measure invasion in real time by generating an endothelial cell monolayer, and challenging the monolayer with cancer cells.
Initial steps of the experiment involving HU XL monolayer formation can be used to evaluate cell proliferation by impedance.Two.
