Three-Chambered Array-Based Impedance Assay: A Real-Time Analysis Technique to Assess Invasive Potential of Cancer Cells by Measuring Electrical Impedance

Cancer-associated stromal cells secrete chemical factors that can trigger the metastatic tumor cells residing on the tissue basement membrane. Consequently, the cancer cells invade the basement membrane and migrate toward the tissue's extracellular space.

To measure the invasive capacity of cancer cells in vitro, begin with a multiwell plate with multiple three-chambered arrays. In each array, the bottom chamber has an adherent culture of stromal cells. The top chamber with fused impedance-measuring microelectrodes - miniature electrodes that measure a cell's electrical activity - is overlaid by cancer cells, which adhere to the extracellular matrix-coated microporous membrane present above the electrodes.

During incubation on an impedance-based analyzer, the stromal cells release chemotactic factors into the medium, which move toward the cancer cells. These factors move through the semi-permeable membrane present at the bottom side of the middle chamber.

The stromal cell-secreted factors signal the adherent cancer cells to transform into invasive phenotypes. In response, the invasive cells migrate through the microporous membrane reaching the interdigitated electrodes on the opposite side of the membrane.

Over time, as more cancer cells stick to the electrodes, they cause cellular impedance - hindrance to the flow of current by cells. The measurement of cellular impedance is indicative of the invasive potential of cancer cells.

Place all three sterile chambers in the tissue culture hood. Locate the knob on the short side of the lower chamber, and orient the lower chamber so that the knob is facing the experimenter.

Add 30,000 to 50,000 cells in 90 microliters of media to each well from the lower chamber without forming any bubbles. Use 5% of fetal bovine serum-supplemented media in two lower chambers as a positive control for cell motility, and use 0% serum-supplemented media as a negative control.

Rotate the lower chamber at 90 degrees after letting it settle for 10 to 15 minutes in the hood. Then, place the middle chamber on top, so that the knob on the lower chamber slides into the notch on the middle chamber. Push the chamber vertically downward, until a click is heard from each of the long sides of the assembly.

Add 160 microliters of serum-free media to all the wells of the middle chamber. Make sure a dome-shaped meniscus is visible after wells are filled, and place the top chamber, with electrodes facing down, onto the middle chamber to align the blue dots on the middle and top chambers. Push the chamber vertically down until a click sound is heard from each of the long sides of the assembly.

Add 20 to 50 microliters of serum-free media to the top chamber. Mount the assembly on the dual-purpose cell analyzer in the tissue culture incubator, and wait 30 minutes before measuring the background.

Open the cell analyzer software, then select the cradle to be used, followed by a click on the 'Message' tab, and make sure it says 'Connections OK' to ensure the array is well-placed in the cradle and the electrodes are well-aligned with the sensors.

Click on the 'Experiment Notes' tab and fill in all the possible information about the experiment. Then, click on the 'Layout' tab and fill in the description of the array layout. Then, click on the 'Schedule' tab and add two steps from the 'Steps' menu; a background step with one sweep, and a test step with 100 sweeps.

Once the array has been in the dual-purpose cell analyzer incubator for 30 minutes, click on the 'Play' button to start background measurement.

After the background measurement is done, remove the array from the cradle, and place it back in the cell culture hood. Then, add 30,000 to 50,000 cells in 100 microliters of serum-free media to each well of the top chamber. These are the cells that the electrode will detect once they successfully migrate through the membrane.

Let the assembly stand in the hood for 30 minutes before mounting on the dual-purpose cell analyzer for impedance measurement.

Place the array back into the dual-purpose cell analyzer, and check the 'Message' tab for the 'Connections OK' message. Click on the 'Play' button to start impedance measurement, and then click on the 'Plot' tab to monitor the progress of the signal.

If the endpoint is reached before 25 hours, click on the 'Abort' step from the 'Execute' dropdown menu. To export data, right-click on the graph, choose 'Copy' in the list format, and then, paste the data into a spreadsheet.