Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells

Working in a laminar flow hood, place a 35-millimeter glass-bottom Petri dish on a paper grid on ice. Using a pre-cooled pipette tip, dispense approximately 10 microliters of growth factor-depleted ECM at the center of the grid. While viewing the dish through a stereomicroscope under 2X to 4X magnification, place the DRG at the center of the ECM, close to the bottom of the dish. After harvesting and washing 40,000 cancer cells of interest from confluent cultures, resuspend the pellet in 40 microliters of ECM on ice.

Once again, view the grid through the stereomicroscope, measure 500 microns from the DRG, and dispense 10 microliters of the cell suspension at this point. Repeat in all directions from the DRG. Leave the dish in the laminar flow hood for 10 to 15 minutes to solidify but not dry out. Once the ECM has solidified, slowly add supplemented DMEM by pipetting against the sidewall of the plate. Add approximately 2 milliliters of media, enough to cover the ECM.

The addition of DMEM should be done very slowly by pipetting against the sidewall of the plate to avoid detachment of the DRG from the plate bottom.

Set the plate in the tissue culture incubator and follow the instructions in the written portion of the protocol to maintain the culture.