3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

First, pour cold basement membrane matrix in a glass-bottom dish and spread it evenly with a pipette tip. Let the membrane solidify at 37 degrees Celsius, with a continuous supply of carbon dioxide. Trypsinize the fluorescently-labeled cancer cells to detach them from the plate surface and resuspend them in a culture medium.

Transfer these resuspended cells into a conical tube and spin. Remove the supernatant and resuspend the cells in the culture medium. After counting, mix this cell suspension containing the desired number of cells with the basement matrix in an equal ratio.

Gently plate the cell matrix mixture onto the solidified basement membrane matrix-coated dish. Let the cell matrix mixture solidify at 37 degrees Celsius, with a continuous supply of carbon dioxide. Once the mixture solidifies, add culture medium to the dish and incubate it for the desired period.

Cancer cells can form actin-rich membrane protrusions, and release proteases to degrade the extracellular matrix, thus invading the matrix. Place the dish under a fluorescent microscope at different time intervals to analyze the cells forming protrusions. In the example protocol, we will culture breast cancer cells on a 3D basement membrane matrix to study the invasion process.

Before beginning the culture procedure, place the basement membrane matrix, a P200 pipette and pipette tips on ice overnight, at 4 degrees Celsius. The next day, use the tip of the ice-cold 200-microliter pipette to spread 50 microliters of the matrix in a spiral pattern over the bottom of a Confocal number 1 glass-bottom dish. Then place the dish in a cell culture incubator at 37 degrees Celsius, with 5% carbon dioxide, for at least 30 minutes.

While the matrix is undergoing solidification, trypsinize a 70% to 80% confluent 100-millimeter plate of cells. Once the cells have begun to detach, inactivate the trypsin with 10 milliliters of medium and then transfer the cell suspension into a 15-milliliter conical tube.

Centrifuge the cells for three minutes at 100 Gs and 4 degrees Celsius. While the cells are spinning down, aliquot 50 microliters of matrix into one 1 milliliter microcentrifuge tube per dish of basement membrane matrix and place the tubes on ice. When the cells have finished spinning, aspirate the supernatant without disturbing the pellet and then resuspend the cells in 1 milliliter of media and count them.

Next, transfer 2.5 times 10 to the 4th cells into a new microcentrifuge tube, topping off the cell suspension with media to a final volume of 50 microliters. Then add the 50 microliters of ice cold basement membrane matrix to the cells at a 1:1 ratio, for a final volume of 100 microliters. Gently plate the matrix to cell mixture onto the solidified basement membrane matrix and allow the cells to become embedded in the matrix in the cell culture incubator.

After 30 minutes, cover the matrix with 2 milliliters of media, and place the dish back into the incubator, changing the media every day for the duration of the experiment. Use the 10x objective of a light microscope once every day for the duration of the experiment to take 20 differential interference contrast images of the colony suspended in the basement membrane matrix. Analyze the images blindly to determine the cell colony stellate formation. A colony is deemed to be stellate if one or more projections from the spheroid of cells are observed.