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