Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

Take a culture of human brain tumor cells.

Treat with a dissociation enzyme to disrupt cell junctions, separating aggregated cells.

Wash with a buffer to stop enzyme activity, then add a culture medium to maintain cell viability.

Introduce methylcellulose, a suspending agent.

Transfer the suspension into a round-bottom microplate and incubate.

The viscous methylcellulose keeps the cells suspended, promoting cell-cell interactions for aggregation into spheroids.

Mix the spheroids with a collagen matrix that mimics the physiological extracellular environment.

Transfer the suspension into a microplate and incubate, allowing the matrix to form a gel that entraps the spheroids.

Add the growth medium on top of the gel.

At the spheroid periphery, tumor cells lose cell-cell adhesion and adopt an invasive phenotype.

These cells secrete proteases to degrade the matrix, extend protrusions to anchor themselves, and move forward, invading the matrix.

The spheroid model exhibiting tumor invasion is ready for analysis.

To generate uniformly-sized tumor spheroids, first wash the tumor cell culture of interest with 5 milliliters of PBS, and treat the cells with 0.5 to 1 milliliter of dissociation enzyme. Afte