This article describes a method for creating tumor spheroids from human brain tumor cells to study tumor invasion. The process involves dissociating cells, suspending them in methylcellulose, and embedding them in a collagen matrix.
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. After 5 minutes at 37 degrees Celsius, stop the reaction with 4 to 4.5 milliliters of PBS, and transfer the detached cells into a 15 milliliter conical tube, containing 10 milliliters of complete growth medium.
After counting resuspend the cells at a 1 times 10 to the 6 cells per 8 milliliters of complete growth medium, supplemented with 2 milliliters of 2% methylcellulose concentration, and transfer the suspension to a sterile system container. Then, add 100 microliters of cells to each well of a 96 well round bottom plate and place the plate into a 37 degrees Celsius, 5% carbon dioxide incubator for three to four days.
To perform an invasion assay, when the tumor cells have formed uniformly-sized spheroids, transfer each cell structure into a 500 microliter tube, and wash the spheroids once with 200 microliters of PBS. After the second wash, transfer the spheroids carefully into 100 microliters of freshly prepared collagen matrix, and add each spheroid suspension into the center of each well of a flat bottom 96 well plate.
After a 30 minute incubation at 37 degrees Celsius, add 100 microliters of complete growth medium on top of the gel in each well.
Be sure to place all of the spheroids into the center of each well for the best imaging of the tumor cell invasion.
繁體中文
