Two-dimensional cell cultures do not adequately mimic in vivo tumor growth. To improve, 3D procedures have been developed using spheroids. Our 3D glioblastoma spheroid-based assay is particularly well-suited to investigate brain tumor invasion into the healthy brain tumor environment.
Three-dimensional culture models can be used to recapitulate a multicellular architecture, heterogeneity and metabolic state of tumors allowing more rigorous evaluation of drug effects. Be sure not to touch the bottom of the well or to completely remove the supernatant, to keep the gel on ice, and to add the cells to the gel quickly. To generate uniformly sized tumor spheroids, first wash the tumor cell culture of interest with five milliliters of PBS and treat the cells with 0.5 to one milliliter of dissociation enzyme.
After five minutes at 37 degrees Celsius, stop the reaction with four 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 one times 10 to the sixth cells per eight milliliters of complete growth medium supplemented with two 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 degree 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. To perform a migration assay, when the tumor cells have formed uniformly sized spheroids, coat a six-well plate with 0.2 milligrams per milliliter of Matrigel in growth medium for 30 minutes at 37 degrees Celsius. At the end of the incubation, remove the Matrigel and add two milliliters of complete growth medium to each well.
Transfer the spheroids from the round bottom plate in 50 microliter volumes into individual wells of the six-well plate and place the plate into the cell culture incubator for 24 hours. The next day, stain the spheroids with 10 nanograms per milliliter of Hoechst for 30 minutes at 37 degrees Celsius. For image acquisition after an invasion or migration assay, place the plate onto the stage of a Brightfield confocal microscope equipped with a video camera and obtain images over the appropriate experimental period.
To analyze the images in a semi-automated manner, import the images into Fiji and click Plugins, Macros, Interactive Interpreter. Copy and paste the adapted purple loop. Keep the purple sentences and add the green sentences of interest as necessary.
Then to measure the area of each spheroid, click Analyze and Measure. To measure hypoxia in distinct areas of the spherical structure, carbonic anhydrase IX staining can be used to determine the hypoxic activity. In this representative analysis, more carbonic anhydrase IX positive cells were observed in the spheroid center.
Hypoxic cells located in the spheroid core tend to be more glycolytic than the cells surrounding the core. Mitochondria can be imaged by electron microscopy for further analysis. The spheroid diameter is directly correlated to the density of the cells that make up the 3D cell structure and Fiji macros can be used to quantify the proliferation, invasion or migration of the cells within each spheroid or migration of the cells within each spheroid.
Increases in the spheroid core reflect the stimulation of cell proliferation. Upon the inhibition of complex I of the mitochondrial respiratory chain, the vast majority of ATP production in the mitochondria is compromised reducing proliferation by 20%after 72 hours. Hydrogen chloride treatment enhances collagen type I invasion over a period of 24 hours but reduces the migratory area of the spheroids 1.5-fold compared to control untreated spheroids.
As assessed by live imaging, the spheroids demonstrate high internal dynamics and move quickly. The size of the spheroids should be relatively uniform and care should be taken to manipulate the spheroids to the center of the wells for the best imaging results. This method can be also used to investigate tumor cell proliferation, migration and death as well as the expression of extracellular matrix, cell receptors or intracellular proteins of interest.
The spheroids may be also encapsulated and the effect of increased cell surface tension can be investigated upon removal of the capsules.
