Reproducible 3D Glioblastoma Migration Assay with Magnetic Nanoparticle Mediated Spheroid Localization Under Hypoxic Conditions

We focused on visually capturing glioblastoma tumor migration, allowing the anti-invasive properties of therapies to be reliably and reproducibly examined. The incredible heterogeneity of glioblastoma is difficult to model. This protocol captures individual glioblastoma tumor invasiveness in a reproducible manner.

To begin obtain the gliomaspheres in a culture flask and keep all the reagents ready under sterile conditions. Using a 10-milliliter serological pipette and pipette aid, collect the gliomaspheres from the flask. Transfer the gliomaspheres into a 15-milliliter conical tube.

Centrifuge at 300g for three minutes, and discard the supernatant without disturbing the cell pellet. Resuspend the pellet in one-milliliter Accutase at room temperature. Use a P-1000 pipette to agitate the solution every two to three minutes and incubate for a total of 10 to 15 minutes.

After incubation, add an equal volume of gliomasphere media to the cell suspension to deactivate the Accutase. Centrifuge the single cell suspension at 300g for three minutes at 37 degrees Celsius. Then, resuspend the cell pellet in three to five milliliters of complete gliomasphere medium, depending on the pellet size.

Note the exact volume used for subsequent cell counting. Take a 10-microliter aliquot of the single cell suspension and gently mix it with 10 microliters of trypan blue in a 1.5-milliliter microcentrifuge tube. Load 10 microliters of this mixture into an automated cell counter.

Prepare a master mix of the glioma cells in the gliomasphere medium containing one microgram per milliliter propidium iodide and 0.05 microliters iron oxide nanoparticles. Seed 500 single glioma cells per well from this master mix into an ultra-low attachment 96-well spheroid microplate. Incubate the plate for 48 hours at 37 degrees Celsius, 5%carbon dioxide, and 21%oxygen in a humidified cell culture incubator to allow gliomasphere formation.

To prepare the Matrigel, dilute the Matrigel extracellular matrix stock 1:10 in ice cold complete neurosphere medium after 48 hours of gliomasphere formation. Perform a second 1:10 dilution to obtain the Matrigel working solution. Keep the diluted Matrigel on ice.

Pre-chill the imaging plate and store it on ice. Dispense 50 microliters of the Matrigel working solution into each well of a 96-well micro clear flat bottom plate. Incubate the plate at 37 degrees Celsius for at least one hour.

Then, remove 30 microliters from each well, leaving 20 microliters of polymerized Matrigel coating the bottom of each well. Spray the magnetic plate with 70%ethanol and wipe dry under sterile conditions. Place the Matrigel-coated microplate onto a magnetic 96-well plate drive.

Pipette 80 microliters of medium containing the single gliomasphere from each well. Gently dispense it onto the Matrigel-coated plate. Treat the spheroids according to the experimental design.

Now, transfer the plate with the magnetic drive to the imaging system for live imaging. Configure a programmable inverted live cell microscope to acquire live cell images. Set the camera to 2x2 Binning.

Gently load the multi-well plate without the magnetic drive into the microscope. Adjust the microscope stage, acceleration, and speed to 10%If applicable, select the plate material in the software so the optics are adjusted to match. Adjust the magnification to encompass the entire invasive field of view.

Bring a gliomasphere into focus and select the 5X objective and 0.5X tube lens to yield 2.5X magnification. Check both the phase gradient contrast, transmitted light, and fluorescence channels. Adjust the exposure time and light intensity to avoid overexposure.

Assign a position that includes the x, y, and z-coordinates. Use hardware auto-focusing to ensure consistent imaging of the same focal plane. Gliomaspheres derived from newly-diagnosed and recurrent patient tumors exhibited dual phenotypes and random cell-to-cell associations.

Transmitted light images of gliomaspheres cultured for 48 hours showed a dark, speckled appearance in gliomaspheres containing iron oxide nanoparticles. Gliomaspheres cultured without nanoparticles showed random localization, whereas those cultured with nanoparticles showed consistent central localization. Representative transmitted light images showed invasion dynamics at zero hours and 72 hours in vehicle-treated gliomaspheres and Drug X-treated gliomaspheres.

Quantification of the invasive area at 72 hours, measured as the total area covered at the time of fixation, revealed significantly decreased invasion in Drug X-treated gliomaspheres compared with vehicle-treated controls. This protocol enables the scalable, high-throughput analysis of glioblastoma tumor-specific migration changes in response to various conditions and our therapies. Optimizing iron oxide concentration ensures minimal use and controlled steroid positioning, greatly enhancing assay consistency, accuracy, and overall performance.

Future studies can develop more representative brain ECM matrices to better mimic glioblastoma migration, and thereby improving translational relevance in in vitro systems.