Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Glioblastoma possesses stem-like cells that migrate along the axonal tracts of myelinated neurons to invade distant locations in the brain. To recapitulate the migration of glioblastoma or GBM cells on neurons in vitro, begin by taking a glass coverslip.

Activate the coverslip by oxygen plasma treatment and prime it with a functionalizing agent. This treatment supports easy access to the chemicals in subsequent steps. Add PEGylated solution to the coverslip and incubate. This solution coats the coverslip preventing neuron adhesion at random places.

Dispense a photoinitiator polymer onto the coverslip and mount it in the imaging chamber. Place the chamber on the stage of a fluorescence microscope. Once inside, the UV illumination activates the photoinitiator molecules locally at the designated areas corresponding to the envisioned micropatterns.

The activated photoinitiator molecules cleave the underlying PEG layer, generating the desired pattern. Now, add the laminin solution and incubate. Laminin - a glycoprotein - gets deposited at specific places where the PEG layer has been removed. Seed the media containing rat hippocampal neurons onto the coverslip.

Incubate to allow adherence of the neurons onto the laminin-coated arrays to obtain neuron-micropatterned coverslips. Deposit a suspension of fluorescently-labeled GBM cells over the micropatterned neurons. Image to see the fluorescent GBM cells migrating on neurons.

To make a substrate for the micropatterning, treat 18-millimeter circular glass coverslips by air or plasma activation for 5 minutes before placing the coverslips in a desiccator with 100 microliters of 3-aminopropyl triethoxysilane for 1 hour. Then, incubate a solution of Peg-SVA at 100 milligrams per milliliter for 1 hour for gel deposition. At the end of the incubation, add 3 microliters of PLPP and 50 microliters of absolute ethanol onto the center of the slide and wait until it dries completely.

For glass slide micropatterning, mount the coverslip in a Ludin chamber and place the chamber onto the stage of a microscope equipped with an auto-focus system. After imaging, load the micropatterned images into the software. After automatic UV-illumination sequencing, use a pipette to wash the PLPP from the coverslip extensively with PBS. Then, incubate the coverslip with 50 micrograms per milliliter of laminin for 30 minutes, followed by another wash with PBS as demonstrated.

To set up an embryonic rat hippocampal neuron culture on the micropatterned coverslips, after the last wash, rehydrate the glass slides with neuronal cell culture medium. Seed 5 times 10 to the fourth rat hippocampal neurons suspended in neurobasal medium enriched with 3% horse serum per square centimeter onto each micropatterned coverslip for a 24-hour incubation in a 5% carbon dioxide incubator at 37 degrees Celsius. Centrifuge dissociated glioblastoma cells for 5 minutes at 1,000 rpm and resuspend the pellet in glioblastoma cell culture medium. Then, deposit 1 times 10 to the third GBM cells over the micropatterned neurons.