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

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.