This article describes a method for studying glioblastoma cell migration in a controlled environment using a polymer matrix. The approach involves co-culturing glioblastoma and microglial cells to observe their interactions and migration patterns.
Begin by adding a cold suspension of glioblastoma and microglial cells, each stained with a distinct fluorescent dye, into a cold polymer matrix solution.
The cold condition prevents the premature polymerization of the matrix.
Next, transfer the mixture into a porous chamber insert within a culture plate.
Incubate to allow matrix polymerization, forming a gel that embeds the cells.
Add serum-free media to the insert and serum-containing growth media to the well below, then incubate.
The chemical factors in the serum promote cell migration toward the serum-rich media.
Additionally, microglia secrete signaling molecules that interact with glioblastoma cells.
This enhances the glioblastoma cell migration to the underside of the porous insert, resembling their migration from a tumor into the surrounding tissue.
Remove the cells from the top of the chamber.
Now, fix the remaining cells with a fixative and wash them with buffer. The cells' distinct fluorescence helps track cell migration.
Thaw the matrix mix at four degrees Celsius overnight. Next, prepare a 10 mg/ml concentration of matrix in the appropriate media on ice. Then, harvest the labeled cells for suspension in matrix as previously shown. Then, pipette 1.5 times 10 to the fifth GL261 cells in a 150-microliter volume, plus five times 10 to the fourth microglia cells in 50 microliters into a 1.5-milliliter microfuge tube.
Add 2 times 10 to the fifth GL261 cells in a 200 microliter volume into a separate 1.5-milliliter microfuge tube. Spin the cells in a microfuge for five minutes at 120 times g. Then, resuspend the cells in 200 microliters of cold matrix on ice.
For this step, it is crucial to have ice tray ready in the hood. Keep tubes on ice tips in the refrigerator. Make sure everything is chilled in order to prevent polymerization of the mature gel prematurely.
After resuspension, plate 50,000 cells in a 50-microliter volume onto the center of the top compartment of an 8-micron pore size chamber insert. Then, incubate it at 37 degrees Celsius for 30 minutes to allow polymerization to occur. After 30 minutes, add 200 microliters of serum-free medium to the upper chamber and 700 microliters of serum containing cell growth medium to the lower well.
After incubating for 48 hours, remove the cells from the top part of the chamber by gentle aspiration. Fix the chambers as before, and replace the fixative with 500 microliters of 1X PBS before imaging the cells.
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