This article describes a method for establishing a 3D co-culture model using mouse brain slices and human brain tumor cells. The procedure involves embedding brain slices in agarose, incubating them with tumor cells, and maintaining the culture for a week.
Begin with an agarose-embedded mouse brain slice.
Place the brain slice on a membrane insert containing a culture medium.
Submerge the slice to ensure uniform exposure to nutrients.
Collect a small volume of the medium from the insert and transfer it to the bottom of the well.
Remove the excess medium from the insert.
Transfer the insert to a Petri dish containing the culture medium.
Carefully remove the agarose edges from the slice to make it accessible for subsequent interactions.
Place the insert in a new well containing the culture medium and incubate it to facilitate slice recovery.
Add human brain tumor cells to the slice and incubate.
Tumor cells interact with brain cells and the microenvironment within the slice, initiating multiplication and establishing a 3D co-culture model.
Using a slotted spoon, place a brain slice into the media on the insert, and gently press the slice so that it is fully submerged. Repeat the procedure for all the slices. Next, draw out one milliliter of slice culture media from the top of the insert, and dispense it into the bottom of the well.
Remove and discard the excess media until the edges of the agarose around the brain slice become visible, and do this for the remaining slices. Then, pick up the insert by the rim with forceps, and tilt to remove any excess media.
Quickly transfer the insert into the 35-millimeter dish containing one milliliter of slice culture media. Remove the agarose around the tissue, taking care not to stretch or damage the slice or poke a hole in the membrane. Subsequently, move the insert back to the six-well plate, and repeat for the remaining slices.
In a tissue culture hood with a blower off, remove agarose fragments from each membrane. After that, transfer the slices to the six-well plate prepared earlier, and store them at 37 degrees Celsius for 24 to 48 hours. In this procedure, transfer the inserts to a new six-well plate containing fresh slice media in a tissue culture hood with a blower off. Then, dispense the tumor cells in 65 microliters of media onto the center of the slice. Maintain the brain slices at 37 degrees Celsius in culture for a week and change the media daily.
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