Establishing a Brain Tumor in an Organotypic Slice 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.