Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

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,