Use an egg candler to trace along the perimeter of the air pocket above the E14 or E15 embryo with a pencil and cover the outlined area with transparent tape. Using curved or fine scissors, gently cut around the traced area. Be careful not to cut into the embryo membrane or blood vessels.
Then discard the top piece of the shell. After decapitating the chick embryo, place the head in a 10 centimeter dish with a cold, sterile CMF solution. Using sterile fine forceps, peel away the skin covering the brain to reveal the underlying dura mater.
Remove the forming skull bones on the left and right sides of the brain. The forming skull bones do not yet cover the majority of the brain. Gently use fine pointed forceps to tear through the meninges, overlying the center of the brain and peel it to each side to uncover the brain.
Using fine curved forceps, scoop the brain up from the bottom front and gently pull it out of its cavity. Dissect the brain into its three main parts forebrain, midbrain, or optic tecta and cerebellum. Remove the overlying pia mater from the tecta using fine forceps and place the dissected brain regions in a small sterile dish on ice.
For embedding and slicing the brain, gently pick up either the optic tectum or forebrain region with curved forceps as a scoop and blot slightly on sterile gauze to remove extra liquid. Prepare a simple mold by forming aluminum foil around an appropriately sized object. Here, a small rectangular metal vibrating tissue slicer block is used as the object.
Use a sterile transfer pipette and fill the mold with low melt agarose. Quickly place the brain region in agarose and let it solidify for approximately four to five minutes on ice. Cut the sections on a vibrating tissue slicer using a sapphire knife.
Use the sterile spatula to scoop up and remove the slice from the tray. Gently slide the section off the spatula and onto a membrane insert using another sterile spatula. Place the six well plate of brain slices on membrane inserts in the cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.
On the day after platting, use a sterile Pasteur pipette to aspirate the media from below the insert and add one milliliter of fresh slice media to each well underneath the membrane insert. Next, to introduce the GBM cells onto the brain slices, remove one to several spheroids from their culture dish using a 20 microliter micro pipette set to five microliters. Gently expel the media with spheroids onto the desired brain slice and allow the spheroids to culture on the slice for two to five days.
The viability results of x vivo optic tectum slices after one week in culture are shown in this figure. The confocal images of a brain slice stained for nuclei with bisbenzimide and immunostained for laminin clearly shows normal and intact blood vessels optically sectioned in various configurations. The maximum projection image of the confocal Z stack of the brain slice stained for SOX 2 transcription factor and total nuclei with bisbenzimide is shown here.
These results suggest that chick embryo brain slices could be cultured on membrane inserts for approximately two weeks and remain viable with normal appearing blood vessels and transcription factor expression.
