A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

The overall goal of this procedure is to grow fluorescent primary human brain tumor cells in a mouse brain slice co-culture in order to investigate the role of brain microenvironment on tumor cell growth and to test possible new drug treatments. This method can help investigate key questions in the cancer biology field, such as what brain areas support tumor growth, and potentially why brain tumors develop in specific brain areas. The main advantage of this technique is that it can also be used as a system to test new drug therapies on human brain tumor cells that do not grow in normal cell culture conditions.

To begin this procedure, hold the head of a postnatal day six mouse by its nose, and make two cuts bilaterally from the posterior skull towards the eye sockets. Keep the tip of the scissors as close to the skull as possible to avoid tissue damage. Then make a third cut across the first cut and second cut.

After that, gently peel off the skull using small spring scissors. Cut the remaining skull along the midline. Subsequently peel off the skull To reveal the olfactory bulbs.

Insert a flat faced spatula moistened with dissection buffer in between the bottom of the brain and the base of the skull. Then gently remove the brain and place it into dissection buffer on ice. In this procedure place 2 35 millimeters square dishes on ice.

Next, pour 3%low melting point agros into the two dishes until a dome forms and wait three minutes. After three minutes, place one brain into the side of an agros filled 35 millimeter dish. Then move it to the center of the dish.

Repeat the procedures for the second brain and wait 10 minutes. After 10 minutes, insert a flat spatula in between the agros and the dish in order to pop out each of the polymerized aros blocks containing a P six brain. Next, trim each aros block into a small cube around the brain, making sure that the edges are as straight as possible.

Subsequently, apply two strips of super glue on the vibram plate. Then gently place the agros mounted brains side by side over the glue For a sagittal cut, make sure that the brains are lined up with one another and are roughly the same height. Position the razor as close to the aros embedded brains as possible, and to make sure the razor is just above the brains.

Then fill the chamber with enough cold dissecting buffer to cover the blade. Now press the single slash cont button once and the light should go on. Set the cutting thickness to 400 to 450 microns vibrating frequency to 5.5 to six, and speed to four.

To set the automatic cutting, press the double arrows button. Once, then press and hold the forward button to manually cut the embedded brain release. After the razor has cut through the aros block immediately press the double arrows button again to define the end of the range for automatic cutting.

Next, press the start stop button once and the automatic cutting should begin. In the meantime, press pause to collect tissue. When it is close to the midline, press pause and set the thickness to 200 microns and the speed to three.

The desired slices of 200 micron thickness will have the olfactory bulb nicely defined through the cerebellum. Transfer each desired slice into a six well plate on ice filled with dissection Buffer, typically around 12 slices with the desired structures can be collected while the slices are on ice. Take out a six well plate with inserts.

Be encoded with laminin from the 37 degree Celsius incubator in the dissection hood. Discard the laminin and take care not to damage the inserts. Then at 3.5 milliliters of slice culture media to the top of each insert.

To form a dome shape 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 aros 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 agros 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 the blower off, remove agros 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 the 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. Next tape a piece of perfil to the lab bench and draw a circle on it. For each of the slices being stained, using a liquid blocker pap pen, put 500 microliters of PBS in the center of each circle in a soft bottom cultured dish, use a razor blade to cut a square around the slice in one milliliter of PBS.

After that, transfer the cutout slice onto the bubble of PBS in the first circle drawn on the param, and to make sure that the slice does not fold over on itself or get flipped upside down during this process, then transfer PBS from underneath the slice to the top of it and add more PBS if needed, to make sure the slice remains submerged. Repeat this step for the rest of the slices. After staining, transfer each stained slice onto a microscope slide.

Make sure the side of the membrane with the slice remains facing up and does not fold over on itself. Draw up order around the slice with a pap pen on the slide. Next place one five millimeter glass cover slip next to each side of the slide without covering the brain slice.

These small glass cover slips prevent the long glass cover slip from pushing down on the slice and damaging it. Then drop two or three drops of floral mount G on top of the slice to just barely cover it. Subsequently covered the slice with a long cover slip.

This figure demonstrates the drug treatment of tumor cells in the slice overlay assay system. Here is the graphical representation of the data collected from a mouse medulloblastoma over one week. In culture.

DMSO was the control condition and one micromolar LDE 2 25 was the treatment condition. These are the representative images of vehicle control treated slices on day one and day seven. And these are the representative images of LDE treated slices on day one and day seven Following this procedure.

Other methods like confocal imaging of the stained brain tumor slice co-culture can be performed to investigate tumor cell proliferation and the effects of drug treatment on tumor cells. This method can provide insight into how human brain tumor cells may respond to targeted drug treatment before a drug goes to clinical trial.