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

Abstract

Source: Chadwick, E. J. et al. A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies. J. Vis. Exp. (2015).

This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Plating the Slices onto the Inserts

1. Fill each well of a 6-well plate with 1,200 µl of slice culture media. Fill any unused wells with 1,200 µl PBS and fill the center space of the plate with 5 ml 1x PBS. Store this plate at 37 °C.
2. Using the slotted spoon tool, place a brain slice into the media on the insert, gently pushing the slice to be fully submerged. Repeat for all slices.
3. Use a p1000 to draw out 1 ml 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. Do this for the remaining slices.
4. Pick up the insert by the rim with forceps, tilt, and remove excess media. Then, quickly transfer the insert into a 35 mm² dish containing 1 ml of slice culture media. Use 2 pairs of sharp forceps to remove the agarose around the tissue, taking care not to stretch/damage, slice, or poke a hole in the membrane (easiest if you make a tear at the very edge of the agarose, then pull apart the agarose from either side). Then, move the insert back to the 6-well plate and repeat for the remaining slices.
5. In a tissue culture hood with the blower off (to prevent slices from drying out), remove agarose fragments from each membrane.
6. Transfer the slices to the 6-well plate prepared in step 1.1 and store the plate at 37 °C. Incubate slices for 24-48 hr.

2. Changing the Slice Culture Media

1. Repeat step 1.1 with a fresh 6-well plate.
2. In a tissue culture hood with the blower off, transfer the inserts to new 6-well plates containing fresh slice media.

3. Plating Tumor Cells on the Slice

1. Sonicate the Cm-DiI dye and spin on medium speed for 2 min.
2. Spin down tumor cells being used for the overlay at 201 x g for 5 min. Then resuspend in 2 ml of slice culture media.
3. Add 7 µl of Cm-DiI into the 2 ml of cells and media and incubate at 37 °C for 20 min.
4. Spin down cells at 201 x g for 5min and resuspend in 200 µl of slicing media. Triturate gently to dissociate the cells (10-20 times or until the pellet is gone) and then add 800 µl slicing media to make 1,000 µl total volume.
5. Count viable tumor cells using Trypan blue. Add green fluorescent microspheres to tumor cells at the same concentration as cells. These inert microspheres will serve as a control. Spin down the cell/microsphere mixture at 201 x g for 5 min and resuspend in the desired amount of slice culture media. Plate cells at a density of 6,000 cells/slice in 65 µl volume. The number of cells plated per slice can be increased depending on preference and availability.
6. In the tissue culture hood, dispense cells in 65 µl of media/slice onto the center of the slice.

Materials

HEPES	Invitrogen	17504044
Glucose	Invitrogen	17502048
Pennicillin Streptomycin	Life Technologies	15140-122
HBSS	Life Technologies	14185-052
B-27	Life Technologies	17504-044
N2	Life Technologies	17502-048
Glutamax	Life Technologies	35050061
Neurobasal-A- Medium minus phenol red	Invitrogen	12349015
Low Melting Point Agarose	Promega	V2111
Slice Culture Inserts	Milipore	PICM0RG50
Laminin	Invitrogen	23017015
Cm-DiI	Invitrogen	V22888
Microspheres	Life Technologies	F-21010