To cast 3D Petri dishes, rinse the micromolds in distilled water and place them in an autoclavable container for autoclaving. Add 0.9%sterile saline to the autoclave agarose powder in a 200 milliliter glass bottle. Screw on the lid loosely to avoid pressure buildup and swirl the bottle to mix the agarose powder.
Boil the agarose powder in a microwave oven with intermittent shaking. Once the agarose solution is cooled down, pipette the molten agarose into the micro mold and wait until it solidifies. Afterward, carefully flex the micro mold to remove the 3D Petri dish and transfer it to a 12-well tissue culture plate.
To equilibrate the 3D Petri dish, add 2.5 milliliters of McCoy's 5A medium per well, and incubate for 15 minutes at 37 degrees Celsius and 5%carbon dioxide. After the final equilibration, completely remove all culture medium surrounding the 3D Petri dish and inside of the dish. Dropwise, seed 190 microliters of cell suspension containing 40, 500 cells into the seeding chamber and allow the cells to settle for 10 minutes.
Add two milliliters of McCoy's 5A medium to the outside of the 3D Petri dish, and incubate at 37 degrees Celsius and 5%carbon dioxide. After four days, the spheroids are compact enough for microscopy. Add 200 microliters of the coating solution to each well of a 96-well U-bottom culture plate, and incubate for one minute.
Remove the excess coating solution and allow the plate to dry for one hour. See 200 microliters of cell suspension containing 500 cells to each well and incubate at 37 degrees Celsius and 5%carbon dioxide for at least four days.
