Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis

To begin, fix the spheroids in a fume hood on day one as outlined in the text protocol. On day two, place the agarose gel into a water-filled beaker in a microwave oven to heat it, and keep the gel warm on a benchtop heating plate set to 60 degrees Celsius until needed.

Wash the spheroids twice in the fume hood using 1 milliliter of ice-cold 1X PBS per wash. Then, aspirate most of the PBS. Prepare a 20-microliter pipette tip by cutting it at an incline to obtain a pointier tip with a larger hole.

After this, make an agarose gel drop on a microscope slide. Place the slide on a warm heating block to prevent the agarose from solidifying. Using the modified pipette tip, catch as many spheroids as possible in a volume of 15 to 20 microliters.

Carefully inject the spheroids into the center of the agarose gel drop, making sure to not touch the microscope slide. Incubate at room temperature or at 4 degrees for 5 to 10 minutes to let the agarose gel drop harden. When the gel drop has solidified somewhat but is still rather soft, use a scalpel to carefully push it from the microscope slide into a plastic tissue cassette. Transfer the tissue cassette to a beaker filled with ethanol and store at room temperature.