This article discusses the process of embedding spheroids, which are three-dimensional cellular aggregates that simulate the tumor microenvironment. The method involves treating spheroids with paraformaldehyde for preservation, followed by embedding in agarose gel for further analysis.
Spheroids are three-dimensional in vitro spherical cellular aggregates that mimic the in vivo tumor microenvironment and help study tumor progression. To preserve these spheroids by embedding, begin by taking a sterile tube containing spheroids in a desired buffer.
Treat the spheroids with paraformaldehyde. Paraformaldehyde crosslinks cellular proteins, resulting in the stabilization and preservation of cell structure. Centrifuge the spheroids and remove the paraformaldehyde-containing supernatant. Resuspend the spheroid pellet in buffer.
Next, prepare the desired concentration of molten agarose gel solution. Make an agarose gel drop on a microscope slide. Maintain the slide on a heating block to avoid the solidification of agarose.
Using a micropipette with a wide orifice tip, collect the spheroids from the tube. Carefully pipette the spheroids into the center of the agarose gel drop without touching the microscope slide. Subsequently, incubate the slide at a suitable temperature to allow the ECM to solidify and entrap the spheroids within.
Gently transfer the semi-solid drop from the microscope slide into a tissue cassette. Store the embedded spheroids in ethanol until further 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.
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