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