Spheroid Generation from Cell Lines: A Three-Dimensional (3D) Cell Culture Method

Grow the desired cell line as an adherent 2D monolayer. Remove the culture media and wash with phosphate-buffered saline or PBS. Then, add trypsin and incubate while the cells detach from the flask surface and become round-shaped. Add fresh media to neutralize the trypsin and suspend the cells. Transfer the mixture to a conical tube, and use a centrifuge to pellet the cells.

Replace the supernatant with fresh media and resuspend the cell's pellet. Use a hemocytometer to count cell numbers and calculate a working concentration. Then, transfer the right amount of cell mix into a round-bottom ultra-low adhesion well plate and incubate. Cells settle to the bottom and aggregate. They will ultimately attach to each other and form a compact 3D cell assembly, the spheroid.

In the example protocol, we will see how to generate 3D spheroids from a colon cancer cell line, and how live imaging is applied to track their formation. Begin by washing the colorectal cancer cell line of interest with five milliliters of PBS, and removing the cells from the flask bottom with 0.5 milliliters of trypsin for five minutes at 37 degrees celsius. After confirming detachment under a microscope, neutralize the