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 cell dissociation enzyme with 10 milliliters of complete medium, and collect the cells by centrifugation. Re-suspend the pellet in five milliliters of fresh complete medium for counting, and re-suspend the cells at a 1.5 times 10 to the third cells per milliliter concentration.

Then seed 20 microliters of cells into each well of a 96-well round-bottom plate, and place the plate in an automated imaging apparatus inside a 37 degree celsius incubator with 5% CO2 and 95% humidity. Log into the acquisition software of the apparatus, and select Schedule to Acquire, Launch Add Vessel, Scan on Schedule, and Create Vessel, New. Next, select Scan Type, Spheroid and select the appropriate Brightfield and Fluorescence channels of interest. Set the magnification to 10 times and select the plate model and its position within the imaging apparatus drawer. Select the position of the wells to be imaged and enter the description of the experiment, including the name, type of cells, and number of cells.

For the Analysis Setup, select Defer Analysis Until Later, right-click on the timeline, and select Set Selected Scan Group Interval, and set Add Scans Every to four hours, and For a Total of to 24 hours. Then set the starting time to at least one hour after the incubation in the automated imaging apparatus. To check the spheroid growth progress, every two days, log into the imaging software and select View Recent Scans. Double-click on the experiment of interest and select Brightfield in the Image Channels panel. Then use the Measure Image Features tool to measure the diameter of the spheroids. Adding 50 microliters of complete medium per well at day four to limit any medium of apparition effects.