Fig. 4

Illustration of day-by-day generation protocol and representative checkpoint images of ATC spheroids using AggreWell plate and Matrigel embedding. (A) Briefly, one day before the generation of tumor spheroids, the ATC cells cultured as colonies are dissociated into single cells and aggregated at a concentration of 2 × 10⁶ cells in 1 mL of Medium A per well in a 24-well AggreWell plate. After centrifugation, the single cells concentrate at the bottom of the AggreWell plate, forming a circular cell cluster layer. On day two, an additional 500 µl of Medium A is added to provide a better proliferative environment. During the process, any dead cells adhered to the aggregates are released so that the surface of the aggregates becomes clean. Starting on day three, all aggregates from the AggreWell plates are collected and transferred to low-attachment six-well plates in Medium B (2 ml/well) and placed on an orbital shaker to provide a floating environment. On day 14, tumor spheroids are individually embedded into the center of the Matrigel coat and transferred back to the six-well plate for further culturing. Afterwards, Matrigel-coated ATC tumor spheroids will continue to be cultured in Medium B. Fresh medium is changed every two to three days to provide nutrition in order to allow the continuous growth of ATC spheroids. The culture plate is placed on an orbital shaker to provide a floating environment, where the aggregates self-organize further. The medium is regularly replenished to provide sufficient nutrition during development. (B) Representative images are taken at 10x or 20x microscope objective magnifications as noted in each image. Scale bars, 200 μm. Medium A = DMEM/F12 medium, 15% FBS, 1% GlutaMAX, 1% antibiotic-Pen/strep, and 5 µM ROCK inhibitor (Y-27,632); Medium B = DMEM/F12 medium, 10% FBS, 1% GlutaMAX