Cell culture and spheroid formation

HeLa cells are cultured in Dulbecco’s modified Eagle’s medium (11–965-092, Fisher Scientific Ltd. Canada), supplemented with 10%, fetal bovine serum (F7524–500ML, Sigma-Aldrich, USA), and 1% penicillin-streptomycin (15–140-122, Thermo Fisher Scientific Inc., USA). During the culture period, the cells are maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide. In the 2D cell culture, the medium is changed every 48 hours. When cells reach 80% confluency, the cells are trypsinized, counted, and resuspended in cell culture medium at a concentration of 2 × 10⁶ cells/ml. In the next step 0.2 mL of cell suspension is then placed on top of an agarose mold, which was previously placed in the well of a 12-well plate for spheroid formation (Fig 2(a)). This results in spheroids which on average contain approximately 270 cells. Half of the medium is replaced daily. For measuring the spheroid morphology, images are taken every day (Fig 2(b)). The measured spheroid perimeter is used to determine the average spheroid diameter by calculating the surface area.

(a) Schematic of cell spheroid formation in microwell array from micropatterned agarose well. (b) After the expansion of HeLa cells in standard tissue culture plates, cells were transferred into agarose microarrays for 3D cell spheroid formation with a diameter of 200 μm. Circularity and diameter of the spheroid versus time are plotted in (b2) and (b3), respectively.