Adherent Cell Sphere Formation and Quantification

The adherent cells were treated with 0.25% trypsin to prepare a single-cell suspension. The cell density was adjusted to 1 × 10³ cells/mL, and then seeded into growth medium containing stem cells (adding 1% N2, 2% B27, 100 ng/mL epidermal derived growth factor, and 1% antifungal agent [Invitrogen, Carlsbad, California, USA]; 20 ng/mL human platelet growth factor, [Sigma-Aldrich, Shanghai, China]) in a low adsorption 6-well plate (Corning Inc., Corning, NY, USA) with 2 mL/well. The cells were then subsequently cultured to obtain suspended cell spheres, and semi-quantitative liquid exchange was performed every 2 days. After such a continuous culture for 10 days, the number of newly formed suspended cell spheres per well were recorded and averaged under a microscope. The experiment was repeated 3 times [20 (link), 21 (link)].