Formation of Multicellular Spheroids

To form spheres in vitro, we mixed the cells at the same ratio as in our previous report [14 (link)], A549-GFP or A549-OKS: HUVECs: MSCs = 10:1:4. A549-GFP or A549-OKS cells (4.0 × 10⁵), HUVECs (4.0 × 10⁴), and MSCs (1.6 × 0⁵) were mixed and resuspended in sphere-forming medium, which contained 10 ng/mL bFGF (WAKO, Osaka, Japan), 10 μg/mL human insulin (Cell Science & Technology Institute, Sendai, Japan), 100 μg/mL human transferrin (Roche, Basel, Switzerland) and 100 μg/mL BSA (Nacalai Tesque, Kyoto, Japan), and seeded on a low-attachment 24-well flat plate or 96-well M bottom plate (Sumitomo Bakelite, Tokyo, Japan) [14 (link)]. After one day of culture, self-organized spheres were formed.