Fabrication of Spheroids with Dome-Shaped Endothelial Tube Networks by an Adhesive Culture System

3D functional tissues, such as spheroids fabricated by mesenchymal stem cells (MSCs), which can mimic parts of tissues and organs, have recently been extensively studied in the fields of regenerative medicine and drug discovery. In this study, spheroids containing endothelial tubular structures are fabricated by use of a novel 3D culture plate, "MicoCell." As MicoCell has a mild cell adhesive surface and multicavity structures, it can provide multiple attached spheroids at the same time (about ≈10² to ≈10⁴ spheroids). Spheroids can be fabricated without using serum, and are easily collected by simple pipetting and no use of enzyme. For the fabrication of spheroids containing endothelial tubular structures, MSCs and endothelial cells are co-cultured with MicoCell. Surprisingly, endothelial tubular structures are found to extend upward from the bottom where the spheroids attach onto, forming a dome-shaped morphology. Notably, some tubular structures in the spheroids have a basement membrane and markedly improved oxygen level of the inner part of spheroids. Moreover, as spheroids attach onto the bottom, they do not require any pre-treatment such as embedding into gel before microscopic observation using an optical clearing reagent. These results indicate that the culture plates will be suitable for clinical and pharmaceutical applications.