Construction of brain-like spheroids containing endothelial tubular networks by an adhesive culture system.

Spheroids which are composed of several types of cells have been widely studied in the pharmaceutical field as their structure and functions are similar to human organs. Three-dimensional brain-like tissues are one of the most important tissues for the development of medicines to treat brain diseases and for in vitro brain models. In this study, spheroids mainly containing neurons, astrocytes, and endothelial cells were fabricated using a novel 3D culture plate, "MicoCell™" to construct a brain mimicking tissue. Due to the multicavity structures of MicoCell, ∼10 of attached spheroids were fabricated in a single plate. Spheroids in MicoCell were attached onto a mild cell adhesive surface, allowing for easy immunostaining and microscopic observation. Spheroid formation was improved by adding a Rho-Kinase inhibitor during cultivation. Endothelial cells formed vascular network structures in spheroids and some parts of the vascular structures attached onto the bottom of a culture plate. Co-culture of multiple cell types required optimization of the culture medium during spheroid formation. The mixture of neural stem cell medium and endothelial growth medium showed good spheroid formation and a vascular network. These results indicated that our culture plates and brain mimicking spheroids would be a suitable candidate for pharmaceutical applications such as drug screening and for in vitro brain models.