Adhesive Culture System Research Articles

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, ∼102 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.

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.