Around 90° contact angle of dish surface is a key factor in achieving spontaneous spheroid formation.

Following the discovery of the primary culture of neural stem cells, the spheroid culture has been recognized as one of the selective culture methods for somatic stem cells. Since then, various methods were reported to generate spheroids, which can enrich the potent stem cell population. However, the fundamental factors affecting spheroid formation remain unclear. In this study, we focused on the surface property of the culture dishes, in particular, hydrophobicity. Primary mouse skin culture cells were prepared with conventional two-dimensional culture, and then, the cells were transferred to culture dishes with varying hydrophobicity, which was confirmed with the water contact angles. Of these, a culture dish possessing an almost 90-degree water contact angle was the only one that successfully exhibited spheroid formation. The spheroid formation was spontaneous, efficient and stable. Since this outcome was achieved with a conventional culture medium with serum but without any additives such as EGF, bFGF and B27, the spheroid formation from this process was not affected by serum and was also not dependent on additives. The results from immunofluorescence and quantitative real-time PCR testing showed the expression of ES cell markers such as SSEA-1, SOX2, OCT4 and Nanog, which confirmed that the spheroids with this method are comparable to those from other methods. This outcome was reproducible and could be applied not only to skin-derived cells but also to oral mucosa-derived cells, cortical bone-derived cells and 3T3 cells, also suggesting the generality and robustness of this phenomenon.