Mesenchymal stem cells (MSCs) are capable of self-renewal and can differentiate into various types of cells for therapeutic purposes. MSCs are frequently cultured in a two-dimensional (2D) system. However, MSCs can lose their differentiation capacity over time in this culture system. In addition, the available surface area for the propagation of cells is limited. Therefore, various three-dimensional (3D) culture systems have been developed. In this study, we developed the scaffold-free 3D culture systems for the expansion of periosteum-derived progenitor cells (PDPCs) as spheres. The spheroid formation of PDPCs was induced using a rotation platform. The spheres maintained their viability and proliferation ability. Moreover, expression levels of the sternness marker genes and proteins were higher in cells grown on 3D culture system than in 2D culture system. In conclusion, a simple and economical 3D culture system has been developed that can increase the potential of PDPCs for clinical use. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.