Cell-based therapy using autologous cells has been suggested as a potential approach for periodontal tissue regeneration. Spheroid systems are a form of three-dimensional cell culture that promotes cell matrix interaction, which could recapitulate the aspect of cell homeostasis in vivo. The aim of this study is to assess the interaction of periodontal fibroblast spheroids with synthetic and collagen-based membranes that have been used in guided tissue regeneration.