In vitro characterization of dental pulp stem cells cultured in two microsphere‐forming culture plates

Abstract

Various three‐dimensional (3D) culture methods have been introduced to overcome the limitations of in vitro culture and mimic in vivo conditions. This study aimed to evaluate two microsphere‐forming culture methods and a monolayer culture method. We evaluated cell morphology, viability, osteo‐, adipo‐, and chondrogenic differentiation potential of dental pulp stem cells (DPSCs) cultured in 3D culture plates: ultra‐low attachment (ULA) and U‐bottomed StemFit 3D (SF) plates, and a two‐dimensional (2D) monolayer plate. RNA sequencing (RNA‐seq) revealed differentially expressed gene (DEG) profiles of the DPSCs. In contrast to an increasing pattern in the 2D group, cell viability in 3D groups (ULA and SF) showed a decreasing pattern; however, high multilineage differentiation was observed in both the 3D groups. RNA‐seq showed significantly overexpressed gene ontology categories including angiogenesis, cell migration, differentiation, and proliferation in the 3D groups. Hierarchical clustering analysis revealed a similar DEG regulation pattern between the 3D groups; however, a comparatively different DEG was observed between the 2D and 3D groups. Taken together, this study shows that DPSCs cultured in microsphere‐forming plates present superior multilineage differentiation capacities and demonstrate higher DEG expression in regeneration‐related gene categories compared to that in DPSCs cultured in a conventional monolayer plate.