Three-dimensional spheroid culture of dental pulp-derived stromal cells enhance their biological and regenerative properties for potential therapeutic applications.

Mesenchymal stem/stromal cell (MSC) spheroids generated in a three-dimensional (3D) culture system serve as a surrogate model that maintain stem cell characteristics since these mimic the in vivo behavior of cells and tissue more closely. Our study involved a detailed characterization of the spheroids generated in ultra-low attachment flasks. The spheroids were evaluated and compared for their morphology, structural integrity, viability, proliferation, biocomponents, stem cell phenotype and differentiation abilities with monolayer culture derived cells (2D culture). The in-vivo therapeutic efficacy of DPSCs derived from 2D and 3D culture was also assessed by transplanting them in an animal model of the critical-sized calvarial defect. DPSCs formed compact and well-organized multicellular spheroids when cultured in ultra-low attachment condition with superior stemness, differentiation, and regenerative abilities than monolayer cells. They maintained lower proliferative state and showed marked difference in the cellular biocomponents such as lipid, amide and nucleic acid between DPSCs from 2D and 3D cultures. The scaffold-free 3D culture efficiently preserves DPSCs intrinsic properties and functionality by maintaining them in the state close to the native tissues. The scaffold free 3D culture methods allow easy collection of a large number of multicellular spheroids of DPSCs and therefore, this can be adopted as a feasible and efficient method of generating robust spheroids for various in-vitro and in-vivo therapeutic applications.