Three‐dimensional culture of dental pulp pluripotent‐like stem cells (DPPSCs) enhances Nanog expression and provides a serum‐free condition for exosome isolation

Stem cell‐derived exosomes have been identified as novel cell‐free therapeutics for regenerative medicine. Three‐dimensional (3D) culture of stem cells were reported to improve their “stemness” and therapeutic efficacy. This work focused on establishing serum‐free 3D culture of dental pulp pluripotent‐like stem cells (DPPSCs)—a newly characterized pluripotent‐like stem cell for exosome production. DPPSCs were expanded in regular 2D culture in human serum‐supplemented (HS)‐medium and transferred to a micropatterned culture plate for 3D culture in HS‐medium (default) and medium supplemented with KnockOut™ serum replacement (KO‐medium). Bright‐field microscopy observation throughout the culture period (24 days) revealed that DPPSCs in KO‐medium formed spheroids of similar morphology and size to that in HS‐medium. qRT‐PCR analysis showed similar Oct4A gene expression in DPPSC spheroids in both HS‐medium and KO‐medium, but Nanog expression significantly increased in the latter. Vesicles isolated from DPPSC spheroids in KO‐medium in the first 12 days of culture showed sizes that fall within the exosomal size range by nanoparticle tracking analysis (NTA) and express the canonical exosomal markers. It is concluded that 3D culture of DPPSCs in KO‐medium provided an optimal serum‐free condition for successful isolation of DPPSC‐derived exosomes for subsequent applications in regenerative medicine.