Defined serum-free three-dimensional culture of umbilical cord-derived mesenchymal stem cells yields exosomes that promote fibroblast proliferation and migration in vitro.

Stem cell-derived exosomes are emerging as novel and clinically relevant cell-free therapeutics for regenerative therapy. This work focused on investigating the stimulation of fibroblasts by exosomes derived from umbilical cord-derived mesenchymal stem cells (ucMSC) in a defined serum-free three-dimensional (3D) culture. 3D culture of ucMSC was carried out in medium supplemented with KnockOut serum replacement (KO-medium) using the Aggrewell system. ucMSC in KO-medium formed spheroids with maintained size and integrity throughout culture. This enabled the isolation of vesicles from ucMSC spheroids in KO-medium with sizes that fall within the exosomal size range and were positive for the expression of canonical exosomal markers CD63, CD9, CD81, Alix, and TSG101. The ucMSC-derived exosomes (Exo ) were shown to significantly increase the migration and proliferation of murine fibroblasts in vitro. To conclude, 3D culture of ucMSC in defined serum-free KO-medium formed viable spheroids which enabled the isolation of Exo with the potential of accelerating wound healing.