Dynamic three-dimensional culture enhances tunneling nanotubes-mediated mitochondrial transfer in mesenchymal stromal cells to accelerate wound healing

Mesenchymal stromal cells (MSCs) have shown promise in treating various diseases, and optimizing their therapeutic potential is a crucial objective in MSCs-based clinical applications. The microenvironment, particularly three-dimensional (3D) culture systems, plays a pivotal role in regulating the fate determination and enhancing the therapeutic potential of MSCs. Currently, the mechanisms governing the interactions between MSCs cultured in a dynamic 3D system and host recipient cells remain incompletely understood. MSCs transfer mitochondria to influence the fate of recipient cells, with tunneling nanotubes (TNTs) being the primary method. However, whether MSCs cultured under dynamic 3D conditions transfer mitochondria via TNTs to exert therapeutic effects remains to be elucidated. This study developed a dynamic 3D culture system for stem cells from human exfoliated deciduous teeth (SHED), a type of MSCs, utilizing gelatin microcryogel microcarriers and stirred tank bioreactor. A mouse model of full-thickness skin defects was employed to validate the enhanced therapeutic efficacy of SHED cultured under dynamic 3D conditions. Co-culture experiments with SHED and endothelial cells demonstrated that the dynamic 3D culture conditions empower the MSCs to transfer mitochondria via TNTs, thereby promoting angiogenesis. This research provides novel insights into the mechanisms underlying wound healing acceleration by SHED cultured under dynamic 3D conditions and offers a new strategy for developing MSCs transplantation applications.