Tuning the Microenvironment to Create Functionally Distinct Mesenchymal Stromal Cell Spheroids

Mesenchymal stromal cells (MSCs) are under investigation for wound healing and tissue regeneration due to their potent secretome. Compared to monodisperse cells, MSC spheroids exhibit increased cell survival and enhanced secretion of endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE₂), two key factors in wound repair. We previously upregulated the proangiogenic potential of homotypic MSC spheroids by manipulating microenvironmental culture conditions. However, this approach depends on the responsiveness of host endothelial cells (ECs)-a limitation when attempting to restore large tissue deficits and for patients with chronic wounds in which ECs are dysfunctional and unresponsive. To address this challenge, we used a Design of Experiments (DOE) approach to engineer functionally distinct MSC spheroids that maximize VEGF production (VEGF_MAX) or PGE₂ production (PGE_2,MAX) while incorporating ECs that could serve as the basic building blocks for vessel formation. VEGF_MAX produced 22.7-fold more VEGF with enhanced endothelial cell migration compared to PGE_2,MAX, while PGE_2,MAX produced 16.7-fold more PGE₂ with accelerated keratinocyte migration compared to VEGF_MAX. When encapsulated together in engineered protease-degradable hydrogels as a model of cell delivery, VEGF_MAX and PGE_2,MAX spheroids exhibited robust spreading into the biomaterial and enhanced metabolic activity. The distinct bioactivities of these MSC spheroids demonstrate the highly tunable nature of spheroids and provide a new approach to leverage the therapeutic potential of cell-based therapies.