Development of a 3D in vitro wound healing model to assess the effect of ADSC-EVs on vascularisation

Angiogenesis is critical for effective wound healing and relies on the successful coordination of various cell types including endothelial cells, macrophages, and fibroblasts. Adipose-derived stem cell extracellular vesicles (ADSC-EVs) have demonstrated pro-angiogenic properties and have been posited as a novel therapeutic to aid wound healing; however, their functional impact within human-derived multicellular models remains largely uncharacterised. This study explores the development and application of a 3D multicellular in vitro model to assess the effects of ADSC-EVs on vascularisation in the context of wound healing. 3D multicellular in vitro models were developed by co-culturing human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages (MDMs), and fibroblasts within Matrigel to recapitulate the in vivo wound healing microenvironment. A five-colour confocal microscopy panel was developed to visualise each cell type and EVs within the models. The optimised models were then treated ADSC-EVs or control to determine their impact on angiogenesis and cell co-localisation. We determined that vessel formation was significantly enhanced when HUVECs were co-cultured in multicellular models compared to monocultures, with the greatest effect observed in the full three-cell-type model. This effect was even more pronounced with the addition of ADSC-EVs. ADSC-EV treatment also enhanced macrophage co-localisation within endothelial structures. This study developed a multicellular model that can be used for future work assessing wound healing in vitro and will be additive to currently used single-cell and in vivo models. We have applied these models to demonstrate that ADSC-EVs significantly enhance tube formation in HUVECs, and the development of tissue-like structures in multicell systems, highlighting their potential as a promising therapeutic approach for improving wound healing.