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July 18th, 2025
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Department of Surgery and Anaesthesia, University of Otago Wellington, Wellington, New Zealand
cell biology
biorxiv

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

Symonds, E. K. C.Open in Google Scholar•Schmidt, A. J.Open in Google Scholar•Brown, A. W.Open in Google Scholar•Currie, M. J.Open in Google Scholar•Herst, P. M.Open in Google Scholar•Hally, K. E.Open in Google Scholar•Danielson, K. M.Open in Google Scholar

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. 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.

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