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Mesenchymal stem cell (MSC)-derived exosomes (EXs) have emerged as promising therapeutic agents for wound healing. However, the optimal conditions for manufacturing MSC-derived EXs that maximize their wound-healing potential have yet to be established. Hence, we compared the efficacy of five different MSC culture media, including three different serum-free, a platelet-supplemented, and a fetal bovine serum-supplemented media, in exosome manufacturing for wound healing applications. Although umbilical cord-derived MSCs (UCMSCs) cultured in these media exhibited similar proliferation, morphology, MSC surface marker expression, and stemness, EXs derived from UCMSCs cultured in different culture media displayed varying levels of growth factors and cytokines. Notably, EXs derived from platelet-supplemented media (DM-PLT_EXs) exhibited significantly higher concentrations of keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF-A), platelet-derived growth factor (PDGF-BB), interleukin 6 (IL-6), interleukin 7 (IL-7), and interleukin 8 (IL-8) than EXs from other media. These differences correlated with the superior capability of DM-PLT_EXs to promote human skin fibroblast proliferation and stimulate angiogenesis of human umbilical vein endothelial cells, making them a more suitable choice for wound healing applications. Our findings emphasize the significance of the culture medium selection in tailoring the therapeutic potential of UCMSC-derived EXs for wound healing.