<p>Human mesenchymal stromal cells (MSCs) possess extensive therapeutic potential, and their biological characteristics and functions can be modulated by culture media. This study aimed to systematically evaluate the effects of two basal media, DMEM/F12(DF12) and low-glucose Dulbecco’s Modified Eagle Medium (LG-DMEM), on the biological characteristics and functions of umbilical cord-derived MSCs. The influences of basal medium on crucial quality parameters of MSCs, including identity, differentiation potential, proliferation, replicative senescence, genetic stability, gene expression profile, and immunomodulatory capacity, were assessed. MSCs cultured in DF12 exhibited smaller cell size, faster proliferation, higher expression levels of genes associated with growth factor activity, cytokine activity, and extracellular matrix binding, but showed more notable replicative senescence with serial passages. MSCs cultured in LG-DMEM expressed higher levels of indoleamine 2,3-dioxygenase (IDO), had a stronger inhibitory effect on T cell proliferation in vitro, especially on Th17 cells, and showed a better therapeutic effect on alleviating skin lesions in imiquimod (IMQ)-induced psoriasis-like mice. These results suggest that DF12 is more suitable for the early passages of MSCs, potentially favoring applications in injury repair, while LG-DMEM is more suitable for the culture of higher passages of MSCs and enhances efficacy in immune-related diseases. These findings provide insights for selecting the appropriate basal media, a critical process parameter during expansion, contributing to the optimization of MSCs manufacturing bioprocesses to meet the requisite quantity and quality for therapeutic applications.</p>