Fine-tuning licensing strategies to boost MSC-based immunomodulatory secretome.

BACKGROUND: Immune-mediated inflammatory diseases (IMIDs) are a major global health challenge, affecting millions of people and often lacking effective treatments. The mesenchymal stromal cell (MSC)-derived secretome has emerged as a promising therapeutic approach owing to its potent immunomodulatory properties. However, progress has been hindered by the lack of standardized protocols for inducing a robust immunomodulatory MSC phenotype. METHODS: In this study, we focused on optimizing the MSC-derived secretome to enhance its ability to suppress activated immune cells. Specifically, we examined (1) the effects of IFN-γ and TNF-α, individually and in combination, to uncover potential synergy; (2) the ideal cytokine ratio and (3) concentration; (4) the best production time for the secretome; and (5) the impact of cellular confluence. These factors were systematically evaluated to assess their influence on cell behavior, viability, cytosolic content release, and the secretion of key immunomodulatory and regenerative factors. RESULTS: Our results demonstrate that overnight licensing with a 1:1 ratio of IFN-γ and TNF-α at 60 ng/mL, followed by 48 h of incubation at 90% confluence, yields an optimized conditioned media (CM) with significantly enhanced immunomodulatory properties. Functional assays showed that this CM can inhibit human peripheral blood mononuclear cell (PBMC) activation with more than twice the effectiveness of suboptimal protocols. Additionally, we found that direct cell-cell contact was critical for inducing regulatory T cells (Tregs), highlighting the complex dynamics of immune regulation. CONCLUSIONS: These findings establish a robust and standardized MSC licensing protocol, paving the way for the development of innovative and effective therapies to combat IMIDs. CLINICAL TRIAL NUMBER: Not applicable.