This project characterizes the proteomic landscape of small extracellular vesicles (sEVs) derived from human adipose-, foreskin-, and umbilical cord-derived mesenchymal stromal cells (MSCs) cultured under single-donor or pooled-donor conditions in conventional 2D culture and 3D microcarrier-based bioreactors. DIA-based quantitative proteomics was used to determine how donor pooling and 3D culture reshape sEV cargo composition, reduce batch-level heterogeneity, and establish conserved protein signatures across tissue sources. The dataset identifies manufacturing-responsive molecular programs related to mitophagy, immune communication, phagocytosis, membrane repair, wound healing, and mitochondrial metabolism. Foreskin-derived MSC-sEVs showed the strongest response to pooled-donor and 3D culture conditions, supporting their potential as a platform for engineered immuno-mitochondrial and reparative vesicle products.