Tuning Hydrogel Mechanics and Microstructure to Maximize Extracellular Vesicle Production from Mesenchymal Stem Cells

The secretory output from mesenchymal stem cells (MSCs) have emerged as promising therapeutics with extracellular vesicles (EVs) gaining prominence due to solution stability and optimal size for overcoming biological barriers during delivery. However, reproducible and scalable production of EVs for therapeutic use remains a challenge in biotechnology. Here we demonstrate optimization of EV production from MSCs using soft hydrogel microcarriers.

Gelatin methacryloyl (GelMA) hydrogels were prepared at a range of concentrations for the culture of two sources of MSCs: adipose derived stem cells (ADSCs) and induced pluripotent stem cell derived MSCs (iMSCs). The mechanical properties of the hydrogels were evaluated using shear rheology. EVs were isolated and analyzed for physical and biological characteristics using electron microscopy, nanoparticle tracking, proteomics, and functional assays for wound healing and angiogenesis.

Both cell types were responsive to hydrogel stiffness (0.3-16 KPa), showing optimal EV secretion from cultures on 10 KPa hydrogels, with a further 18-fold increase when formulated as microcarriers compared to traditional monolayer culture. Proteomics analysis and functional assays revealed that EVs from microcarrier culture displayed increased wound healing and regenerative properties.

This study demonstrates the advantages of hydrogel microcarriers in the production of cell-derived products, with optimized design parameters to guide scaleup and translation to manufacturing, in support of biotechnology and biomedical applications.

The online version contains supplementary material available at 10.1007/s12195-026-00917-x.