Hydrogel Embedding of Mesenchymal Stem Cells Supports Extracellular Vesicle Production

Extracellular vesicles (EVs) derived from adherent cells are promising therapeutics for a wide variety of diseases. Previous studies have shown that mesenchymal stem cell (MSC)-derived EVs have many applications in wound healing and regenerative medicine. Specifically, MSC-derived EVs are safer than cell-based therapies because EV treatments do not involve the administration of live cells to patients. However, a lack of scalable workflows for producing EVs from 2D adherent sources is a major current limitation of the field. One proposed method for culture scale-up is to encapsulate MSCs in a gelatin methacryloyl (GelMA) hydrogel, which provides a 3D matrix that better mimics the in vivo microenvironment of human cells and can be shaped into spherical beads or thin films to support the growth of shear-sensitive cells inside bioreactors. To establish proof of concept, we embedded MSCs in a layer of GelMA hydrogel to assess the production rate, molecular properties, and functional characteristics of EVs collected from 3D cultures. Hydrogel-encapsulated MSCs yielded a greater number of EVs per volume of culture compared to traditionally grown unencapsulated MSCs, and 3D cultures produced EVs with improved functionality in a scratch assay relative to vehicle treatment. These findings support the hypothesis that GelMA can be used to support scalable manufacturing of bioactive EVs from adherent cell sources.