Extracellular Vesicle Production from hTERT-MSCs Embedded in Hydrogel

Extracellular vesicles (EVs) are lipid membrane-bound nanoparticles secreted by cells that contain nucleic acids, proteins, and metabolites derived from their cells of origin. While there is much to be learned about their structure and function, they are known to play key roles in intercellular communication and are involved in many physiological and pathological processes. EVs have wide therapeutic potential given their abilities to to cross membrane barriers and deliver cargo from their cells of origin to other locations throughout the body. Although they have much potential as therapeutics, current GMP that focus on the large-scale biomanufacturing of EVs is very limited. The use of hydrogel microspheres has been recognized as a potential way to scale up the culture of adherent cell lines, and thus the production of EVs, as they would provide a 3D model to culture cells at an increased seeding density and allow for extended culturing time. Though the pores in hydrogel should be large enough to allow for the secretion of EVs, in order to further confirm that hydrogel is a medium sufficient for cell growth and EV production, hTERT mesenchymal stem cells (MSCs), a cell type known to produce therapeutically relevant EVs, were cultured in 3D hydrogel layers so their EVs could be purified, counted, and compared to EV counts of parallel experiments done in traditional 2D cultures. Gelatin methacrylate (GelMA) and GelMA-Cad were the two types of hydrogel used for these experiments. From EV counts done on the Zetaview Nanoparticle Counter, the concentrations reflected an increase in EV secretion in hydrogel cultures while maintaining similar size distributions. EV protein characterization revealed that Flotillin-1, a common MSC EV marker, was not present in the EV counts for experiments done in hydrogel. Staining for other common EV surface markers should be done to better characterize the EVs and ensure their quality. Additional replicates of EV counts and EV protein characterization need to be done in hydrogel to further confirm the presence of EVs in hydrogel cultures. Ultimately, the goal of this project is to develop a scalable platform to produce EVs from tissue-specific cell types.