3D Culture of Mesenchymal Stem Cells in PEG-4MAL Hydrogels Increases Exosome Production with Bacterial Sphingomyelinase Treatment

Abstract

Exosomes are becoming increasingly popular in the fields of regenerative medicine and tissue engineering for their immunomodulatory potential to enhance tissue regeneration. Exosomes are a membrane-bound extracellular vesicle with a diameter between 30-150nm that act as a non-immunogenic delivery method for biological molecules to communicate between cells, regulating cell function and activity. Despite the growing interest in exosomes, the lack of an established production method limits further research. Our lab has previously modulated sphingolipid signaling in human mesenchymal stem cells (MSCs) via sphingomyelinase (SMase), an enzyme that converts cell membrane sphingomyelin to ceramide, increasing cell membrane curvature and subsequent endosome production, the initiating step in exosome biogenesis. We showed that SMase treated MSCs cultured in tissue culture plastic increases overall exosome production compared to untreated MSCs, likely from SMase-mediated sphingomyelin to ceramide conversion.

Here, we demonstrate increased exosome production in a 3D microenvironment by encapsulating MSCs into 4-arm polyethylene glycol-maleimide (PEG-4MAL) hydrogels with and without SMase compared to tissue culture plastic controls. We utilize nanoparticle tracking analysis to quantify changes in exosome production induced by a 3D biogenesis. We confirmed the immunomodulatory potential of these exosomes by evaluating their effect on TNF-a levels in LPS-stimulated Macrophages and found anti-inflammatory effects among all exosomes. Not only do these findings contribute to more effective methods for extracellular vesicle production, but further, this platform can be leveraged as a delivery vehicle of exosome-producing-MSCs to a range of pre-clinical injury and disease models as a novel immunotherapy.