Assistant Professor National University of Singapore Singapore, Singapore
Abstract: Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) hold great promise as therapeutic agents due to their low immunogenicity and ability to deliver bioactive molecules. However, the endogenous cargo of MSC EVs is not always sufficient to confer therapeutic effects. Additional payloads and modifications may enhance the potency and specificity of MSC EVs, allowing for effective therapeutic action at lower doses. In this study, we generated immortalized MSCs from umbilical cord tissue and scaled up MSC culture using a 3D culture system with the CelCradle® Benchtop Bioreactor. This system facilitates cell growth on macroporous carriers or biomesh scaffolds suspended in a dynamic culture environment, enabling efficient cell attachment, proliferation, and nutrient exchange. These optimized conditions support high-yield MSC expansion and EV production. Among the three immortalized MSC lines evaluated, MSCs expressing hTERT with P53 knockdown exhibited the highest MSC EV yield. We further tested several methods for loading nucleic acids into MSC EVs and labeling the EVs with fluorescent dyes. Our results demonstrated that REG1-mediated plasmid loading into MSC EVs enabled successful delivery and gene expression. Additionally, MemGlow was identified as the most effective fluorescent dye for labeling MSC EVs without aggregation, facilitating further studies on EV uptake and biodistribution. We are also working on the conjugation of MSC EVs with proteins using click chemistry for the addition of protein payloads and targeted delivery. This is a post-isolation modification method that does not require genetic engineering. It is a gentle yet efficient method for EV modification that can be scaled up for GMP production.
Funding Source: Singapore Agency for Science, Technology and Research EVANTICA program
