(71d) Heparin/Collagen Multilayers Affect Extracellular Vesicle Production in Human Mesenchymal Stem Cells

Human mesenchymal stromal cells (hMSCs) are multipotent stem cells with significant regenerative and immunomodulatory capabilities, making them an attractive candidate for various therapeutic applications. These activities are mediated by the extracellular vesicles (EVs) that hMSCs produce. EVs are small, non-replicating subcellular compartments secreted by cells as part of the complex cellular signaling system. These vesicles carry bioactive molecules, including proteins, lipids, RNA, and other signaling components, and have emerged as promising candidates for therapeutic use. Given their ability to facilitate intercellular communication, EV-based therapies hold the potential to replace traditional synthetic drug delivery systems, reduce the need for invasive diagnostic and prognosis procedures, and improve the effectiveness of various immunotherapies.

Despite their therapeutic potential, the development of EV-directed therapies remains limited. One of the major obstacles is the labor-intensive methods required to isolate EVs in sufficient quantities and purity. Moreover, the molecular content of EVs is not fully understood, which hinders their standardization and optimization for clinical applications. To address these challenges, a deeper understanding of the factors that influence EV production and composition is essential.

This project aimed to investigate the effect of culturing hMSCs on heparin/collagen surfaces on the production and content of hMSC-derived EVs. Previous studies have demonstrated that culturing cells on multilayer surfaces, such as heparin and collagen, can enhance cell proliferation and improve cell viability when compared to conventional tissue culture plastic. These findings suggest that the physical and biochemical properties of the substrate may have a significant impact on cellular behavior and, potentially, the production and content of the EVs they produce.

In this study, hMSCs were cultured on polyionic bilayers consisting of heparin and collagen. To isolate the EVs, a chemical isolation method was employed, ensuring that the vesicles could be separated from other cellular debris and components in the media. Once isolated, the EVs were characterized by several parameters, including size, concentration, total protein content, and molecular composition. These characteristics were then compared to EVs derived from hMSCs cultured on standard tissue culture plastic.

Preliminary results indicate that the layer-by-layer technique, which involves the deposition of heparin and collagen to create a polyionic bilayer, has a positive effect on the production of hMSC-derived EVs. Specifically, the EVs produced on these surfaces exhibited differences in their molecular profiles and physical characteristics compared to those derived from hMSCs cultured on standard plastic. This suggests that culturing hMSCs on heparin/collagen surfaces may enhance the overall quality and quantity of EVs produced, which could be beneficial for downstream applications, such as EV-based therapeutics.

These findings have important implications for the future of EV-directed therapies. By optimizing the conditions under which cells are produced, it may be possible to increase the yield and enhance the therapeutic potential of EV-based treatments. The layer-by-layer technique could serve as a valuable tool for improving EV production, making it easier to scale up the production process while maintaining high-quality EVs. Further research is needed to better understand the molecular mechanisms that underlie these effects.