Plant suspension culture derived extracellular vesicles

Although extracellular vesicles (EVs) are an ever-present part of life, research of non-mammalian EVs have been severely limited compared to the studies of human EVs. Initial studies of plant EVs already report promising results, such as therapeutic effects for various types of cancers, colitis, immunomodulation, and wound healing. However, there are still many unknowns in plant EV research. A large majority of the articles published in the field fail in adhering to the standards and definitions set in international guidelines of EV research. The abundant contaminants created due to the harsh isolation procedures, lack of adequate controls, and incomplete characterization of the isolated EVs makes these findings questionable. The issues are exacerbated due to a lack of a plant EV isolation method or biomarkers agreed upon by the scientific community. This dissertation attempts to address this issue by providing a complete workflow for the isolation and characterization of true plant EVs using plant cell suspension cultures. Using this workflow, we have isolated and characterized EVs from Nicotiana tabacum, Vitis vinifera and Stevia rebaudiana cell cultures. Proteomic analysis of N. tabacum sEVs revealed the presence of eight proteins suggested as plant EV biomarker candidates. Furthermore, we ran an ortholog protein discovery analysis using proteomes of N. tabacum and seven other plant EV proteomes against top human EV proteins. We identified 116 human proteins with plant orthologues, with 24 of these proteins being present in more than five or more plants. Our findings prove that plant cell suspension cultures can be used for the production of highly pure plant EVs. Plant EVs have considerable potential as therapeutic agents or drug delivery devices, which can be easily, and efficiently produced in a large scale manner. Finally, the biomarkers identified in this study can pave the way for future studies in the field of plant EV research.