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Figure 3. Current challenges and technical limitations hindering the therapeutic utility of probiotic EVs. Schematic illustration to depict challenges and limitations facing the utility of probiotic EVs in clinical applications. Several technical constraints such as EV cargo and yield variations due to different isolation methods, the presence of secretory factors and contaminants in the preparation, and the use of inappropriate preclinical models to study the effect and variation in phenotype based on the route of administration could prevent a detailed understanding and widespread acceptance of the role of probiotic EVs in pathophysiology. Additionally, current technical limitations prevent a better understanding of the EV cargo component responsible for the observed phenotypes. The annotated bibliography is shown in the Supplementary Materials. EVs: Extracellular vesicles.