Unlocking the potential of extracellular vesicles: one stimulus away from clinical implementation.

Extracellular vesicles (EVs) are cell-derived, phospholipid bilayer vesicles that hold growing potential in therapeutic applications, as demonstrated by encouraging preclinical and clinical trial results. Despite this potential, translating EV-based therapies from bench to bedside remains challenging. A key bottleneck lies in the development of robust biomanufacturing platforms capable of producing enough EVs to meet clinical demand. Establishing reliable EVs-producing cell lines is a critical step in this process. Mammalian cell lines are often preferred for generating "customized" EVs, particularly for drug delivery, however they typically yield low quantities and require carefully optimized culture conditions. Alternative sources, such as red blood cells (RBCs) or even non-human sources, like plants, bacteria, and food-derived EVs, offer promising alternatives that bypass the need for large-scale culture, but they are unlikely to fully replace human-derived EVs across all therapeutic contexts. Therefore, strategies that elevate EV production are essential. Shifting from traditional two-dimensional (2D) culture systems to more advanced three-dimensional (3D) platforms has emerged as a key approach to enhance EV yield, improve process monitoring, and reduce labour and batch-to-batch variability. Additionally, alternative methods, such as the use of specific treatments and supplements, are currently being explored to further boost cellular productivity and promote EV secretion. In this review, we summarize the various cell lines currently being evaluated in EV studies and describe the strategies designed to increase EV secretion from cells, discussing the importance of EV quantification strategies and how they are being applied throughout these studies.