Innovative technologies for the production and tracking of Extracellular Vesicles

Extracellular vesicles (EVs) have rapidly moved from being considered simple cellular by-products to becoming key mediators of intercellular communication with significant therapeutic and diagnostic potential. As their clinical potential expands, two parallel needs have become increasingly evident: the ability to produce EVs in a standardized, scalable and regulatory-compliant manner, and the availability of reliable tools to study how EVs are generated and released by cells. This work explores two complementary technological approaches designed to advance both the production and the tracking of EVs. The first study focused on osteoarthritis (OA), which was the main part of my work. OA is the leading cause of disability characterized by cartilage degeneration and chronic inflammation. In this context, EVs derived from mesenchymal stem cells (MSCs) have gained interest as cell-free therapeutic agents due to their ability to influence immune and regenerative processes. Here, adipose-derived MSCs (AD-MSCs) were selected as an optimal EVs source because they can be obtained through minimally invasive procedures and display stable biological properties, making them suitable for translational manufacturing. Cell expansion was transitioned to xeno-free conditions by comparing exosome-depleted fetal bovine serum with human platelet lysate, the latter proving more appropriate for good manufacturing practice (GMP) culture. After, the production was transferred into a fully automated bioreactor system, enabling controlled and reproducible large-scale EVs generation under clinical-grade conditions. Once a stable platform was established, AD-MSCs were stimulated with IFN-γ and TNF-α to enhance the immunomodulatory features of the resulting EVs. Native and activated EVs populations were then applied to primary osteoarthritic chondrocytes to assess biocompatibility, their capacity to regulate inflammatory pathways, and their impact on cellular homeostasis. The findings indicate that automated GMP-compatible EVs production is technically feasible and that both EVs populations exhibit favorable safety and functional activity relevant to OA pathophysiology. This work provides a technological basis for future GMP-aligned preclinical development of EV-based regenerative and anti-inflammatory strategies for osteoarthritis. The second project focused on cardiovascular disease, where cellular senescence plays a central role in driving cardiac dysfunction. Senescent cardiomyocytes exhibit marked alterations in EVs biogenesis and release, contributing to chronic inflammation and fibrosis of heart tissue. Understanding how senescence reshapes EVS characteristics and secretion dynamics is therefore essential for developing new diagnostic and therapeutic strategies. To study how senescence influences EVS biology, a high-density-induced senescence model was established in the HL-1 mouse cardiomyocyte cell line, which retains essential cardiac properties. Senescence was validated through the upregulation of canonical markers, ensuring a reliable experimental system. The onset of the senescent phenotype was confirmed through the upregulation of canonical markers. Building on this model, the project then developed and validated a dual-fluorescent CD63-based reporter system, one component of which incorporated a pH-sensitive probe. The construct was delivered via lentiviral transduction, generating stable HL-1 reporter cells. Subsequently, the EVs produced were quantified revealing that senescent-like cultures released notably higher EVs numbers. This innovative tool enabled real-time visualization of EVs biogenesis and secretion in living cells. Overall, the use of fluorescently labeled EVs proved to be an innovative and quantitative approach for monitoring vesicle secretion. Together, these two complementary projects highlight how innovative technological approaches can advance both the large-scale production and the mechanistic understanding of EVs.