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However, their stability, appropriate storage, and handling conditions are largely unexplored, which raises questions related to their biotechnological applicability. Here, we evaluated the physical and functional stability of\n                      <jats:italic toggle=\"yes\">C. albicans<\/jats:italic>\n                      EVs under long-term storage. Furthermore, we conducted a comparative analysis of these properties in\n                      <jats:italic toggle=\"yes\">C. albicans<\/jats:italic>\n                      EVs obtained through three commonly utilized isolation protocols documented in the literature. After identifying the most efficient isolation method for optimal yield, we devised a potential quality control for EV isolation based on protein and sterol ratios. Subsequently, we investigated the impact of drying EVs using vacuum centrifugation at room temperature or \u22124\u00b0C and the effect of freeze-thaw cycles on EV stability. Transmission electron microscopy (TEM) revealed that EVs maintained morphological stability after long-term (up to 4 years) storage at \u221280\u00b0C, as well as storage at room temperature, 4\u00b0C, and \u221220\u00b0C for 7 days with or without vacuum centrifugation, with a tendency of higher recovery when a lower temperature is used. Remarkably, all of the\n                      <jats:italic toggle=\"yes\">C. albicans<\/jats:italic>\n                      EV suspensions maintained their biological properties as demonstrated by their ability to protect\n                      <jats:italic toggle=\"yes\">Galleria mellonella<\/jats:italic>\n                      against\n                      <jats:italic toggle=\"yes\">C. albicans<\/jats:italic>\n                      infection. However, the number of freeze-thaw cycles significantly impacted the protective effect of the EVs. Overall, our findings demonstrate that\n                      <jats:italic toggle=\"yes\">C. albicans<\/jats:italic>\n                      EVs maintain notable morphological and biological stability under several conditions, enabling their efficient and reproducible utilization in research and potentially as therapeutic agents.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Extracellular vesicles (EVs) released by\n                      <jats:italic toggle=\"yes\">Candida albicans<\/jats:italic>\n                      are promising vaccine prototypes due to their multi-antigenic nature. However, their storage and handling conditions are not well understood, raising concerns about their biotechnological use. This study evaluated the long-term physical and functional stability of\n                      <jats:italic toggle=\"yes\">C. albicans<\/jats:italic>\n                      EVs. We compared three isolation methods to identify the most effective one and suggested a quality control measure based on protein and sterol ratios. We also examined the effects of vacuum drying and freeze-thaw cycles on EV stability. Our findings show that\n                      <jats:italic toggle=\"yes\">C. albicans<\/jats:italic>\n                      EVs maintain their biological function after long-term storage at \u221280\u00b0C and under various conditions. Notably, their protective effect in an insect model was reduced through repeated freeze-thaw cycles. This research provides valuable insights for the efficient use of these vesicles in future studies.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01692-25","type":"journal-article","created":{"date-parts":[[2025,11,28]],"date-time":"2025-11-28T12:50:21Z","timestamp":1764334221000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Impact of storage on the stability and the protective effect of extracellular vesicles released by\n                    <i>Candida albicans<\/i>"],"prefix":"10.1128","volume":"91","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0370-5682","authenticated-orcid":true,"given":"Leandro","family":"Honorato","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03490as77","id-type":"ROR","asserted-by":"publisher"}],"name":"Laborat\u00f3rio de Glicobiologia de Eucariotos, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de G\u00f3es, Universidade Federal do Rio de Janeiro","place":["Rio de Janeiro, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jhon J. 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