ISOLATION METHODS OF LARGE AND SMALL EXTRACELLULAR VESICLES DERIVED FROM CARDIOVASCULAR PROGENITORS: A COMPARATIVE STUDY

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Posted: 2021-12-30 20:00:00
Eur J Pharm Biopharm . 2021 Dec 27;S0939-6411(21)00361-1. doi: 10.1016/j.ejpb.2021.12.012. Online ahead of print. Affiliations Expand Affiliations 1 Department of Pharmaceutical Technology and Chemistry, School of Pharmacy and Nutrition, University of Navarra, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. 2 Hematology Service and Area of Cell Therapy, Clinic Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain; Department of Animal Biology, Institute of Biomedicine of Málaga (IBIMA), Faculty of Science, University of Málaga, Málaga, Spain. Andalusian Centre for Nanomedicine and Biotechnology (BIONAND), Málaga, Spain. 3 Hematology Service and Area of Cell Therapy, Clinic Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain. 4 Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands. 5 Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain; Hematology Service and Area of Cell Therapy, Clinic Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain; Hematology Department, Clínica Universidad de Navarra and Foundation for Applied Medical Research (CIMA), Pamplona, Spain and Centro de Investigacion en Red de Oncologia (CIBERONC). Electronic address: fprosper@unav.es. 6 Department of Pharmaceutical Technology and Chemistry, School of Pharmacy and Nutrition, University of Navarra, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. Electronic address: mjblanco@unav.es. Item in Clipboard Laura Saludas et al. Eur J Pharm Biopharm. 2021. Show details Display options Display options Format Eur J Pharm Biopharm . 2021 Dec 27;S0939-6411(21)00361-1. doi: 10.1016/j.ejpb.2021.12.012. Online ahead of print. Affiliations 1 Department of Pharmaceutical Technology and Chemistry, School of Pharmacy and Nutrition, University of Navarra, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. 2 Hematology Service and Area of Cell Therapy, Clinic Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain; Department of Animal Biology, Institute of Biomedicine of Málaga (IBIMA), Faculty of Science, University of Málaga, Málaga, Spain. Andalusian Centre for Nanomedicine and Biotechnology (BIONAND), Málaga, Spain. 3 Hematology Service and Area of Cell Therapy, Clinic Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain. 4 Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands. 5 Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain; Hematology Service and Area of Cell Therapy, Clinic Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain; Hematology Department, Clínica Universidad de Navarra and Foundation for Applied Medical Research (CIMA), Pamplona, Spain and Centro de Investigacion en Red de Oncologia (CIBERONC). Electronic address: fprosper@unav.es. 6 Department of Pharmaceutical Technology and Chemistry, School of Pharmacy and Nutrition, University of Navarra, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. Electronic address: mjblanco@unav.es. Item in Clipboard CiteDisplay options Display options Format Abstract Since the discovery of the beneficial therapeutical effects of extracellular vesicles (EVs), these agents have been attracting great interest as next-generation therapies. EVs are nanosized membrane bodies secreted by all types of cells that mediate cell-cell communication. Although the classification of different subpopulations of EVs can be complex, they are broadly divided into microvesicles and exosomes based on their biogenesis and in large and small EVs based on their size. As this is an emerging field, current investigations are focused on basic aspects such as the more convenient method for EV isolation. In the present paper, we used cardiac progenitor cells (CPCs) to study and compare different cell culture conditions for EV isolation as well as two of the most commonly employed purification methods: ultracentrifugation (UC) and size-exclusion chromatography (SEC). Large and small EVs were separately analysed. We found that serum starvation of cells during the EV collecting period led to a dramatic decrease in EV secretion and major cell death. Regarding the isolation method, our findings suggest that UC and SEC gave similar EV recovery rates. Separation of large and small EV-enriched subpopulations was efficiently achieved with both purification protocols although certain difference in sample heterogeneity was observed. Noteworthy, while calnexin was abundant in large EVs, ALIX and CD63 were mainly found in small EVs. Finally, when the functionality of EVs was assessed on primary culture of adult murine cardiac fibroblasts, we found that EVs were taken up by these cells, which resulted in a pronounced reduction in the proliferative and migratory capacity of the cells. Specifically, a tendency towards a larger effect of SEC-related EVs was observed. No differences could be found between large and small EVs. Altogether, these results contribute to establish the basis for the use of EVs as therapeutic platforms, in particular in regenerative fields. Keywords: Large and small extracellular vesicles; cardiovascular progenitors; size-exclusion chromatography; ultracentrifugation. Copyright © 2021. Published by Elsevier B.V. Conflict of interest statement Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. [x] Cite Copy Format: Send To [x]

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