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Comparison of the Efficiency of Ultrafiltration, Precipitation, and Ultracentrifugation Methods for Exosome Isolation Publisher



Ansari FJ1 ; Tafti HA2 ; Amanzadeh A3 ; Rabbani S2 ; Shokrgozar MA3 ; Heidari R4 ; Behroozi J5 ; Eyni H6 ; Uversky VN7 ; Ghanbari H1, 2, 8
Authors
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Authors Affiliations
  1. 1. Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
  2. 2. Research Center for Advanced Technologies in Cardiovascular Medicine, Cardiovascular Disease Research Institute, Tehran Heart Center Hospital, Tehran University of Medical Sciences, Tehran, Iran
  3. 3. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
  4. 4. Medical Biotechnology Research Center, AJA University of Medical Sciences, Tehran, 1411718541, Iran
  5. 5. Research Center for Cancer Screening and Epidemiology, AJA University of Medical Sciences, Tehran, Iran
  6. 6. Stem Cell and Regenerative Medicine Research Center, Department of Anatomy, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
  7. 7. Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, 33612, FL, United States
  8. 8. Institute for Biomaterials, University of Tehran & Tehran University of Medical Sciences, Tehran, Iran

Source: Biochemistry and Biophysics Reports Published:2024


Abstract

Extracellular vesicles (EVs) are enclosed by a lipid-bilayer membrane and secreted by all types of cells. They are classified into three groups: apoptotic bodies, microvesicles, and exosomes. Exosomes play a number of important roles in the intercellular communication and crosstalk between tissues in the body. In this study, we use three common methods based on different principles for exosome isolation, namely ultrafiltration, precipitation, and ultracentrifugation. We use field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS) analyses for characterization of exosomes. The functionality and effect of isolated exosomes on the viability of hypoxic cells was investigated by alamarBlue and Flow-cytometry. The results of the FESEM study show that the ultrafiltration method isolates vesicles with higher variability of shapes and sizes when compared to the precipitation and ultracentrifugation methods. DLS results show that mean size of exosomes isolated by ultrafiltration, precipitation, and ultracentrifugation methods are 122, 89, and 60 nm respectively. AlamarBlue analysis show that isolated exosomes increase the viability of damaged cells by 11%, 15%, and 22%, respectively. Flow-cytometry analysis of damaged cells also show that these vesicles increase the content of live cells by 9%, 15%, and 20%, respectively. This study shows that exosomes isolated by the ultracentrifugation method are characterized by smaller size and narrow size distribution. Furthermore, more homogenous particles isolated by this method show increased efficiency of the protection of hypoxic cells in comparison with the exosomes isolated by the two other methods. © 2024
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