Exosome isolation from distinct biofluids using precipitation and column-based approaches

Abstract

The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.

Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid

“…The detected size of the vesicles and their concentration evaluated using NTA was in accordance with previous data [7,23]. It should be noted that the obtained concentration of CSF vesicles is two orders of magnitude lower than that described for peripheral blood plasma [22,24].…”

CNS-Derived Blood Exosomes as a Promising Source of Biomarkers: Opportunities and Challenges

“…Exosomes have been isolated successfully from human serum and plasma (Caby et al, 2005;Fiandaca et al, 2015), cerebrospinal fluid (CSF) (Vella et al, 2008), saliva (Ogawa et al, 2008;Michael et al, 2010), and urine (Pisitkun et al, 2004). Characterization of exosomes isolated from human serum and plasma has demonstrated similarities in vesicle size, shape, concentration, and presence of exosomal markers suggesting that serum and plasma are equally useful for isolation of blood exosomes (Soares Martins et al, 2018). Multiple exosomal surface markers have been reported and are routinely used to identify exosomes, including the tetraspanins CD9, CD63, and CD81, ALG 2-interacting protein X (ALIX), tumor susceptibility gene 101 protein (TSG101) and ESCRT proteins (Théry et al, 2002;Dutta et al, 2015;Thompson et al, 2016).…”

Exosomes as a Source of Cancer Biomarkers: Advances in Electrochemical Biosensing of Exosomes

“…Exosomes are released from the cell by the fusion of multivesicular bodies with a plasma membrane. They are present in many body fluids, including blood, plasma and serum, urine, semen, amniotic and cerebrospinal fluids . These biovesicles are enriched in a variety of biological molecules, such as RNA (mRNAs‐messenger RNAs, miRNAs‐micro RNAs), DNA, lipids, proteins (including enzymes) and glycoconjugates, and also carry various metabolites (Figure ).…”

“…Since exosomes can be produced by many different cell types, they can be found in a wide range of biological fluids ‐ including more “exotic” ones, such as synovial fluid or breast milk. Affinity columns and commercially available kits, used as an alternative to differential centrifugation, provide a high‐purity exosome fraction with a relatively high yield . Similarly, a comparison of four exosome enrichment kits with ultracentrifugation revealed quite different results taking into account yield, zeta potential (ζ), marker expression, size distribution (most probably the broad size distribution is caused by microvesicle co‐precipitation or by the presence of impurities, such as non‐vesicular aggregates) and RNA/protein quality .…”