Exosomes isolated from two different cell lines using three different isolation techniques show variation in physical and molecular characteristics

Abstract

Exosomes are the nanoscopic lipid bi-layered extracellular vesicles that can deliver molecular medicine in the form of targeted therapeutics with great fidelity demonstrating enhanced permeability and high retention effect. In our investigation, we focused on the comparison of three major exosome isolation techniques based on the biophysical and physicochemical characteristics of exosomes isolated from COLO 205 and MCF-7 cancer cell's conditioned media. Commercially available Total Exosome Isolation reagent (TEI), Protein organic solvent precipitation (PROSPR) and differential ultracentrifugation are the three methods used for isolation with an aim to select a suitable method for clinical translation.HRTEM images of exosomes are subjected to 3D image analysis and particle size distribution of exosomes depicted the morphological differences. Molecular and analytical characterization of exosomes using western blotting, Raman and ATR-FTIR spectroscopy and the multivariate analysis on the spectral data obtained, assessed for better molecular specifications and purity of particle. TEI method isolated exosomes with higher exosomal yield, purity, and recovery directly translatable into drug delivery and targeted therapeutics whereas ultracentrifuge had good recovery of particle morphology but showed particle aggregation and yielded exosomes with smaller mean size. PROSPR technique isolated a mixture of EVs and showed the least recovery of exosomes in particle size distribution. This comparative study should help in choosing a specific exosome isolation technique required for the objective of downstream applications. IntroductionExosomes are the minuscule ultrafine bioparticles, the lipid vesicles with a potential to carry cellular cargo from one cell to another 1,2 . They are involved in cell-cell communication playing a major role in growth and development, immune response and even in regulating the tumor microenvironment and disease progression. Exosomes are efficient and robust in their function and have versatile cargo composed of uniquely sorted material carrying it from the cytosol of parent cell to the target cell throughout the body 3 . They are highly efficient due to their nonimmunogenic delivery, their ability to easily fuse with the cell membrane, avoid phagocytosis and circumventing the lysosomes via bypassing the engulfment 4,5 .Exosomes are the ideal drug delivery system with the applications of targeted therapeutics, prognosis and diagnostics of the diseases 6 . This is because the synthetic lipid nanoparticles like lipoplexes can only be used in vitro and not in vivo as a delivery system for gene therapy and transfection 7-9 . Also, in animal models, these lipoplexes elucidate a strong immune response with increased toxicity levels and accumulation in the liver, hindering the drug activity in the target 10 .The efficiency, biocompatibility, low accumulation of exosomes in organs and tissues with low toxicity levels are been harnessed for multiple novel therapeutic strategies [11][12][13] .During the dev...

Exosome Processing and Characterization Approaches for Research and Technology Development

“…EVs are also distinguished by their physicochemical properties, allowing the detection and isolation of specific EV subtypes based on differences in properties. [ 16 ] The databases Vesiclepedia (http://microvesicles.org/) and Exocarta (http://www.exocarta.org) are collections of published data on EV properties that facilitate EV isolation and analysis. [ 17 ] Here we discuss the biochemical compositions of different EV subtypes and how these properties can be used to distinguish exosomes from other EVs such as microvesicles and apoptotic bodies.…”

Label‐Free Identification of Exosomes using Raman Spectroscopy and Machine Learning

“…Molecular identification is essential since many EV characterization methods are size‐based, and there are still fundamental questions about how the size distributions of the EVs are determined by the cell and what makes the difference between healthy and diseased states. Characterizing EV preparations and comparing individually characterized EVs are essential tasks already demonstrated by others [ 23,24,27 ] and were achieved in our work. However, there are remaining challenges in the characterization of mixed preparations of EVs whose Raman spectra are similar.…”

“…Recently, there has been increasing attention to using Raman spectroscopy and multivariate analysis/machine learning methods to characterize EVs. [ 22–30 ] Most of these studies applied traditional spectral analysis methods or statistical data exploration/reduction methods like principal components analysis (PCA) and bayesian classification algorithms like linear discriminant analysis (LDA). [ 31 ] The emergence of machine learning algorithms has dramatically improved spectral analysis methods, and they found application in EV investigations.…”

The biogenesis of large extracellular vesicles occurs in mitosis

“…All of these markers were localized at the midbody region with MKLP1 in late telophase and MBR stages. 38,41,54,[76][77][78] Newly developed EV isolation methods are ideal for isolating large EVs or MBRs. There are several common and commercial isolation methods used to isolate EVs, including ultracentrifugation followed by sucrose gradient [39][40][41] , total exosome isolation reagent (TEIR) [76][77][78][79][80] , immune capture by nanoparticles (Dynabeads) [81][82][83][84] , and 8% PEG6000 (Polyethylene Glycol) treatment 85 .…”

“…38,41,54,[76][77][78] Newly developed EV isolation methods are ideal for isolating large EVs or MBRs. There are several common and commercial isolation methods used to isolate EVs, including ultracentrifugation followed by sucrose gradient [39][40][41] , total exosome isolation reagent (TEIR) [76][77][78][79][80] , immune capture by nanoparticles (Dynabeads) [81][82][83][84] , and 8% PEG6000 (Polyethylene Glycol) treatment 85 . These methods have been used extensively to isolate all sizes of EVs; however, they are expensive and often lengthy protocols and are not specific enough for isolating either large or small EVs.…”