Abstract
BackgroundExosomes are nanovesicles (30–120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Currently, having a standard method for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. The most commonly used method is differential ultracentrifugation but it has its own disadvantages, which include high time consumption, low yield due to disruption of exosome integrity, and high protein contaminants. In this study, we have identified an improved method addressing these problems for exosome isolation using ultracentrifugation since it is cost-effective and used worldwide.MethodWe have compared differential ultracentrifugation with the modified method called one-step sucrose cushion ultracentrifugation for exosome isolation. The conditioned serum-free media from human mesenchymal stem cells cultured for 48 h was collected for exosome isolation. The cellular debris was removed by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to remove microvesicles. Equal volumes of pre-processed conditioned media were used for exosome isolation by direct ultracentrifugation and one-step sucrose cushion ultracentrifugation. The exosomes isolated using these methods were characterized for their size, morphology, concentration, and surface marker protein expression.ResultIt was observed that the recovery of exosomes with cup-shaped morphology from one-step sucrose cushion ultracentrifugation was comparatively high as estimated by nanoparticle tracking analysis and electron microscopy. These results were confirmed by Western blotting and flow cytometry.ConclusionWe conclude that this one-step sucrose cushion ultracentrifugation method provides an effective and reproducible potential standard method which could be used for various starting materials for isolating exosomes. We believe that this method will have a wide application in the field of extracellular vesicle research where exosome isolation with high yield and purity is an imperative step.Graphical abstractSchematic representation of comparison of UC and SUC exosome isolation methods for tissue-specific human mesenchymal stem cells. The SUC isolation method yields a greater number of cup-shaped exosomes with a relatively homogenous population for mass-scale production of exosomes for downstream analysis. Abbreviations: SUC One-step sucrose cushion ultracentrifugation, UC Direct ultracentrifugation.
Highlights
Exosomes are nanovesicles (30–120 nm) of endosomal origin
In the present study based on density and cushioning property of sucrose, we have developed a modified onestep sucrose cushion ultracentrifugation (SUC) method for isolation of exosomes and compared it with UC for better yield, exosome integrity, and purity from protein contaminants
Characterization of mesenchymal stem cells and their differentiation potential Mesenchymal stem cells (MSCs) isolated from bone marrow and adipose tissue of adult donors (18–35 years old) that were cryopreserved during previous studies were used
Summary
Exosomes are nanovesicles (30–120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Having a standard method for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. Mesenchymal stem cells (MSCs) are multi-potent selfrenewing cells having remarkable potential in regenerative medicine. These MSCs used in clinical trials for regeneration studies are derived from various sources like bone marrow (BMSCs), dental pulp, adipose tissue (ADSCs), and Wharton’s jelly [1,2,3]. The exosomes are small (30- to 120-nm) nanovesicles originating from endosomes and have been shown to elicit similar biological activity as stem cells themselves [5, 6]. There are contraindicated reports for ultracentrifugation-based methods of exosome isolation for low yield and purity, loss of membrane integrity, and several other parameters [8,9,10]
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