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Protocols: A Deep Dive into Ultracentrifugation Techniques</span></div></div></header><div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-3082 post type-post status-publish format-standard has-post-thumbnail category-centrifugation entry" aria-label="Unveiling Exosome Isolation and Purification Protocols: A Deep Dive into Ultracentrifugation Techniques"><header class="entry-header"><h1 class="entry-title">Unveiling Exosome Isolation and Purification Protocols: A Deep Dive into Ultracentrifugation Techniques</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/lifeinthelabstaff/" title="Posts by Life in the Lab Staff" rel="author">Life in the Lab Staff</a> </span> <span class="post-info-date"> 06.20.2024 </span> </header><div class="entry-content"><p><img decoding="async" class="alignnone wp-image-3083" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/Exosome-image-teal-banner-300x141.jpg" alt="" width="600" height="282" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/Exosome-image-teal-banner-300x141.jpg 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/Exosome-image-teal-banner-1024x480.jpg 1024w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/Exosome-image-teal-banner-768x360.jpg 768w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/Exosome-image-teal-banner-1536x719.jpg 1536w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/Exosome-image-teal-banner-2048x959.jpg 2048w" sizes="(max-width: 600px) 100vw, 600px" /></p> <p>Exosomes, a subtype of extracellular vesicles (EVs), have been drawing significant attention in the scientific community due to their unique properties and biogenesis pathways. They serve as transporters of genetic information between cells and are being explored for their potential use as disease biomarkers and drug delivery vehicles. The isolation and purification of exosomes are vital steps in leveraging their potential, and ultracentrifugation has emerged as a preferred technique for this purpose.</p> <p><strong>Differential Ultracentrifugation (DUC): A Go-to Technique for Exosome Isolation</strong></p> <p>Ultracentrifugation, recognized as an excellent method for exosome isolation, offers several unique advantages. It allows for a high purity of isolation, separating exosomes from other cellular debris and contaminants, yielding pure exosome samples. The technique functions based on size and density, making it particularly suited for exosome isolation due to its typically smaller size compared to other cellular vesicles.</p> <p>This method begins with various centrifugation steps that effectively remove larger vesicles, debris, and cells. This is followed by exosome precipitation with differential ultracentrifugation at 100,000 x g for more than 70 minutes to pellet exosome vesicles. For the past three decades, differential ultracentrifugation has been extensively used for the isolation of exosomes from a wide variety of sources including cell culture medium, serum, saliva, urine, and cerebrospinal fluid. This is largely attributed to its ease of use, minimal requirement for technical expertise, and its applicability to larger volume preparations without the need for complex sample pre-processing procedures.</p> <p>A combination of sucrose gradient separation and ultracentrifugation has been proven to yield high-quality exosomes.</p> <p><strong>High Purity Exosome Isolation: Density Gradient Ultracentrifugation (DGC)</strong></p> <p>Density gradient ultracentrifugation is an advanced technique that helps ensure the isolation of high-purity exosomes. The process begins by filling a tube with layers of a biocompatible medium of varied densities, such as iodoxinol or sucrose, designed to cover the spectrum of particle densities in the sample. These layers are arranged in order of descending density from bottom to top. After setting up the density-gradient medium, the desired sample is layered on the top, succeeded by an extended centrifugation phase (e.g. 100,000 x g for 16 hours). After completing the centrifugation, the extracellular components, including exosomes, apoptotic bodies, and protein aggregates, gradually reach a static position (isopycnic position) in the layer of the same density.</p> <p>For density gradient ultracentrifugation, the <a href="https://www.thermofisher.com/order/catalog/product/75000100">Thermo Scientific Sorvall WX+ Ultracentrifuge</a> with a <a href="https://www.thermofisher.com/order/catalog/product/75003032">SureSpin 632</a> Swinging Bucket Rotor can be used.</p> <p><strong> </strong><strong>Large-Scale Exosome Isolation: Zonal Density Gradient Ultracentrifugation</strong></p> <p>For therapeutic applications and large-scale isolation, zonal density gradient ultracentrifugation is a suitable technique that can provide pure exosome in large scale. This method uses a bowl rotor and iodixanol density gradient for the isolation of EVs at a large scale with high purity and high recovery.</p> <p>For zonal density gradient ultracentrifugation, the <a href="https://www.thermofisher.com/order/catalog/product/75000080">Thermo Scientific Sorvall WX 80+ Ultracentrifuge</a> and <a href="https://www.thermofisher.com/order/catalog/product/75000008">Thermo Scientific TZ-32 Zonal Rotor</a> are suitable.</p> <p><a href="https://www.thermofisher.com/us/en/home/global/forms/life-science/exosomes-ultracentrifugation.html">Download the latest whitepaper</a> from Thermo Fisher Scientific and EVOX Therapeutics to read about the scale-up method.</p> <p><strong>Final Thoughts</strong></p> <p>The isolation and purification of exosomes are critical steps in leveraging their potential in various scientific and medical applications. Ultracentrifugation techniques, from differential to density gradient methods, help provide reliable and efficient ways to isolate these vesicles. However, the choice of method should be based on the specific requirements of the study or application, such as the need for purity, yield, or scalability.</p> <p>The journey to uncover the full potential of exosomes is still ongoing, but with advanced isolation and purification techniques, we are one step closer to harnessing their capabilities for scientific and medical breakthroughs.</p> <p><a href="https://www.thermofisher.com/us/en/home/global/forms/life-science/exosomes-ultracentrifugation.html">Watch our on-demand webinar: “Demystifying exosome isolation: Best practices and centrifuge selection strategies”</a> to learn more.</p> <aside class="mashsb-container mashsb-main "><div class="mashsb_above_buttons">Share this article </div><div class="mashsb-box"><div class="mashsb-buttons"><a class="mashicon-facebook mash-large mashsb-noshadow" 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