High-Performance Exosome Purification and Characterization Via Density Gradient Ultracentrifugation and Dynamic Light Scattering

Exosomes purification and analyses comprise a fast evolving research area; more than 70% of published research on exosomes has been done within the last six years.

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Faster, more accurate exosome analysis

Challenges to researchers working with exosomes include setting up density gradients by hand, because it is tedious, time-consuming and subject to user, lab, and method variability. There also is a need for greater accuracy in size and concentration analysis. At the same time, experts in the field have called for the establishment of standard protocols.1 We offer solutions to those challenges through cost-effective, large-scale purification, and fast analysis of exosomes. Specifically, the Biomek 4000 Workstation helps overcome human variables and provides a consistent, reproducible, high-throughput method for gradient setup, representing a breakthrough solution to scale-up problems. Optima Ultracentrifugation Series helps researchers maintain reliability between runs, making outcomes highly reproducible. The DelsaMax CORE saves time and cost of TEM analysis for size and concentration.

Although scientists have known about extracellular vesicles for decades, only recently have techniques been able to distinguish exosomes from microvesicles and apoptotic bodies. Classification of membrane vesicles— and the most appropriate, and effective protocols for their isolation—continue to be intense areas of investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. At the same time, there is a need for increased size and concentration accuracy, as well as enhanced workflow. Exosomes are membrane vesicles (~ 30–120 nm in diameter) released by almost all cell types.2,3 They are freely available in plasma as well as other body fluids and contain proteins, mRNA and miRNA, representing the cells they are secreted from.4,5 Exosomes have come into focus as diagnostic as well as therapeutic biomarkers. Additionally, exosomes have been shown to be part of intercellular communication functions, with implications toward both anti-tumor and pro-tumor activity.6 Previous work has provided insight to the isolation of exosomes using density gradient ultracentrifugation,2,7 although there is an effort to gain more concrete confirmation of the size and concentration after purification.

Beckman Coulter offers products covering the entire exosome workflow using automation, centrifugation and dynamic light scattering (DLS), to purify and analyze exosome samples. The Biomek 4000 Laboratory Automation Workstation helps overcome the human variable and provides a consistent, reproducible, high-throughput method for density gradient setup— an elegant solution to scale-up hurdles. Preparative ultracentrifugation helps to maintain reliability between runs and high reproducibility. Importantly, preparative ultracentrifugation, using the Optima MAX-XP and Optima XPN, reaches the g-force necessary for timely separation of biological macromolecule samples to their isopycnic point in density gradients. The DelsaMax CORE DLS platform is used for size analysis of the fractions, because exosome particles can be: (1) analyzed in solution, (2) with statistical significance, (3) with less cost and time, compared to Electron Microscopy. Instead of taking several hours to analyze a few hundred particles, the DelsaMax CORE is able to analyze and size thousands of exosomes in one minute.

The entire exosome preparation workflow is outlined below.

Step 1 - Jurkat cells grown in log phase cell density -1 x 10 cells/mL

Step 2 - Tabletop centrifuge (Allegra X-15R) to remove cells and cell debris/

Step 3 - Ultracentrifuge (Optima XPN) to remove smaller cellular debris.

Step 4 - Ultracentrifuge (Optima XPN) to pellet exosomes.

Step 5 - Biomek 4000 Workstation to set up density gradients

Step 6 - Density gradient ultracentrifugation (Optima XPN) to isolate exosome from co-purified proteins and other mebrane vesicles.

Step 7 - Ultracentrifuge (Optima MAX-XP) to exchange solvent from Iodixanol and sucrose to Phosphate Buffered Saline

Step 8 - DelsaMax CORE for size determination and analysis

References

1. Simpson R J and Mathivanan S. Extracellular microvesicles: the need for internationally recognised nomenclature and stringent purification criteria. J Proteomics Bioinform. 5: (2012).

2. Van Der Pol E et al. Optical and non-optical methods for detection and characterization of microparticles and exosomes. Journal of Thrombosis and Haemostasis. 8.12; 2596–2607: (2010).

3. Simons M and Raposo G. Exosomes—vesicular carriers for intercellular communication. Current opinion in cell biology. 21.4; 575–581: (2009).

4. Keller S et al. Exosomes: from biogenesis and secretion to biological function. Immunology letters. 107.2; 102-108: (2006).

5. Pegtel, Michiel D et al. Functional delivery of viral miRNAs via exosomes. Proceedings of the National Academy of Sciences. 107.14; 6328-6333: (2010).

6. Duijvesz D et al. Exosomes as biomarker treasure chests for prostate cancer. European urology. 59.5; 823-831: (2011).

7. Tauro B J et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods. 56.2; 293-304: (2012).

For more information visit www.optimaxpn.com  

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Future Labs

Published: July 13, 2015

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