Sample Preparation & Processing
Exosome Isolation and Characterisation: A Standardised, Automated Approach with Ultracentrifugation
Dr Chad Schwartz and Zach Smith, Beckman Coulter Life Sciences, Indianapolis, IN 46268.
Centrifugation using automated liquid handling now offers an improved, more effi cient and standardised exosome isolation protocol, as demonstrated by work reported by Chad Schwartz PhD and Zach Smith M.S.
Exosomes are small microvesicles, derived from late endosome, released by all cell type and are already implicated in cancer metastasis [1-3]. They are found in nearly all bodily fl uids, cell types and species, and are involved in cell-to-cell communication, with the information encapsulated within their lipid-derived shell critical to homeostasis.
Recent studies suggest they serve as biomarkers not only for clinical and diagnostic use in cancer but in many other diseases [4]. New fi ndings have implicated exosomes in cardiovascular diseases [5-7], autoimmune syndromes [8] and neurodegenerative disorders such as Alzheimer’s [9] and Parkinson’s [10] disease, in addition to tuberculosis [11], diphtheria [12] and human immunodefi ciency virus (HIV) [13].
Exosome characterisation and analysis is therefore a fast-evolving research area. However, an improved and more effi cient isolation and characterisation protocol for exosomes and other extracellular vesicles (EVs) is critical to advancing this fi eld, and experts have recently called for the establishment of standardised methods.
Integrated Workfl ow *
Density gradient, combined with differential centrifugation, is an important step in the enrichment and isolation of exosomes from cell culture media. Challenges involve several rounds of differential centrifugation and development of a standardised method for genetic profi ling of exosome encapsulated miRNA.
To maximise the benefi ts of this technique, the authors explained the advantages of a workfl ow protocol incorporating automated Biomek (Beckman Coulter) methods for centrifugal layering and fractionation, total RNA extraction and cDNA amplifi cation and clean-up for next-generation sequencing, with results reported on benign and malignant colonic cell lines.
Figure 1. Typical centrifugation workfl ow and iodixanol gradient setup for stringent purifi cation of exosomes from cell culture.
The seven-step methodology included*: • preparation of ultracentrifuged exosome-depleted media • cell culture • differential centrifugation for exosomes • density gradient centrifugation • particle characterisation • RNA extraction • NGS library preparation.
Not all the steps can be discussed in this article. Full details of the materials and methods used may be found at
http://info.beckmancoulter.com/ExosomeIsolation [14].
Centrifugation and Isolation
For the preparation of exosome-depleted media, 500 mL of standard HI-FBS was added equally to six 94 mL centrifuge tubes with an adapter and then spun in an ultracentrifuge at 120,000 x g, 18 hours, 4˚C. The supernatant of each tube was recovered and aliquoted to 50 mL and stored at -20˚C for future use. 50 mL of the centrifugally-depleted FBS was then added to 450 mL of both MEM and RPMI 1640 media. The media was fi nally supplemented with 10 mM HEPES and 100 U/mL Penicillin-Streptavidin.
While cell culture remains a popular approach to study EVs, the researchers used an automated viability counter (Beckman Coulter’s Vi-Cell) to assess the number and viability
Figure 2. Representative plot of DLS data acquired from purifi ed exosomes.
between runs and decrease hands-on time. Following isolation, the exosomes were sized by dynamic light scattering, and in all cases the purifi ed exosomes were between 30 nm and 150 nm in size, but with some residual proteins or other particles of around 5 nm. Data for HCT 116 crude exosomes are shown in Figure 2.
of HCT 116 (normal colon) and CCD 841 CoN (colorectal carcinoma) cells, with levels of 97.3% and 98.4%, and densities of 1.52 x 108 and 0.82 x 108, respectively.
Several differential centrifugation steps and a density gradient are required to separate whole cells, cell debris, large aggregates, and soluble proteins from the vesicles of interest. Figure 1. The experimental workfl ow outlined by the authors follows two paths, one in which exosome isolation is terminated following the fourth centrifugation (100,000 xg pellet) and the other that includes density gradient separation and two additional pelleting steps; however, the starting material for both protocols was the same.
The Biomek 4000 workstation (Beckman Coulter) was utilised in the density gradient workfl ow to layer the separation medium and fractionate the samples to reduce variability
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