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Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum.

ABSTRACT: Exosomes are nanosized vesicles that are abundant in biological fluids. In recent years, exosomes have attracted increasing attention as their cargo may provide promising biomarkers for the early diagnosis of and therapy for many diseases, such as cancer. In addition to ultracentrifugation (UC), many alternative methods including size-exclusion chromatography (SEC) have been developed for isolating exosomes. It has been reported that the SEC method provided improved performance relative to the UC method in isolating exosomes from plasma, where the former contained less residual blood protein contamination. We have compared the SEC method with an optimized UC method in isolating exosomes from human serum. This was based on dilution of the serum to reduce the viscosity and a prolonged cycle of UC, followed by another four cycles. We found that >95% of serum proteins were removed without a significant loss of exosome proteins relative to SEC. We also combined one cycle of UC with SEC and found that this method provided improved results relative to the SEC method, although the serum protein contamination was several times higher than that of our optimized UC method. The TEM showed that the size distribution of exosomes isolated from each of the three methods was similar.

SUBMITTER: An M

PROVIDER: S-EPMC6292670 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum.

An Mingrui M Wu Jing J Zhu Jianhui J Lubman David M DM

Journal of proteome research 20180919 10

Exosomes are nanosized vesicles that are abundant in biological fluids. In recent years, exosomes have attracted increasing attention as their cargo may provide promising biomarkers for the early diagnosis of and therapy for many diseases, such as cancer. In addition to ultracentrifugation (UC), many alternative methods including size-exclusion chromatography (SEC) have been developed for isolating exosomes. It has been reported that the SEC method provided improved performance relative to the U ...[more]

PMID: 30192545

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Project description:Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Project description:Lignins are valuable renewable resources for the potential production of a large array of biofuels, aromatic chemicals and biopolymers. Yet native and industrial lignins are complex, highly branched and heterogenous macromolecules, properties that have to date often undermined their use as starting materials in lignin valorisation strategies. Reliable knowledge of weight average molar mass, conformation and polydispersity of lignin starting materials can be proven to be crucial to and improve the prospects for the success of such strategies. Here we evaluated the use of commonly-used size exclusion chromatography (SEC)-calibrated with polystyrene sulphonate standards-and under-used analytical ultracentrifugation-which does not require calibration-to characterise a series of lignin fractions sequentially extracted from soda and Kraft alkaline lignins using ethyl acetate, methyl ethyl ketone (MEK), methanol and acetone:water (fractions F01-F04, respectively). Absolute values of weight average molar mass (Mw) determined using sedimentation equilibrium in the analytical ultracentrifuge of (3.0 ± 0.1) kDa and (4.2 ± 0.2) kDa for soda and Kraft lignins respectively, agreed closely with previous SEC-determined Mws and reasonably with the size exclusion chromatography measurements employed here, confirming the appropriateness of the standards (with the possible exceptions of fraction F05 for soda P1000 and F03 for Indulin). Both methods revealed the presence of low (~ 1 kDa) Mw material in F01 and F02 fractions followed by progressively higher Mw in subsequent fractions. Compositional analysis confirmed > 90% (by weight) total lignins successively extracted from both lignins using MEK, methanol and acetone:water (F02 to F04). Considerable heterogeneity of both unfractionated and fractionated lignins was revealed through determinations of both sedimentation coefficient distributions and polydispersity indices. The study also demonstrates the advantages of using analytical ultracentrifugation, both alongside SEC as well as in its own right, for determining absolute Mw, heterogeneity and conformation information for characterising industrial lignins.

Project description:BackgroundCirculating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins and plasma proteins. Previous studies suggested combinations of different sEVs isolation methods could improve the yield and purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC), and their combination in a proteomic perspective.ResultsPlasma samples were collected from healthy individuals, and sEVs were separated by one-step SEC, one-step UC, and combining SEC with UC, respectively. Here we exhibited that the purity of sEVs was improved by SEC in contrast to traditional UC. Furthermore, by conducting a SEC procedure followed by UC, we separated sEVs with the highest purity. In the proteomic analysis, 992 protein species were identified in the plasma sEVs isolated by our novel separation method, of which several proteins are sEVs-associated proteins but hitherto never been identified in the previous studies and database, much more than plasma sEVs isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, plasma sEVs isolated by the new procedure kept 584 previously identified sEVs-associated proteins and 360 other proteins that have not been detected before. Detailed analysis suggested that more kinds of sEVs biomarkers, such as CD9, ALIX, and FLOT1, could be identified in plasma sEVs isolated by the novel isolation method as compared to one-step UC/SEC. Furthermore, the lower abundance ranks of common contaminants, such as lipoproteins and IgG chains, in the sEVs fractions obtained by our new method as compared to one-step UC/SEC also demonstrated the purity of sEVs had been improved.ConclusionsCombining SEC with UC could significantly improve the performance of mass spectrometry-based proteomic profiling in analyzing plasma-derived sEVs.

Project description:BACKGROUND:Plasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC. METHODS:By using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science). RESULTS:Our data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit. CONCLUSIONS:We demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.

Dataset Information

Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum.

Publications

Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum.

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