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Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

Gallart-Palau X, Serra A, Wong AS, Sandin S, Lai MK, Chen CP, Kon OL, Sze SK - Sci Rep (2015)

Bottom Line: Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR).PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension.We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, 637551.

ABSTRACT
Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

No MeSH data available.


Related in: MedlinePlus

Cryo-immunogold EM micrographs of PROSPR isolated EVs.Presence of tetraspanin CD9 (10 nm colloidal gold particles) was observed as attached to the internal/external face of the membrane in single layered exosome-like vesicles (a,b) and in multilayered exosome-like vesicles (c). Presence of the ESCRT protein Alix was also confirmed (10 nm colloidal gold particles) in exosome-like vesicles isolated by PROSPR from blood plasma (d–f). Scale bars in all images represent 40 nm.
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f4: Cryo-immunogold EM micrographs of PROSPR isolated EVs.Presence of tetraspanin CD9 (10 nm colloidal gold particles) was observed as attached to the internal/external face of the membrane in single layered exosome-like vesicles (a,b) and in multilayered exosome-like vesicles (c). Presence of the ESCRT protein Alix was also confirmed (10 nm colloidal gold particles) in exosome-like vesicles isolated by PROSPR from blood plasma (d–f). Scale bars in all images represent 40 nm.

Mentions: Having established that PROSPR is an efficient method for the separation of EVs from human blood plasma, we next proceeded to assess the structural integrity and diversity of the vesicles obtained using this method. Cryo-electron microscopy was used to determine the ultra-structural morphology of PROSPR-separated EVs for comparison with vesicles isolated by a standard ultra-cushion protocol. Using this approach, we observed that both PROSPR and ultra-cushion techniques generated a heterogeneous mix of circular, membrane-encapsulated structures with diameters ranging from 20–300 nm (Fig. 3). The morphologies and sizes of the structures we detected were consistent with those widely reported for exosomes (50–100 nm) and microvesicles (100–1000 nm)14. In addition, both the PROSPR and ultra-cushion methods yielded multi-layered and elongated vesicles of various sizes15, and separated non-membranous particles of ~30 nm diameter that resembled lipoproteins in size and morphology15161718. Although there was no difference in EV size distribution between the two methodologies tested here (data not shown), PROSPR separation appeared to generate a higher density of EVs. In order to obtain definitive evidence that PROSPR is an efficient method of EVs purification, we next conducted a confirmatory experiment using cryo-immunogold labeling to confirm the presence of characteristic EV markers (Alix and CD9) in PROSPR-separated EVs. Visualization of the immunogold-labeled EVs obtained by PROSPR confirmed and validated the presence of membrane-associated Alix and CD9 proteins on both multi-layered and single-layer EVs (Fig. 4). These data indicate that the plasma EVs obtained by PROSPR separation exhibit size distribution and biochemical features previously shown by those of EVs isolated using conventional techniques.


Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

Gallart-Palau X, Serra A, Wong AS, Sandin S, Lai MK, Chen CP, Kon OL, Sze SK - Sci Rep (2015)

Cryo-immunogold EM micrographs of PROSPR isolated EVs.Presence of tetraspanin CD9 (10 nm colloidal gold particles) was observed as attached to the internal/external face of the membrane in single layered exosome-like vesicles (a,b) and in multilayered exosome-like vesicles (c). Presence of the ESCRT protein Alix was also confirmed (10 nm colloidal gold particles) in exosome-like vesicles isolated by PROSPR from blood plasma (d–f). Scale bars in all images represent 40 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588595&req=5

f4: Cryo-immunogold EM micrographs of PROSPR isolated EVs.Presence of tetraspanin CD9 (10 nm colloidal gold particles) was observed as attached to the internal/external face of the membrane in single layered exosome-like vesicles (a,b) and in multilayered exosome-like vesicles (c). Presence of the ESCRT protein Alix was also confirmed (10 nm colloidal gold particles) in exosome-like vesicles isolated by PROSPR from blood plasma (d–f). Scale bars in all images represent 40 nm.
Mentions: Having established that PROSPR is an efficient method for the separation of EVs from human blood plasma, we next proceeded to assess the structural integrity and diversity of the vesicles obtained using this method. Cryo-electron microscopy was used to determine the ultra-structural morphology of PROSPR-separated EVs for comparison with vesicles isolated by a standard ultra-cushion protocol. Using this approach, we observed that both PROSPR and ultra-cushion techniques generated a heterogeneous mix of circular, membrane-encapsulated structures with diameters ranging from 20–300 nm (Fig. 3). The morphologies and sizes of the structures we detected were consistent with those widely reported for exosomes (50–100 nm) and microvesicles (100–1000 nm)14. In addition, both the PROSPR and ultra-cushion methods yielded multi-layered and elongated vesicles of various sizes15, and separated non-membranous particles of ~30 nm diameter that resembled lipoproteins in size and morphology15161718. Although there was no difference in EV size distribution between the two methodologies tested here (data not shown), PROSPR separation appeared to generate a higher density of EVs. In order to obtain definitive evidence that PROSPR is an efficient method of EVs purification, we next conducted a confirmatory experiment using cryo-immunogold labeling to confirm the presence of characteristic EV markers (Alix and CD9) in PROSPR-separated EVs. Visualization of the immunogold-labeled EVs obtained by PROSPR confirmed and validated the presence of membrane-associated Alix and CD9 proteins on both multi-layered and single-layer EVs (Fig. 4). These data indicate that the plasma EVs obtained by PROSPR separation exhibit size distribution and biochemical features previously shown by those of EVs isolated using conventional techniques.

Bottom Line: Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR).PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension.We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, 637551.

ABSTRACT
Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

No MeSH data available.


Related in: MedlinePlus