Size Exclusion HPLC Detection of Small-Size Impurities as a Complementary Means for Quality Analysis of Extracellular Vesicles

Huang, Tao; Banizs, Anna B.; Shi, Weibin; Klibanov, Alexander L.; He, Jiang

doi:10.5772/61148

Cited by 10 publications

(12 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A number of reports suggest that lipoproteins may co-purify with exosomes when using standard methodologies to isolate exosomes from plasma (Huang, Banizs, Shi, Klibanov, & He, 2015;Karimi et al, 2018). Because exosomes are secreted vesicles that are in the same size range as lipoproteins (Table 1), their co-purification is not surprising.…”

Section: Downstream Analysis Of Exosomes Using High-performance Liquimentioning

confidence: 99%

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

et al. 2020

View full text Add to dashboard Cite

Exosomes are 50‐ to 150‐nm‐diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation‐ and column‐based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek®) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream®). High‐performance liquid chromatography followed by nano‐flow liquid chromatography–mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5‐fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Pre‐analytic fluid collection and processing Basic Protocol 2: Exosome isolation by ultracentrifugation Alternate Protocol 1: Exosome isolation by precipitation Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry Basic Protocol 4: Downstream analysis of exosomes using high‐performance liquid chromatography Basic Protocol 5: Downstream analysis of the exosome proteome using nano‐flow liquid chromatography–mass spectrometry

show abstract

Section: Downstream Analysis Of Exosomes Using High-performance Liquimentioning

confidence: 99%

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Several protocols, which are faster, easier and less expensive than ultracentrifugation, are available . Filtration by size and molecular weight are usually coupled in the same protocol . Filters with pore sizes of 0.8, 0.45, and 0.22 µm and membranes with a molecular weight cut‐off ranging from 10 to 100 kDa are commonly used .…”

Section: Exosome Recovery and Purificationmentioning

confidence: 99%

“…Nowadays, lab‐on‐a‐chip devices that involve the integration of functional components explore the behavior of exosomes at the microscale. Exosome manipulation techniques based on microdevices include approaches like electrophoresis, dielectrophoresis, acoustics, magnetism, and immunoaffinity . Furthermore, devices that combine two or more separation techniques have been designed.…”

Section: Exosome Recovery and Purificationmentioning

confidence: 99%

Recent advances and challenges in the recovery and purification of cellular exosomes

et al. 2019

View full text Add to dashboard Cite

Exosomes are nanovesicles secreted by most cellular types that carry important biochemical compounds throughout the body with different purposes, playing a preponderant role in cellular communication. Because of their structure, physicochemical properties and stability, recent studies are focusing in their use as nanocarriers for different therapeutic compounds for the treatment of different diseases ranging from cancer to Parkinson's disease. However, current bioseparation protocols and methodologies are selected based on the final exosome application or intended use and present both advantages and disadvantages when compared among them. In this context, this review aims to present the most important technologies available for exosome isolation while discussing their advantages and disadvantages and the possibilities of being combined with other strategies. This is critical since the development of novel exosome‐based therapeutic strategies will be constrained to the effectiveness and yield of the selected downstream purification methodologies for which a thorough understanding of the available technological resources is needed.

show abstract

“…High-purity exosomes could be obtained, but this method had a low yield and was time-consuming. 7 – 10 Commercial kits had the advantages of quickly obtaining EVs and a high yield, but these methods were associated with low purity and high cost. 11 Recently developed immunocapture methods could be used to isolate specific subgroups of EVs, but these methods also had a high cost.…”

Section: Introductionmentioning

confidence: 99%

<p>A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles from Culture Supernatant of Human Ovarian Cancer Cell Line A2780 and Body Fluids of High-Grade Serous Carcinoma Patients</p>

Jiao¹,

Sun²,

Gao³

et al. 2020

CMAR

View full text Add to dashboard Cite

This study tried to evaluate whether 8% polyethylene glycol (PEG) 6000 precipitation combined with differential ultracentrifugation (PPDU) was an efficient and practical method for the enrichment and purification of extracellular vesicles (EVs) derived from the culture supernatant of human ovarian cancer cell line A2780 and from body fluids of patients with high-grade serous carcinoma (HGSC). Methods: PPDU was used to enrich and purify the EVs derived from body fluids of patients with HSGC and cell culture supernatant of subclones of human ovarian cancer cell line A2780 with high/low invasive capacity (named as A-H/A-L, respectively). Transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) were used to identificate the EVs size and distribution. Western blots (WB) were used to detect the expression of CD9, CD63, Alix and Calnexin. The high-purity EVs derived from the cell culture supernatant of A-H/A-L were detected by the protein profile. Expression of integrins (ITGs) αV, β1 and β3 in the EVs derived from body fluids of HGSC patients was also evaluated. Results: The diameter of EVs was about 30-260 nm observed under the TEM. Under the NTA identification, the peak size of EVs was ranged from 70 to 159nm. EVs derived from different specimens did not significantly differ in mean size and peak size. Presence of CD9, CD63 and Alix and absence of Calnexin were confirmed in the EVs. The protein concentrations of EVs' sample extracted from A-H/A-L cell culture supernatant were 0.36µg/µL and 0.20µg/µL, respectively. The total amount of protein obtained from 300ul EVs was 108.02ug and 61.44ug, respectively. Totally, 2397 peptides and 952 proteins were identified by isobaric tags for relative and absolute quantitation (ITRAQ). The expression of ITGαV, β1, and β3 in the EVs from plasma and ascites of HGSC patients was significantly higher than the control group (plasma: all P<0.0001; ascites: P=0.036, 0.001 and 0.004, respectively). The expression level of ITGαV and β1 in EVs of HGSC's ascites was significantly higher than that in plasma (P= 0.004, 0.001, respectively). The expression of ITGβ3 was also slightly elevated in EVs-derived HGSC patients' ascites (P=0.492). Conclusion: PPDU was an efficient and practical method to enrich EVs from body fluids and cell culture supernatant. The characteristic expression of ITGαV, β1 and β3 in ascites and plasma EVs of patients with HGSC provided useful information on the development of EVs in HGSC.

show abstract

Size Exclusion HPLC Detection of Small-Size Impurities as a Complementary Means for Quality Analysis of Extracellular Vesicles

Cited by 10 publications

References 27 publications

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

Recent advances and challenges in the recovery and purification of cellular exosomes

<p>A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles from Culture Supernatant of Human Ovarian Cancer Cell Line A2780 and Body Fluids of High-Grade Serous Carcinoma Patients</p>

Contact Info

Product

Resources

About