Framework for rapid comparison of extracellular vesicle isolation methods
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Curated by eLife
Evaluation Summary:
This manuscript describes a framework for rapidly quantifying relative extracellular vesicle (EV) yield and purity across isolation methods, with a focus on using size exclusion chromatography (SEC) for EV isolation from small volumes of pooled plasma and cerebrospinal fluid (CSF) samples. The authors used single molecule array (Simoa) assays for the quantification of EVs using three tetraspanins (CD9, CD63, and CD81), and report the outcomes of assessing EV yields and purity with respect to albumin by various SEC parameters (Sepharose size, column length, fractions collected). This is the first demonstration of the use of Simoa with three commonly used tetraspanins to measure EVs from small volumes of CSF, of great relevance to human CSF biomarker studies, but these methods could also be applied to compare EV isolation methods from other fluids such as cell culture media.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single-molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.
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Evaluation Summary:
This manuscript describes a framework for rapidly quantifying relative extracellular vesicle (EV) yield and purity across isolation methods, with a focus on using size exclusion chromatography (SEC) for EV isolation from small volumes of pooled plasma and cerebrospinal fluid (CSF) samples. The authors used single molecule array (Simoa) assays for the quantification of EVs using three tetraspanins (CD9, CD63, and CD81), and report the outcomes of assessing EV yields and purity with respect to albumin by various SEC parameters (Sepharose size, column length, fractions collected). This is the first demonstration of the use of Simoa with three commonly used tetraspanins to measure EVs from small volumes of CSF, of great relevance to human CSF biomarker studies, but these methods could also be applied to compare EV …
Evaluation Summary:
This manuscript describes a framework for rapidly quantifying relative extracellular vesicle (EV) yield and purity across isolation methods, with a focus on using size exclusion chromatography (SEC) for EV isolation from small volumes of pooled plasma and cerebrospinal fluid (CSF) samples. The authors used single molecule array (Simoa) assays for the quantification of EVs using three tetraspanins (CD9, CD63, and CD81), and report the outcomes of assessing EV yields and purity with respect to albumin by various SEC parameters (Sepharose size, column length, fractions collected). This is the first demonstration of the use of Simoa with three commonly used tetraspanins to measure EVs from small volumes of CSF, of great relevance to human CSF biomarker studies, but these methods could also be applied to compare EV isolation methods from other fluids such as cell culture media.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Ter-Ovanesyan, Dmitry et al. reported a novel strategy for quantifying EV yield and purity through detecting three widely expressed transmembrane proteins in EVs (i.e., the tetraspanins CD9, CD63, and CD81) and one most common contamination protein (i.e., albumin) using Simoa. By applying this method, the authors directly compared the yield and purity of commonly used EV isolation methods, i.e., ultracentrifugation, precipitation and SEC columns. According to the report, SEC columns outperform other methods, and the 10 mL Sepharose CL-6B column is the best choice for EV isolation from plasma or CSF, which showed better performance than the most widely used 10 mL Sepharose CL-2B columns.
This work is interesting as it provided a rapid framework for comparing and improving EV isolation methods from biofluids. …Reviewer #1 (Public Review):
Ter-Ovanesyan, Dmitry et al. reported a novel strategy for quantifying EV yield and purity through detecting three widely expressed transmembrane proteins in EVs (i.e., the tetraspanins CD9, CD63, and CD81) and one most common contamination protein (i.e., albumin) using Simoa. By applying this method, the authors directly compared the yield and purity of commonly used EV isolation methods, i.e., ultracentrifugation, precipitation and SEC columns. According to the report, SEC columns outperform other methods, and the 10 mL Sepharose CL-6B column is the best choice for EV isolation from plasma or CSF, which showed better performance than the most widely used 10 mL Sepharose CL-2B columns.
This work is interesting as it provided a rapid framework for comparing and improving EV isolation methods from biofluids. However, some conclusions are not entirely clear and need further clarification.As both using Simoa to quantify EVs and the different isolations methods will lead to different EV yield and purity are both published previously. The most novelty part of this manuscript is combining these two aspects, i.e., using Sioma to determine the yield and purity of EV samples. However, to address this part, this method should be directly compared with some commonly used techniques such as NTA to test the consistency and different performance between those methods. Especially, the authors indicated in the discussion part that this method is possibly better than commonly used methods.
Interestingly, the authors claimed that lipoproteins are the hardest to be distinguished from EVs due to their similar size but still using albumin as the contamination marker. Due to the smaller size, albumins can be easily excluded in the NTA analysis. The success of using albumin to determine the purity of the EV sample does not support the conclusion that Simoa is better than the NTA.
Some sub-conclusions are not solid enough. For example, whether different centrifuge speeds and time will affect the claim that SEC outperformance ultracentrifuge in regard to EV yield. As it is well known that longer and quicker spinning will increase the yield of EVs. Moreover, whether 10 mL Sepharose CL-6B column still the best choice for EV isolation from plasma or CSF when extending contamination markers to lipoprotein markers. As the size of lipoproteins is much larger, the purity of 10 mL Sepharose CL-6B column may be more affected than 10 mL Sepharose CL-2B columns.
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Reviewer #2 (Public Review):
The authors used a series of sensitive Simoa assays to evaluate EVs and contamination from albumin across common isolation techniques. The experiments were performed rigorously using the same pool of samples from two biofluids, plasma and CSF, for all comparisons. Both biofluids were examined across each isolation method and both the recovery of EVs and contaminants were reported. The number of isolation methods tested was good, though the rationale for choosing each method was missing. The inclusion of albumin as a contaminant was useful information for assessing each isolation method. The additional measurement of lipoprotein contaminants would have strengthened the available data. SEC, once chosen, was very thoroughly examined along with changes such as the volume of the column and the pore sizes.
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Reviewer #3 (Public Review):
There is a lack of consensus about the best way to isolate EVs from biofluids, particularly for human biofluids such as cerebrospinal fluid (CSF) where samples sizes are limited due to the invasive nature of biofluid collection. The authors report that size exclusion chromatography (SEC) is superior for isolation of EVs from 0.5 mL of plasma or CSF versus using ultracentrifugation or commercial precipitation kits. They report the outcomes of isolating EVs using various SEC parameters (Sepharose size, column length, fractions collected) and using single molecule array (Simoa) assays and three commonly used tetraspanins (CD9, CD63, and CD81) to quantify EVs. They report the EV yields and purity with respect to albumin by the various SEC parameters for both plasma and CSF, and the authors conclusions are …
Reviewer #3 (Public Review):
There is a lack of consensus about the best way to isolate EVs from biofluids, particularly for human biofluids such as cerebrospinal fluid (CSF) where samples sizes are limited due to the invasive nature of biofluid collection. The authors report that size exclusion chromatography (SEC) is superior for isolation of EVs from 0.5 mL of plasma or CSF versus using ultracentrifugation or commercial precipitation kits. They report the outcomes of isolating EVs using various SEC parameters (Sepharose size, column length, fractions collected) and using single molecule array (Simoa) assays and three commonly used tetraspanins (CD9, CD63, and CD81) to quantify EVs. They report the EV yields and purity with respect to albumin by the various SEC parameters for both plasma and CSF, and the authors conclusions are justified by their data. This is the first demonstration of the use of Simoa with three commonly used tetraspanins to measure EVs from small volumes of CSF, of great relevance to human CSF biomarker studies. Their findings also support that SEC isolation of EVs combined with Simoa using common tetraspanins and additional selective markers could be applied to compare EV isolation methods from other fluids such as cell culture media.
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