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Comparison of the yield and purity of plasma exosomes extracted by ultracentrifugation, precipitation, and membrane-based approaches

Li, Wei-Jian, Chen, Hong, Tong, Man-Li, Niu, Jian-Jun, Zhu, Xiao-Zhen, Lin, Li-Rong·2022·Open Chemistry
DOI 10.1515/chem-2022-0139
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Abstract

Exosomes were enriched from plasma by ultracentrifugation, precipitation, and membrane-based approaches for yield and purity. Using the four isolation approaches, particles with mode sizes within the expected range (50–200 nm) can be isolated. By protein estimation, polymer precipitation resulted in a maximum yield (5610.59 ± 51.189 µg/mL), followed by membrane affinity (471.57 ± 12.16 µg/mL), ultracentrifugation (440.22 ± 11.71 µg/mL) and filter + ultracentrifugation (235.47 ± 13.27 µg/mL). By total RNA estimation, the yield of polymer precipitation (3.26 ± 0.42 ng/mL) was higher than that of ultracentrifugation (1.52 ± 0.06 ng/mL), filter + ultracentrifugation (1.21 ± 0.25 ng/mL) and membrane affinity (1.44 ± 0.14 ng/mL). The purity of exosomal preparations was determined as the ratio of the particle number to protein and of protein to RNA. According to the ratio of the particle number to protein concentration, the “purity” of the polymer precipitation method was similar to that of the membrane affinity method and higher than that of ultracentrifugation and filter + ultracentrifugation. When the ratio of RNA to protein was used, the “purity” of the polymer precipitation method was lower than that of the membrane affinity method. Differential methods can be employed to enrich specific exosome subpopulations. The steps of the methods affect the particle number, protein content, and even exosomal purity. The best extraction and evaluation methods for exosomes need to be selected in the laboratory according to their experimental needs.

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Figures

Figure 1 Exosome isolation fl ow diagram.
Figure 1 Exosome isolation fl ow diagram.CC-BY · From the PDF
Figure 2 Electron microscopic image of exosomes following di ff erent isolation methods. Morphology of exosomes ( white arrow indicate ) isolated using ( a ) ultracentrifugation, ( b ) fi lter + ultracentrifugation, ( c ) membrane affi nity, ( d ) and polymer precipitation. Cup -shaped vesicles with a heterogeneous size ( 50 -200 nm diameter ) were clearly visualized in all preparations. The bars indicate 100 nm.
Figure 2 Electron microscopic image of exosomes following di ff erent isolation methods. Morphology of exosomes ( white arrow indicate ) isolated using ( a ) ultracentrifugation, ( b ) fi lter + ultracentrifugation, ( c ) membrane affi nity, ( d ) and polymer precipitation. Cup -shaped vesicles with a heterogeneous size ( 50 -200 nm diameter ) were clearly visualized in all preparations. The bars indicate 100 nm.CC-BY · From the PDF
Figure 3 Size distribution and concentration of exosomes enriched with di ff erent isolation procedures. ( a ) Particle size for plasma exosomes measured by nanoparticle tracking analyses ( NTA ) . Insets depict higher magni fi cations of the black dot box areas. ( b ) Size distribution as a percentage of vesicles larger than 195 nm ( black ) , between 35 and 195 nm ( gray ) and smaller than 35 nm ( white ) based on the same data as in ( a ) . ( c ) Particle concentration of exosomes extracted by di ff erent isolation procedures. The results are the mean ± standard error of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences between multiple groups, followed by Tukey ' s post hoc test. ** P < 0.01. NS, P > 0.05.
Figure 3 Size distribution and concentration of exosomes enriched with di ff erent isolation procedures. ( a ) Particle size for plasma exosomes measured by nanoparticle tracking analyses ( NTA ) . Insets depict higher magni fi cations of the black dot box areas. ( b ) Size distribution as a percentage of vesicles larger than 195 nm ( black ) , between 35 and 195 nm ( gray ) and smaller than 35 nm ( white ) based on the same data as in ( a ) . ( c ) Particle concentration of exosomes extracted by di ff erent isolation procedures. The results are the mean ± standard error of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences between multiple groups, followed by Tukey ' s post hoc test. ** P < 0.01. NS, P > 0.05.CC-BY · From the PDF
Figure 4 The yield of exosomes from di ff erent isolation procedures. ( a ) The exosomal yield presented as the protein estimation. ( b ) The exosomal yield presented as the total RNA. ( c ) Representative bioanalyzer pro fi les of RNA analyzed by an Agilent 2100 Bioanalyzer; the y -axis shows fl uorescence units ( FU ) , and the x -axis shows the nucleotide length ( nt ) of the RNA. The results are the mean ± standard error of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences among multiple groups, followed by Tukey ' s post hoc test. **, P < 0.01; ***, P < 0.001. NS, P > 0.05.
Figure 4 The yield of exosomes from di ff erent isolation procedures. ( a ) The exosomal yield presented as the protein estimation. ( b ) The exosomal yield presented as the total RNA. ( c ) Representative bioanalyzer pro fi les of RNA analyzed by an Agilent 2100 Bioanalyzer; the y -axis shows fl uorescence units ( FU ) , and the x -axis shows the nucleotide length ( nt ) of the RNA. The results are the mean ± standard error of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences among multiple groups, followed by Tukey ' s post hoc test. **, P < 0.01; ***, P < 0.001. NS, P > 0.05.CC-BY · From the PDF
Figure 5 Surface marker expression pro fi les in exosome preparations. ( a ) Western blot for Galectin -3 BP, Tsg101, and CD9 expression in each exosome preparation ( 20 µL of total exosomal protein was loaded ) . ( b ) Quanti fi cation of the expression levels of Galectin -3 BP, Tsg101, and CD9 in each exosome preparation. The results are the mean ± SE of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences among multiple groups, followed by Tukey ' s post hoc test. * P < 0.05, *** P < 0.001.
Figure 5 Surface marker expression pro fi les in exosome preparations. ( a ) Western blot for Galectin -3 BP, Tsg101, and CD9 expression in each exosome preparation ( 20 µL of total exosomal protein was loaded ) . ( b ) Quanti fi cation of the expression levels of Galectin -3 BP, Tsg101, and CD9 in each exosome preparation. The results are the mean ± SE of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences among multiple groups, followed by Tukey ' s post hoc test. * P < 0.05, *** P < 0.001.CC-BY · From the PDF
Figure 6 Purity of exosomes from di ff erent isolation procedures. ( a ) The exosomal purity presented as a ratio between particle concen -tration and protein. ( b ) Exosomal purity presented as the RNA ( µg ) to protein ( µg ) ratio. ( c ) Western blots for calnexin and albumin expression in each exosome preparation ( 20 µL of total exosomal protein was loaded ) . The results are the mean ± SE of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences among multiple groups, followed by Tukey ' s post hoc test. *, P < 0.05. **, P < 0.01. NS, P > 0.05.
Figure 6 Purity of exosomes from di ff erent isolation procedures. ( a ) The exosomal purity presented as a ratio between particle concen -tration and protein. ( b ) Exosomal purity presented as the RNA ( µg ) to protein ( µg ) ratio. ( c ) Western blots for calnexin and albumin expression in each exosome preparation ( 20 µL of total exosomal protein was loaded ) . The results are the mean ± SE of the mean of three independent experiments. One -way analysis of variance ( ANOVA ) was used to assess the di ff erences among multiple groups, followed by Tukey ' s post hoc test. *, P < 0.05. **, P < 0.01. NS, P > 0.05.CC-BY · From the PDF

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Metadata

Authors
Li, W.-J., Chen, H., Tong, M.-L., Niu, J.-J., Zhu, X.-Z., & Lin, L.-R.
Venue
Open Chemistry
Publisher
Walter de Gruyter GmbH
Published
2022-01-01
Volume
20
Issue
1
Pages
182-191
ISSN
2391-5420
DOI
10.1515/chem-2022-0139
Cited by
60
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0 (unspecified)

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CoChat. (2026). Citation check: Comparison of the yield and purity of plasma exosomes extracted by ultracentrifugation, precipitation, and membrane-based approaches. https://app.cochat.ai/papers/10.1515/chem-2022-0139
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Li, W.-J., Chen, H., Tong, M.-L., Niu, J.-J., Zhu, X.-Z., & Lin, L.-R. (2022). Comparison of the yield and purity of plasma exosomes extracted by ultracentrifugation, precipitation, and membrane-based approaches. Open Chemistry. https://doi.org/10.1515/chem-2022-0139