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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190046 1/2 Homo sapiens Cell culture supernatant (d)(U)C Deville, Sarah 2021 56%

Study summary

Full title
All authors
Sarah Deville, Pascale Berckmans, Rebekka Van Hoof, Ivo Lambrichts, Anna Salvati, Inge Nelissen
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major (show more...)Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day. (hide)
EV-METRIC
56% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
THP-1
Sample origin
LPS-stimulated
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: / TSG101/ Flotillin1/ CD9
non-EV: Cytochrome C
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
LPS-stimulated
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101
Not detected contaminants
Cytochrome C
Flow cytometry
Type of Flow cytometry
Hardware adjustments
Calibration bead size
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
182
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
A BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection was used for analysis. The device utilizes a highly sensitive fluorescence trigger to measure the EVs.
Calibration bead size
0.1
EV concentration
Yes
EV200093 2/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Total Exosome Isolation
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PC3
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Total Exosome Isolation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
110.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.42E+08
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
59.75
EV concentration
Yes
EV200093 3/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Exodisc
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PC3
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Exodisc
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
115.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.19E+08
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
57.25
EV concentration
Yes
EV200093 4/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
SEC and UF
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PC3
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC and UF
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
114.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6.73E+07
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
59.25
EV concentration
Yes
EV200093 5/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
DG
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PC3
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
DG
Protein markers
EV: CD81/ Flotillin1
non-EV: None
Proteomics
no
EV density (g/ml)
1.10-1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
38
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
4.75
Fraction processing
Centrifugation
Pelleting: volume per fraction
28
Pelleting: duration (min)
60
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD81
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
EV200093 7/10 Homo sapiens Blood plasma (d)(U)C
Filtration
Total Exosome Isolation
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Total Exosome Isolation
Protein markers
EV: CD81/ Flotillin1/ None/ CD63/ CD9
non-EV: ApoA1/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD81/ Flotillin1
Detected contaminants
ApoA1
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
127.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.70E+11
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
61.75
EV concentration
Yes
EV200093 8/10 Homo sapiens Blood plasma (d)(U)C
Filtration
Exodisc
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Exodisc
Protein markers
EV: CD81/ Flotillin1/ None/ CD63/ CD9
non-EV: ApoA1/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD81/ Flotillin1
Detected contaminants
ApoA1
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
129.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.29E+11
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
62.25
EV concentration
Yes
EV200093 9/10 Homo sapiens Blood plasma (d)(U)C
Filtration
SEC and UF
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC and UF
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: ApoA1/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD81
Detected contaminants
ApoA1
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
128.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.05E+11
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
58.25
EV concentration
Yes
EV200092 2/5 Homo sapiens Urine (d)(U)C
Filtration
Total Exosome Isolation
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Total Exosome Isolation
Protein markers
EV: CD81/ Flotillin1/ None/ CD63/ CD9
non-EV: Tamm-Horsfall protein/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD81/ Flotillin1/ CD63
Not detected contaminants
Calreticulin/ Tamm-Horsfall protein
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9
Not detected EV-associated proteins
CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
107.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6.85E+08
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
59.75
EV concentration
Yes
EV200092 3/5 Homo sapiens Urine (d)(U)C
Filtration
Exodisc
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Exodisc
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Tamm-Horsfall protein/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin/ Tamm-Horsfall protein
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9
Not detected EV-associated proteins
CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
110.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 3.64E+09
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
59.75
EV concentration
Yes
EV200092 4/5 Homo sapiens Urine (d)(U)C
Filtration
SEC and UF
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC and UF
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Tamm-Horsfall protein/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin/ Tamm-Horsfall protein
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9
Not detected EV-associated proteins
CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
110.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.77E+09
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
60.75
EV concentration
Yes
EV200092 5/5 Homo sapiens Urine (d)(U)C
Filtration
DG
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
DG
Protein markers
EV: CD81/ Flotillin1
non-EV: None
Proteomics
no
EV density (g/ml)
1.10-1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
48
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
4.75
Fraction processing
Centrifugation
Pelleting: volume per fraction
28
Pelleting: duration (min)
60
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD81
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan
Calibration bead size
0.2
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
EV190046 2/2 Homo sapiens Cell culture supernatant exoEasy (Qiagen) Deville, Sarah 2021 50%

Study summary

Full title
All authors
Sarah Deville, Pascale Berckmans, Rebekka Van Hoof, Ivo Lambrichts, Anna Salvati, Inge Nelissen
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major (show more...)Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
THP-1
Sample origin
LPS-stimulated
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
exoEasy (Qiagen)
Protein markers
EV: / TSG101/ Flotillin1/ CD9
non-EV: Cytochrome C/ Cytochrome c
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
LPS-stimulated
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Commercial kit
Other;exoEasy (Qiagen)
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101
Not detected contaminants
Cytochrome C
Flow cytometry
Type of Flow cytometry
Hardware adjustments
Calibration bead size
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
210
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
A BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection was used for analysis. The device utilizes a highly sensitive fluorescence trigger to measure the EVs.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EV210003 1/3 Homo sapiens Cell culture supernatant (d)(U)C
DC
Qinyu, Ma 2021 44%

Study summary

Full title
All authors
Qinyu Ma, Mengmeng Liang, Yutong Wu, Ce Dou, Jianzhong Xu, Shiwu Dong, Fei Luo
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment thro (show more...)Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis. (hide)
EV-METRIC
44% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PC3
Sample origin
prostate cancer cell line
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density cushion
Protein markers
EV: TSG101/ Alix/ CD63/ CD9/ CD81
non-EV: Argonaute2/ Histone 3/ LaminA/C
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
prostate cancer cell line
EV-producing cells
PC3
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
39
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
110000
Density cushion
Density medium
Sucrose
Sample volume
30
Cushion volume
9
Density of the cushion
1,21
Centrifugation time
120
Centrifugation speed
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix/ CD81
Not detected contaminants
LaminA/C/ Histone 3/ Argonaute2
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
70-140
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 418000
EV210003 2/3 Homo sapiens Cell culture supernatant (d)(U)C
DC
Qinyu, Ma 2021 44%

Study summary

Full title
All authors
Qinyu Ma, Mengmeng Liang, Yutong Wu, Ce Dou, Jianzhong Xu, Shiwu Dong, Fei Luo
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment thro (show more...)Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis. (hide)
EV-METRIC
44% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
C4
Sample origin
prostate cancer cell line
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density cushion
Protein markers
EV: TSG101/ Alix/ CD63/ CD9/ CD81
non-EV: Argonaute2/ Histone 3/ LaminA/C
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
prostate cancer cell line
EV-producing cells
C4
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
39
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
110000
Density cushion
Density medium
Sucrose
Sample volume
30
Cushion volume
9
Density of the cushion
1,21
Centrifugation time
120
Centrifugation speed
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix/ CD81
Not detected contaminants
LaminA/C/ Histone 3/ Argonaute2
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
70-150
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 461000
EV210003 3/3 Homo sapiens Cell culture supernatant (d)(U)C
DC
Qinyu, Ma 2021 44%

Study summary

Full title
All authors
Qinyu Ma, Mengmeng Liang, Yutong Wu, Ce Dou, Jianzhong Xu, Shiwu Dong, Fei Luo
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment thro (show more...)Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis. (hide)
EV-METRIC
44% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
C4-2
Sample origin
prostate cancer cell line
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density cushion
Protein markers
EV: Alix/ TSG101/ CD63/ CD9/ CD81
non-EV: Argonaute2/ Histone 3/ LaminA/C
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
prostate cancer cell line
EV-producing cells
C4-2
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
39
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
110000
Density cushion
Density medium
Sucrose
Sample volume
30
Cushion volume
9
Density of the cushion
1,21
Centrifugation time
120
Centrifugation speed
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101/ CD81
Not detected contaminants
LaminA/C/ Histone 3/ Argonaute2
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
70-150
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 445000
EV200111 1/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Tortolici, Flavia 2021 43%

Study summary

Full title
All authors
Flavia Tortolici, Simone Vumbaca, Bernadette Incocciati, Renu Dayal, Katia Aquilano, Anna Giovanetti, Stefano Rufini
Journal
Cells
Abstract
Radiation therapy is one of the most effective methods of tumor eradication; however, in some forms (show more...)Radiation therapy is one of the most effective methods of tumor eradication; however, in some forms of neuroblastoma, radiation can increase the risk of secondary neoplasms, due to the ability of irradiated cells to transmit pro-survival signals to non-irradiated cells through vesicle secretion. The aims of this study were to characterize the vesicles released by the human neuroblastoma cell line SH-SY5Y following X-ray radiations and their ability to increase invasiveness in non-irradiated SH-SY5Y cells. We first purified the extracellular vesicles released by the SH-SY5Y cells following X-rays, and then determined their total amount, dimensions, membrane protein composition, and cellular uptake. We also examined the effects of these extracellular vesicles on viability, migration, and DNA damage in recipient SH-SY5Y cells. We found that exposure to X-rays increased the release of extracellular vesicles and altered their protein composition. These vesicles were readily uptaken by non-irradiated cells, inducing an increase in viability, migration, and radio-resistance. The same results were obtained in an MYCN-amplified SK-N-BE cell line. Our study demonstrates that vesicles released from irradiated neuroblastoma cells stimulate proliferation and invasiveness that correlate with the epithelial to mesenchymal transition in non-irradiated cells. Moreover, our results suggest that, at least in neuroblastomas, targeting the extracellular vesicles may represent a novel therapeutic approach to counteract the side effects associated with radiotherapy. (hide)
EV-METRIC
43% (70th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
SH-SY5Y
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD81/ CD63/ Flotillin1/ CD9
non-EV: BAX
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
SH-SY5Y
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
150
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Other;absorbance of 215 nm, using known protein concentrations of commercial freeze-dried exosomes
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ CD81
Not detected contaminants
BAX
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Cytoflex
Hardware adjustment
The single vesicles dimensional analysis was performed by the Cytoflex high-resolution flow cytometer based on the side scatter of the violet laser (405 nm) that allows the detection of particles below 300 nm of diameter, corresponding to the size-range of exosomes.
Calibration bead size
3
EV concentration
Yes
EV190107 3/3 Homo sapiens Cell culture supernatant IAF
IAF
qEV
Martin-Jaular, Lorena 2021 38%

Study summary

Full title
All authors
Lorena Martin-Jaular, Nathalie Nevo, Julia P Schessner, Mercedes Tkach, Mabel Jouve, Florent Dingli, Damarys Loew, Kenneth W Witwer, Matias Ostrowski, Georg H H Borner, Clotilde Théry
Journal
EMBO J
Abstract
Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surro (show more...)Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re-routed to non-viral EVs in a Nef-dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release. (hide)
EV-METRIC
38% (68th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Jurkat
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
IAF
Immunoaffinity capture (non-commercial)
qEV
Protein markers
EV: "MOV10/ CD63/ SPN/ SERINC3"
non-EV:
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Jurkat
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Commercial kit
qEV
Other
Name other separation method
Immunoaffinity capture (non-commercial)
EV-subtype
Distinction between multiple subtypes
Used subtypes
Characterization: Protein analysis
Protein Concentration Method
"Other;Gel stain free assay"
Western Blot
Detected EV-associated proteins
"CD63/ SERINC3/ SPN/ MOV10"
Other 1
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV200158 2/4 Escherichia coli Cell culture supernatant (d)(U)C
Filtration
Tangential flow filtration
Zanella, Ilaria 2021 33%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)DompA
Sample origin
Control condition
Focus vesicles
Other / OMVsDompA
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Tangential flow filtration
Protein markers
EV: ompF
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
E. coli BL21(DE3)DompA
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Detected EV-associated proteins
ompF
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
66.95
EV concentration
Yes
Particle yield
particle number per 100 ng of total OMV protein;Yes, other: 3.21E+08
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200158 4/4 Escherichia coli Cell culture supernatant (d)(U)C
Filtration
Tangential flow filtration
Zanella, Ilaria 2021 33%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)D60
Sample origin
genetically modified strain
Focus vesicles
Other / OMVsD60
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Tangential flow filtration
Protein markers
EV: ompF
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
genetically modified strain
EV-producing cells
E. coli BL21(DE3)D60
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Detected EV-associated proteins
ompF
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
69.9
EV concentration
Yes
Particle yield
particle number per 100 ng of total OMV protein;Yes, other: 3.09E+08
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190081 1/3 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Haghighitalab, Azadeh 2021 33%

Study summary

Full title
All authors
Azadeh Haghighitalab, Maryam M Matin, Ahmad Amin, Shima Minaee, Hamid Reza Bidkhori, Thorsten R Doeppner, Ahmad Reza Bahrami
Journal
Sci Rep
Abstract
The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawb (show more...)The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
hTERT-immortalized adipose tissue derived mesenchymal stromal cells
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63
non-EV:
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
hTERT-immortalized adipose tissue derived mesenchymal stromal cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
An-50Ti
Pelleting: speed (g)
70
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD63/ TSG101
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
40-400
NTA
Report type
Mean
Reported size (nm)
176
EV concentration
Yes
EM
EM-type
Atomic force-EM/ Transmission-EM
Image type
Wide-field
EV190081 2/3 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Haghighitalab, Azadeh 2021 33%

Study summary

Full title
All authors
Azadeh Haghighitalab, Maryam M Matin, Ahmad Amin, Shima Minaee, Hamid Reza Bidkhori, Thorsten R Doeppner, Ahmad Reza Bahrami
Journal
Sci Rep
Abstract
The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawb (show more...)The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
hTERT-immortalized adipose tissue derived mesenchymal stromal cells
Sample origin
Chemically primed
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63
non-EV:
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Chemically primed
EV-producing cells
hTERT-immortalized adipose tissue derived mesenchymal stromal cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
An-50Ti
Pelleting: speed (g)
70
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD63/ TSG101
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
40-400
NTA
Report type
Mean
Reported size (nm)
176-180
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
EV190081 3/3 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Haghighitalab, Azadeh 2021 29%

Study summary

Full title
All authors
Azadeh Haghighitalab, Maryam M Matin, Ahmad Amin, Shima Minaee, Hamid Reza Bidkhori, Thorsten R Doeppner, Ahmad Reza Bahrami
Journal
Sci Rep
Abstract
The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawb (show more...)The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases. (hide)
EV-METRIC
29% (55th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
hTERT-immortalized adipose tissue derived mesenchymal stromal cells
Sample origin
Genetically engineered
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV:
non-EV:
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Genetically engineered
EV-producing cells
hTERT-immortalized adipose tissue derived mesenchymal stromal cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
An-50Ti
Pelleting: speed (g)
70
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Protein Concentration Method
microBCA
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
40-400
EM
EM-type
Atomic force-EM/ Transmission-EM
Image type
Wide-field
EV200158 1/4 Escherichia coli Cell culture supernatant Filtration
(d)(U)C
Zanella, Ilaria 2021 14%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
14% (36th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)DompA
Sample origin
Control condition
Focus vesicles
Other / OMVsDompA
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
(Differential) (ultra)centrifugation
Protein markers
EV: ompF
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
E. coli BL21(DE3)DompA
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
132000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
ompF
Characterization: Particle analysis
EV200158 3/4 Escherichia coli Cell culture supernatant Filtration
(d)(U)C
Zanella, Ilaria 2021 14%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
14% (36th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)D60
Sample origin
genetically modified strain
Focus vesicles
Other / OMVsD60
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
(Differential) (ultra)centrifugation
Protein markers
EV: ompF
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
genetically modified strain
EV-producing cells
E. coli BL21(DE3)D60
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
121
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
132001
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
ompF
Characterization: Particle analysis
EV190053 1/2 Homo sapiens Cell culture supernatant SEC
SEC (non-commercial)
UF
Benet, Susana 2021 0%

Study summary

Full title
All authors
Susana Benet, Cristina Gálvez, Francis Drobniewski, Irina Kontsevaya, Lilibeth Arias, Marta Monguió‐Tortajada, Itziar Erkizia, Victor Urrea, Ruo‐Yan Ong, Marina Luquin, Maeva Dupont, Jakub Chojnacki, Judith Dalmau, Paula Cardona, Olivier Neyrolles, Geanncarlo Lugo‐Villarino, Christel Vérollet, Esther Julián, Hansjakob Furrer, Huldrych F. Günthard, Paul R. Crocker, Gustavo Tapia, Francesc E. Borràs, Jacques Fellay, Paul J. McLaren, Amalio Telenti, Pere‐Joan Cardona, Bonaventura Clotet, Cristina Vilaplana, Javier Martinez‐Picado, Nuria Izquierdo‐Useros
Journal
J Extracell Vesicles
Abstract
The identification of individuals with null alleles enables studying how the loss of gene function a (show more...)The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non‐functional variant in SIGLEC1, which encodes the myeloid‐cell receptor Siglec‐1/CD169 implicated in HIV‐1 cell‐to‐cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb‐infected Siglec‐1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec‐1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec‐1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination. (hide)
EV-METRIC
0% (median: 23% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
THP-1-derived macrophages
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
SEC
Size-exclusion chromatography (non-commercial)
UF
Protein markers
EV: CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
Function/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
THP-1-derived macrophages
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
0.25
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Protein Concentration Method
Nanodrop Absorbance at 280nm
Flow cytometry aspecific beads
Detected EV-associated proteins
CD9/ CD63
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
EV190053 2/2 Homo sapiens Cell culture supernatant SEC
SEC (non-commercial)
UF
Benet, Susana 2021 0%

Study summary

Full title
All authors
Susana Benet, Cristina Gálvez, Francis Drobniewski, Irina Kontsevaya, Lilibeth Arias, Marta Monguió‐Tortajada, Itziar Erkizia, Victor Urrea, Ruo‐Yan Ong, Marina Luquin, Maeva Dupont, Jakub Chojnacki, Judith Dalmau, Paula Cardona, Olivier Neyrolles, Geanncarlo Lugo‐Villarino, Christel Vérollet, Esther Julián, Hansjakob Furrer, Huldrych F. Günthard, Paul R. Crocker, Gustavo Tapia, Francesc E. Borràs, Jacques Fellay, Paul J. McLaren, Amalio Telenti, Pere‐Joan Cardona, Bonaventura Clotet, Cristina Vilaplana, Javier Martinez‐Picado, Nuria Izquierdo‐Useros
Journal
J Extracell Vesicles
Abstract
The identification of individuals with null alleles enables studying how the loss of gene function a (show more...)The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non‐functional variant in SIGLEC1, which encodes the myeloid‐cell receptor Siglec‐1/CD169 implicated in HIV‐1 cell‐to‐cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb‐infected Siglec‐1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec‐1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec‐1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination. (hide)
EV-METRIC
0% (median: 23% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
THP-1-derived macrophages
Sample origin
Mycobacterium tuberculosis infection
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
SEC
Size-exclusion chromatography (non-commercial)
UF
Protein markers
EV: CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
Function/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Mycobacterium tuberculosis infection
EV-producing cells
THP-1-derived macrophages
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
0.25
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Protein Concentration Method
Nanodrop Absorbance 280nm
Flow cytometry aspecific beads
Detected EV-associated proteins
CD9/ CD63
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
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