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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
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200010 4/4 Homo sapiens Blood plasma DG
(d)(U)C
SEC
Kuypers, Sören 2021 100%

Study summary

Full title
All authors
Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani
Journal
Small
Abstract
Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide)
EV-METRIC
100% (99th 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
DG
(d)(U)C
SEC
Protein markers
EV: ICAM/ CD63/ CD9/ ANXA2
non-EV: APOA1
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
New methodological development/Biomarker/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
Below or equal to 800 g
Between 800 g and 10,000 g
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 28.1
Speed (g)
100000
Duration (min)
1451
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1-6
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ ANXA2
Not detected contaminants
APOA1
Detected EV-associated proteins
CD9/ CD63/ ICAM
Not detected contaminants
APOA1
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100-200
EV200159 2/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 89%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
89% (98th 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
Expi293F
Sample origin
CD63-mCherry
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Alix/ Flotillin1/ CD9/ mcherry
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
CD63-mCherry
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ mCherry/ Alix/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 5,00E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
56
EV200159 3/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 89%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
89% (98th 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
Expi293F
Sample origin
CD63-FLuc
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Alix/ Flotillin1/ CD9
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
CD63-FLuc
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Firely luciferase/ CD9/ CD63/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 1,50E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
80
EV200118 1/1 Homo sapiens Cell culture supernatant (d)(U)C
ExoQuick
Filtration
UF
Tu, Chenggong 2021 75%

Study summary

Full title
All authors
Chenggong Tu, Zhimin Du, Hui Zhang, Yueyuan Feng, Yujun Qi, Yongjiang Zheng, Jinbao Liu, Jinheng Wang
Journal
Theranostics
Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal cells (BMSCs) have been demonstrated as key factors in the progression and drug resistance of multiple myeloma (MM). EV uptake involves a variety of mechanisms which largely depend on the vesicle origin and recipient cell type. The aim of the present study was to identify the mechanisms involved in the uptake of BMSC-derived small EVs (sEVs) by MM cells, and to evaluate the anti-MM effect of targeting this process. Methods: Human BMSC-derived sEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. The effects of chemical inhibitors and shRNA-mediated knockdown of endocytosis-associated genes on sEV uptake and cell apoptosis were analyzed by flow cytometry. The anti-MM effect of blocking sEV uptake was evaluated in vitro and in a xenograft MM mouse model. Results: sEVs derived from BMSC were taken up by MM cells in a time- and dose-dependent manner, and subsequently promoted MM cell cycling and reduced their chemosensitivity to bortezomib. Chemical endocytosis inhibitors targeting heparin sulphate proteoglycans, actin, tyrosine kinase, dynamin-2, sodium/proton exchangers, or phosphoinositide 3-kinases significantly reduced MM cell internalization of BMSC-derived sEVs. Moreover, shRNA-mediated knockdown of endocytosis-associated proteins, including caveolin-1, flotillin-1, clathrin heavy chain, and dynamin-2 in MM cells suppressed sEV uptake. Furthermore, an endocytosis inhibitor targeting dynamin-2 preferentially suppressed the uptake of sEV by primary MM cells ex vivo and enhanced the anti-MM effects of bortezomib in vitro and in a mouse model. Conclusion: Clathrin- and caveolin-dependent endocytosis and macropinocytosis are the predominant routes of sEV-mediated communication between BMSCs and MM cells, and inhibiting endocytosis attenuates sEV-induced reduction of chemosensitivity to bortezomib, and thus enhances its anti-MM properties. (hide)
EV-METRIC
75% (95th 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
primary bone marrow 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
(Differential) (ultra)centrifugation
ExoQuick
Filtration
Ultrafiltration
Protein markers
EV: CD63/ Flotillin1/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary bone marrow stromal cells
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63
Not detected contaminants
Calreticulin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-210
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 9.00E+07
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-120
EV200013 1/2 Bos taurus Bovine ampulla oviductal fluid DG
qEV
Asaadi, Anise 2021 75%

Study summary

Full title
All authors
Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani
Journal
Int J Biol Sci
Abstract
Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide)
EV-METRIC
75% (50th 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
Bovine ampulla oviductal fluid
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
Density gradient
qEV
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Function
Sample
Species
Bos taurus
Sample Type
Bovine ampulla oviductal fluid
Sample Condition
Control condition
Separation Method
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
14
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
165 +/- 5.8 nm
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.80E+12
EM
EM-type
Transmission-EM
Image type
Close-up
EV200013 2/2 Bos taurus Bovine ovarian follicular fluid DG
qEV
Asaadi, Anise 2021 75%

Study summary

Full title
All authors
Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani
Journal
Int J Biol Sci
Abstract
Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide)
EV-METRIC
75% (50th 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
Bovine ovarian follicular fluid
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
Density gradient
qEV
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Function
Sample
Species
Bos taurus
Sample Type
Bovine ovarian follicular fluid
Sample Condition
Control condition
Separation Method
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
14
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
166.9 +/- 9.7
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6.44E+12
EM
EM-type
Transmission-EM
Image type
Close-up
EV200010 1/4 Homo sapiens Cell culture supernatant (d)(U)C
SEC
UF
Kuypers, Sören 2021 75%

Study summary

Full title
All authors
Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani
Journal
Small
Abstract
Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide)
EV-METRIC
75% (95th 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
HUVEC
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
(d)(U)C
SEC
UF
Protein markers
EV: ANXA2/ CD81/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HUVEC
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
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
ANXA2/ CD81/ CD9
Not detected contaminants
GM130
Detected EV-associated proteins
CD9/ CD63/ ICAM1
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100-200
EV200010 2/4 Homo sapiens Cell culture supernatant (d)(U)C
SEC
UF
Kuypers, Sören 2021 75%

Study summary

Full title
All authors
Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani
Journal
Small
Abstract
Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide)
EV-METRIC
75% (95th 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
HUVEC
Sample origin
TNFalpha
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
(d)(U)C
SEC
UF
Protein markers
EV: ANXA2/ CD81/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
TNFalpha
EV-producing cells
HUVEC
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
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
ANXA2/ CD81/ CD9
Not detected contaminants
GM130
Detected EV-associated proteins
CD9/ CD63/ ICAM1
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100-200
EV200010 3/4 Homo sapiens Cell culture supernatant (d)(U)C
SEC
UF
Kuypers, Sören 2021 75%

Study summary

Full title
All authors
Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani
Journal
Small
Abstract
Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide)
EV-METRIC
75% (95th 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
HUVEC
Sample origin
IL1beta
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
(d)(U)C
SEC
UF
Protein markers
EV: ANXA2/ CD81/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
IL1beta
EV-producing cells
HUVEC
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
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
ANXA2/ CD81/ CD9
Not detected contaminants
GM130
Detected EV-associated proteins
CD9/ CD63/ ICAM1
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100-200
EV210044 1/1 Homo sapiens Serum qEV Newman, Lauren 2021 71%

Study summary

Full title
All authors
Lauren A. Newman, Alia Fahmy, Michael J. Sorich, Oliver G. Best, Andrew Rowland, Zivile Useckaite
Journal
Cells
Abstract
Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human bl (show more...)Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease. (hide)
EV-METRIC
71% (98th 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
Serum
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
qEV
Protein markers
EV: CD81/ ASGR1/ CD63/ CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Flow cytometry
Type of Flow cytometry
CytoFlex S
Hardware adjustments
1. Filter configuration as per Beckman Coulter website 2. Violet filter used for small particle detection 3. Calibration beads were used to determine particle size gating strategy 4. Callibration beads used Megamix Plus SSC and Megaamix Plus FSC
Calibration bead size
0.1-0.9
Detected EV-associated proteins
CD63/ CD9/ CD81/ ASGR1
Proteomics
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
85.8
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.25E+11
Particle analysis: flow cytometry
Flow cytometer type
CytoFlex S
Hardware adjustment
1. Laser configuration to detect small particles, violet laser use, hardware set up according to Beckman Coulter website 2. Use of MegaMix beads to determine particle size for gating purposes 3. MegaMix plus beads were used: MegaMix Plus SSC and MegaMix Plus Forward FSC
Calibration bead size
0.1-0.9
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200159 1/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 67%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
67% (92nd 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
Expi293F
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ Flotillin1/ CD9/ CD81
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
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)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ Alix/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
No NA
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
56
EV200076 1/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Dinoflagellate
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
(d)(U)C
Protein markers
EV: Alix/ HSP70/ beta-actin/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Dinoflagellate
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
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
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
beta-actin/ enolase/ HSP70/ Alix
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
92
NTA
Report type
Size range/distribution
Reported size (nm)
125
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 6.00E+09
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200076 2/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Diatom
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
(d)(U)C
Protein markers
EV: Alix/ HSP70/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Diatom
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
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
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
enolase/ HSP70/ Alix
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
135
NTA
Report type
Size range/distribution
Reported size (nm)
90
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 2.40E+08
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200076 3/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Glaucophyte
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
(d)(U)C
Protein markers
EV: Alix/ HSP70/ beta-actin/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Glaucophyte
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
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
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ HSP70/ beta-actin/ enolase
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
125
NTA
Report type
Size range/distribution
Reported size (nm)
122
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 2.00E+09
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200076 4/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Haptophyte
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
(d)(U)C
Protein markers
EV: Alix/ HSP70/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Haptophyte
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
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
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ enolase
Not detected EV-associated proteins
HSP70
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
87
NTA
Report type
Size range/distribution
Reported size (nm)
165
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 1.30E+08
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200076 5/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Chlorophyte
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
(d)(U)C
Protein markers
EV: Alix/ beta-actin/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Chlorophyte
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
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
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
beta-actin/ enolase/ Alix
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
75
NTA
Report type
Size range/distribution
Reported size (nm)
137
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 2.60E+08
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200047 2/2 Homo sapiens Cell culture supernatant UF
DG
Yildizhan, Yagmur 2021 67%

Study summary

Full title
All authors
Yagmur Yildizhan, Venkata Suresh Vajrala, Edward Geeurickx, Charles Declerck, Nevena Duskunovic, Delphine De Sutter, Sam Noppen, Filip Delport, Dominique Schols, Johannes V. Swinnen, Sven Eyckerman, An Hendrix, Jeroen Lammertyn, Dragana Spasic
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including (show more...)Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well‐characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO‐SPR) bioassay. In this context, EV binding on the FO‐SPR probes was achieved only with EV‐specific antibodies (e.g. anti‐CD9 and anti‐CD63) but not with non‐specific anti‐IgG. To increase detection sensitivity, we tested six different combinations of EV‐specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti‐CD9/Banti‐CD81 and anti‐CD63/Banti‐CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti‐CD63/Banti‐CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti‐EpCAM antibody on the FO‐SPR surface. The obtained results combined with FO‐SPR real‐time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis. (hide)
EV-METRIC
67% (92nd 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
MCF7
Sample origin
Rab27B-GFP
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
Ultrafiltration
Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Rab27B-GFP
EV-producing cells
MCF7
EV-harvesting Medium
Serum free medium
Separation Method
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
11
Lowest density fraction
6
Highest density fraction
18
Total gradient volume, incl. sample (mL)
15
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
186700
Duration (min)
116
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200100 1/1 Homo sapiens colostrum (d)(U)C
ExoQuick
Civra, Andrea; Francese, Rache 2021 63%

Study summary

Full title
All authors
Andrea Civra, Rachele Francese, Manuela Donalisio, Paola Tonetto, Alessandra Coscia, Stefano Sottemano, Raffaella Balestrini, Antonella Faccio, Laura Cavallarin, Guido E Moro, Enrico Bertino, David Lembo
Journal
J hum Lact
Abstract
Background: It is known that breastfeeding protects the infant from enteric and respiratory infectio (show more...)Background: It is known that breastfeeding protects the infant from enteric and respiratory infections; however, the antiviral properties of human milk against enteric and respiratory viruses are largely unexplored. Research aims: To explore the antiviral activity of human preterm colostrum against rotavirus and respiratory syncytial virus and to assess whether the derived extracellular vesicle contribute to this activity. Methods: We used a cross-sectional, prospective two-group non-experimental design. Colostra were collected from mothers of preterm newborns (N = 10) and extracellular vesicles were purified and characterized. The antiviral activity of colostra and derived extracellular vesicles were tested in vitro against rotavirus and respiratory syncytial virus and the step of viral replication inhibited by extracellular vesicles was investigated. Results: Each sample of colostrum and colostrum-derived extracellular vesicles had significant antiviral activity with a wide interpersonal variability. Mechanism of action studies demonstrated that extracellular vesicles acted by interfering with the early steps of the viral replicative cycle. Conclusion: We demonstrated the intrinsic antiviral activity of human colostrum against rotavirus and respiratory syncytial virus and we showed that extracellular vesicles substantially contribute to the overall protective effect. Our results contribute to unravelling novel mechanisms underlying the functional role of human milk as a protective and therapeutic agent in preterm infants. (hide)
EV-METRIC
63% (50th 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
colostrum
Sample origin
preterm mothers
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
(d)(U)C
ExoQuick
Protein markers
EV: CD81/ CD63/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
colostrum
Sample Condition
preterm mothers
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected contaminants
calnexin
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
258
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 7.40E+11
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200093 10/10 Homo sapiens Blood plasma (d)(U)C
Filtration
DG
Dong, Liang 2021 63%

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
63% (93rd 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
(d)(U)C
Filtration
DG
Protein markers
EV: CD81/ Flotillin1
non-EV: ApoA1
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
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
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
Not detected contaminants
ApoA1
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
Report type
Not Reported
EV200159 4/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 57%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
57% (88th 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
Expi293F
Sample origin
CD63-NLuc
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
CD63-NLuc
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 2,00E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
75
EV200047 1/2 Homo sapiens Cell culture supernatant UF
DG
Yildizhan, Yagmur 2021 57%

Study summary

Full title
All authors
Yagmur Yildizhan, Venkata Suresh Vajrala, Edward Geeurickx, Charles Declerck, Nevena Duskunovic, Delphine De Sutter, Sam Noppen, Filip Delport, Dominique Schols, Johannes V. Swinnen, Sven Eyckerman, An Hendrix, Jeroen Lammertyn, Dragana Spasic
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including (show more...)Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well‐characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO‐SPR) bioassay. In this context, EV binding on the FO‐SPR probes was achieved only with EV‐specific antibodies (e.g. anti‐CD9 and anti‐CD63) but not with non‐specific anti‐IgG. To increase detection sensitivity, we tested six different combinations of EV‐specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti‐CD9/Banti‐CD81 and anti‐CD63/Banti‐CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti‐CD63/Banti‐CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti‐EpCAM antibody on the FO‐SPR surface. The obtained results combined with FO‐SPR real‐time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis. (hide)
EV-METRIC
57% (88th 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
HEK293T
Sample origin
transfected with gag-EGFP
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
Ultrafiltration
Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
transfected with gag-EGFP
EV-producing cells
HEK293T
EV-harvesting Medium
Serum free medium
Separation Method
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
108.6
EV concentration
Yes
Particle yield
No NA
Particle analysis: flow cytometry
Flow cytometer type
High resolution flow cytometry
Hardware adjustment
200 mW 488 nm laser (Sapphire; Coherent, Santa Clara, CA, USA) and a large-bore nozzle (140 m) were used, sheath pressure was permanently monitored and kept within 4.89 to 5.02 psi, and the sample pressure was set at 4.29 psi, to assure an identical diameter of the core in the jet stream. Forward scattered light was measured with a collection angle of 1525 (reduced wide-angle forward scatter [rw-FSC]).
Calibration bead size
0.102
EV concentration
Yes
EM
EM-type
Immuno-EM/ Transmission-EM
Proteïns
CD63
Image type
Close-up, Wide-field
EV200125 1/2 Homo sapiens Cell culture supernatant (d)(U)C Aminzadeh, Mark 2021 56%

Study summary

Full title
All authors
Mark A. Aminzadeh, Mario Fournier, Akbarshakh Akhmerov, K. Candis Jones‐Ungerleider, Jackelyn B. Valle, Eduardo Marbán
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from cardiac stromal cells, developed as therapeutic candidates, improv (show more...)Extracellular vesicles (EVs) from cardiac stromal cells, developed as therapeutic candidates, improve dystrophic muscle function when administered parenterally, but oral delivery remains untested. We find that casein, the dominant protein in breast milk, enhances the uptake and bioactivity of ingested heart‐derived EVs, altering gene expression in blood cells and enhancing muscle function in mdx mice with muscular dystrophy. Thus, EVs, administered orally, are absorbed and exert disease‐modifying bioactivity in vivo. Formulating EVs with casein enhances uptake and markedly expands the range of potential therapeutic applications. (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
cardiosphere-derived 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
cardiosphere-derived cells
EV-harvesting Medium
Serum free medium
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)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
150
Used for determining EV concentration?
Yes
EM
EM-type
Immuno-EM/ Transmission-EM
Proteïns
CD63
Image type
Close-up, Wide-field
EV200125 2/2 Homo sapiens Cell culture supernatant (d)(U)C Aminzadeh, Mark 2021 56%

Study summary

Full title
All authors
Mark A. Aminzadeh, Mario Fournier, Akbarshakh Akhmerov, K. Candis Jones‐Ungerleider, Jackelyn B. Valle, Eduardo Marbán
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from cardiac stromal cells, developed as therapeutic candidates, improv (show more...)Extracellular vesicles (EVs) from cardiac stromal cells, developed as therapeutic candidates, improve dystrophic muscle function when administered parenterally, but oral delivery remains untested. We find that casein, the dominant protein in breast milk, enhances the uptake and bioactivity of ingested heart‐derived EVs, altering gene expression in blood cells and enhancing muscle function in mdx mice with muscular dystrophy. Thus, EVs, administered orally, are absorbed and exert disease‐modifying bioactivity in vivo. Formulating EVs with casein enhances uptake and markedly expands the range of potential therapeutic applications. (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
cardiosphere-derived cells
Sample origin
Hypoxia
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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Hypoxia
EV-producing cells
cardiosphere-derived cells
EV-harvesting Medium
Serum free medium
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)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
150
Used for determining EV concentration?
Yes
EM
EM-type
Immuno-EM/ Transmission-EM
Proteïns
CD63
Image type
Close-up, Wide-field
EV200111 2/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Tortolici, Flavia 2021 56%

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
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
SH-SY5Y
Sample origin
Irradiated with 0.1 Gy (X-Ray)
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
(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
Irradiated with 0.1 Gy (X-Ray)
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
Report type
Size range/distribution
Reported size (nm)
3
EV concentration
Yes
EV200111 3/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Tortolici, Flavia 2021 56%

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
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
SH-SY5Y
Sample origin
Irradiated with 1 Gy (X-Ray)
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
(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
Irradiated with 1 Gy (X-Ray)
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
Report type
Size range/distribution
Reported size (nm)
3
EV200111 4/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Tortolici, Flavia 2021 56%

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
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
SH-SY5Y
Sample origin
Irradiated with 10 Gy (X-Ray)
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
(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
Irradiated with 10 Gy (X-Ray)
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
Report type
Size range/distribution
Reported size (nm)
3
EV concentration
Yes
EV200093 1/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Dong, Liang 2021 56%

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
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
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
(d)(U)C
Filtration
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
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
28
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
120000
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)
113.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 9.05E+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
EV concentration
Yes
EV200093 6/10 Homo sapiens Blood plasma (d)(U)C
Filtration
Dong, Liang 2021 56%

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
56% (90th 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
(d)(U)C
Filtration
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
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
28
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
120000
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)
121.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.42E+09
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 1/5 Homo sapiens Urine (d)(U)C
Filtration
Dong, Liang 2021 56%

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
56% (89th 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
(d)(U)C
Filtration
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
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
28
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
120000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Detected contaminants
Tamm-Horsfall protein
Not detected contaminants
Calreticulin
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)
83.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 3.48E+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
EV concentration
Yes
EV200014 1/4 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Wallis, Ryan 2021 56%

Study summary

Full title
All authors
Ryan Wallis, Natasa Josipovic, Hannah Mizen, Arturo Robles‐Tenorio, Eleanor J. Tyler, Argyris Papantonis, Cleo L. Bishop
Journal
J Extracell Vesicles
Abstract
A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediato (show more...)A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents – the senescence‐associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co‐isolate with EVs following differential ultracentrifugation (dUC). We present size‐exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non‐cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co‐isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP. (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
IMR90 ER:STOP
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
(d)(U)C
qEV
Protein markers
EV: TSG101/ CD9
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
IMR90 ER:STOP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell number specification
No
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)
90
Pelleting: rotor type
T-865
Pelleting: speed (g)
99582
Commercial kit
qEV
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
microBCA
Western Blot
Not detected EV-associated proteins
TSG101/ CD9
Not detected contaminants
Calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
92
EV concentration
Yes
Particle yield
total concentration;Yes, other: 1.60E+10
EV200014 2/4 Homo sapiens Cell culture supernatant (d)(U)C Wallis, Ryan 2021 56%

Study summary

Full title
All authors
Ryan Wallis, Natasa Josipovic, Hannah Mizen, Arturo Robles‐Tenorio, Eleanor J. Tyler, Argyris Papantonis, Cleo L. Bishop
Journal
J Extracell Vesicles
Abstract
A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediato (show more...)A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents – the senescence‐associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co‐isolate with EVs following differential ultracentrifugation (dUC). We present size‐exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non‐cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co‐isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP. (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
IMR90 ER:STOP
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
(d)(U)C
Protein markers
EV: TSG101/ IL-8/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
IMR90 ER:STOP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell number specification
No
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)
90
Pelleting: rotor type
T-865
Pelleting: speed (g)
99582
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Calnexin
ELISA
Detected EV-associated proteins
IL-8
Proteomics
Proteomics database
No
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
85.2
EV concentration
Yes
Particle yield
total concentration;Yes, other: 2.20E+11
EV200014 3/4 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Wallis, Ryan 2021 56%

Study summary

Full title
All authors
Ryan Wallis, Natasa Josipovic, Hannah Mizen, Arturo Robles‐Tenorio, Eleanor J. Tyler, Argyris Papantonis, Cleo L. Bishop
Journal
J Extracell Vesicles
Abstract
A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediato (show more...)A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents – the senescence‐associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co‐isolate with EVs following differential ultracentrifugation (dUC). We present size‐exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non‐cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co‐isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP. (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
IMR90 ER:RAS
Sample origin
HRas:ER Oncogene Induced Senescence Model
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
(d)(U)C
qEV
Protein markers
EV: TSG101/ IL-8/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HRas:ER Oncogene Induced Senescence Model
EV-producing cells
IMR90 ER:RAS
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell number specification
No
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)
90
Pelleting: rotor type
T-865
Pelleting: speed (g)
99582
Commercial kit
qEV
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Not detected contaminants
Calnexin
ELISA
Detected EV-associated proteins
IL-8
Proteomics
Proteomics database
No
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
107
EV concentration
Yes
Particle yield
total concentration;Yes, other: 8.80E+10
EV200014 4/4 Homo sapiens Cell culture supernatant (d)(U)C Wallis, Ryan 2021 56%

Study summary

Full title
All authors
Ryan Wallis, Natasa Josipovic, Hannah Mizen, Arturo Robles‐Tenorio, Eleanor J. Tyler, Argyris Papantonis, Cleo L. Bishop
Journal
J Extracell Vesicles
Abstract
A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediato (show more...)A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents – the senescence‐associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co‐isolate with EVs following differential ultracentrifugation (dUC). We present size‐exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non‐cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co‐isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP. (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
IMR90 ER:RAS
Sample origin
HRas:ER Oncogene Induced Senescence Model
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
(d)(U)C
Protein markers
EV: TSG101/ IL-8/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HRas:ER Oncogene Induced Senescence Model
EV-producing cells
IMR90 ER:RAS
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell number specification
No
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)
90
Pelleting: rotor type
T-865
Pelleting: speed (g)
99582
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Calnexin
ELISA
Detected EV-associated proteins
IL-8
Proteomics
Proteomics database
No
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
102
EV concentration
Yes
Particle yield
total concentration;Yes, other: 4.50E+11
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
(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% (81st 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
(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% (81st 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
(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% (81st 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
(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