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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV220127 1/2 Homo sapiens lung tissue (d)(U)C
DG
Filtration
Liu, Bowen/ Jin, Yuan 2022 100%

Study summary

Full title
All authors
Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide)
EV-METRIC
100% (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
lung tissue
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
20553 (pelleting) / 17842 (washing)
Protein markers
EV: Alix/ CD9/ CD81
non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.1
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
lung tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
20553
Wash: volume per pellet (ml)
1.5
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
110,000
Wash: adjusted k-factor
17842
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
0.25 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.45
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
180,000
Duration (min)
780
Fraction volume (mL)
0.45
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
17842
Pelleting-wash: volume per pellet (mL)
1.5
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
TLA-55
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
0.8
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ CD81
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
131.7
Particle analysis: flow cytometry
Flow cytometer type
BD LSRFortessa
Hardware adjustment
use calibration beads
Calibration bead size
0.05/ 0.1/ 0.2/ 0.3/ 0.5
Report type
Size range/distribution
Reported size (nm)
100 - 200
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV220127 2/2 Mus musculus lung tissue (d)(U)C
DG
Filtration
Liu, Bowen/ Jin, Yuan 2022 100%

Study summary

Full title
All authors
Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide)
EV-METRIC
100% (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
lung tissue
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
20553 (pelleting) / 17842 (washing)
Protein markers
EV: Alix/ CD9/ Flotillin-1/ TSG101
non-EV: GM130/ Calnexin/ Albumin/ Argonaute-2/ Calreticulin/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.1
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
lung tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
20553
Wash: volume per pellet (ml)
1.5
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
110,000
Wash: adjusted k-factor
17842
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
0.25 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.45
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
180,000
Duration (min)
780
Fraction volume (mL)
0.45
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
17842
Pelleting-wash: volume per pellet (mL)
1.5
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
TLA-55
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
0.7
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ Flotillin-1/ TSG101
Not detected contaminants
GM130/ Calnexin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
124.9
Particle analysis: flow cytometry
Flow cytometer type
BD LSRFortessa
Hardware adjustment
use calibration beads
Calibration bead size
0.05/ 0.1/ 0.2/ 0.3/ 0.5
Report type
Size range/distribution
Reported size (nm)
100 - 200
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210151 1/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2
Not detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 2.23E7 +- 1.35E7
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 2/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CPC (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2/ TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 8.27E6 +- 3.53E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 3/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMi (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2/ TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 2.95E7 +- 1.19E7
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 4/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMm (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2
Not detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250 nm
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 4.10E7 +- 9.75E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200030 1/2 Homo sapiens Cell culture supernatant (d)(U)C
Other/ ExoSpin
UF
Filtration
DG
Knight R 2022 100%

Study summary

Full title
All authors
Knight R, Board-Davies E, Brown H, Clayton A, Davis T, Karatas B, Burston J, Tabi Z, Falcon-Perez JM, Paisey S, Stephens P
Journal
Stem Cells Transl Med
Abstract
Scar formation during wound repair can be devastating for affected individuals. Our group previously (show more...)Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings/ hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Control condition
Focus vesicles
Other/ small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Commercial method
Ultrafiltration
Filtration
Density gradient
Protein markers
EV: CD81/ CD63/ CD9
non-EV: CD105/ CD90/ CD166
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
oral mucosa lamina propria progenitor cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0.2M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
16
Fraction volume (mL)
0.3
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Commercial kit
Other/ ExoSpin
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected contaminants
CD90/ CD105/ CD166
Flow cytometry specific beads
Antibody details provided?
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
94
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 7.7E+12
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV200030 2/2 Homo sapiens Cell culture supernatant (d)(U)C
Other/ ExoSpin
UF
Filtration
DG
Knight R 2022 100%

Study summary

Full title
All authors
Knight R, Board-Davies E, Brown H, Clayton A, Davis T, Karatas B, Burston J, Tabi Z, Falcon-Perez JM, Paisey S, Stephens P
Journal
Stem Cells Transl Med
Abstract
Scar formation during wound repair can be devastating for affected individuals. Our group previously (show more...)Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings/ hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Control condition
Focus vesicles
Other/ small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Commercial method
Ultrafiltration
Filtration
Density gradient
Protein markers
EV: CD81/ CD63/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
bone marrow derived mesenchymal stromal cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0.2M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
16
Fraction volume (mL)
0.3
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Commercial kit
Other/ ExoSpin
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected contaminants
CD90/ CD105/ CD166
Flow cytometry specific beads
Antibody details provided?
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
99.5
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 4.27E+12
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210151 5/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
90
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 1.99E7 +- 1.99E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 6/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CPC (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 6.43E06 +- 5.40E05
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 7/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMi (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 3.49E07 +- 2.85E06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 8/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMm (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 2.13E07 +- 2.25E06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV21008 1/2 Homo sapiens Blood plasma DG
(d)(U)C
Annalisa Radeghieri 2022 89%

Study summary

Full title
All authors
Annalisa Radeghieri, Silvia Alacqua, Andrea Zendrini, Vanessa Previcini, Francesca Todaro, Giuliana Martini, Doris Ricotta, Paolo Bergese
Journal
Journal of Extracellular Biology
Abstract
Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clott (show more...)Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the “EV-protein corona.” The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms. (hide)
EV-METRIC
89% (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. For the quantitative method, the reporting of measured EV concentration is expected.
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Adam 10/ Alix/ Antithrombin 3
non-EV: Argonaute2/ Apo A1/ GM130
Proteomics
no
EV density (g/ml)
1.11-1.22
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
8%
Highest density fraction
75%
Total gradient volume, incl. sample (mL)
4,8
Sample volume (mL)
1
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0,4
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Adam 10/ TSG101/ Alix/ CD81
Not detected contaminants
Apo A1/ GM130
Detected EV-associated proteins
CD63/ TSG101
Detected contaminants
Argonaute2
Detected EV-associated proteins
Antithrombin 3
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Atomic force-EM
Image type
Close-up, Wide-field
Report size (nm)
50
EV210024 3/12 Homo sapiens Cell culture supernatant (d)(U)C
DG
Gwennan André-Grégoire 2022 89%

Study summary

Full title
All authors
Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard
Journal
iScience
Abstract
Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: Alix/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Glioblastoma Stem-like cells (GSC)
EV-harvesting Medium
Serum free medium
Cell count
5.00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
11
Wash: time (min)
120
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40
Total gradient volume, incl. sample (mL)
11.5
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
120
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ Alix
Not detected contaminants
GM130
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50
EV210204 1/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PANC-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ANXA5/ EpCAM/ ICAM/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
180.8
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 2/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
UF
qEV
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Commercial method
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PANC-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ICAM/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 4/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PPCL-68
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ANXA5/ EpCAM/ ICAM/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
180.8
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 5/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
UF
qEV
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Commercial method
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PPCL-68
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ICAM/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 7/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hTERT-HPNE
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ANXA5/ EpCAM/ ICAM/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
180.8
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 8/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
UF
qEV
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Commercial method
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hTERT-HPNE
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ICAM/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 10/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HPDE-H6c7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ANXA5/ EpCAM/ ICAM/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
180.8
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 11/12 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
UF
qEV
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Commercial method
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HPDE-H6c7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ ICAM/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV220305 1/2 Homo sapiens Cell culture supernatant (d)(U)C Ngo NH 2022 78%

Study summary

Full title
All authors
Ngo NH, Chang YH, Vuong CK, Yamashita T, Obata-Yasuoka M, Hamada H, Osaka M, Hiramatsu Y, Ohneda O
Journal
Front Cell Dev Biol
Abstract
The therapeutic effects of endothelial progenitor cells (EPC) in neovascularization have been sugges (show more...)The therapeutic effects of endothelial progenitor cells (EPC) in neovascularization have been suggested/ however, to date, few studies have been conducted on the ability of EPC-derived extracellular vesicles (EV) to rescue the ischemic tissues. In order to examine the functional sources of EV for cell-free therapy of ischemic diseases, we compared the functions of EPC-EV and those of Wharton's Jelly-derived mesenchymal stem cell (WJ-EV) in the flap mouse model. Our results demonstrated that in the intravenous injection, EPC-EV, but not WJ-EV, were uptaken by the ischemic tissues. However, EPC-EV showed poor abilities to induce neovascularization and the recovery of ischemic tissues. In addition, compared to EPC-EV, WJ-EV showed a higher ability to rescue the ischemic injury when being locally injected into the mice. In order to induce the secretion of high-functional EPC-EV, EPC were internalized with hypoxic pre-treated WJ-EV, which resulted in a transformed hwEPC. In comparison to EPC, hwEPC showed induced proliferation and upregulation of angiogenic genes and miRNAs and promoted angiogenic ability. Interestingly, hwEPC produced a modified EV (hwEPC-EV) that highly expressed miRNAs related to angiogenesis, such as miR-155, miR-183, and miR-296. Moreover, hwEPC-EV significantly induced the neovascularization of the ischemic tissues which were involved in promoting the proliferation, the expression of VEGF and miR-183, and the angiogenic functions of endothelial cells. Of note, hwEPC-EV were highly uptaken by the ischemic tissues and showed a greater effect with regard to inducing recovery from ischemic injury in the intravenous administration, compared to EPC-EV. Therefore, hwEPC-EV can be considered a functional candidate for cell-free therapy to treat the distal ischemic tissues. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ CD40/ integrin beta-1
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Wharton's Jelly mesenchymal stem cell
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99.9
Cell count
1000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
140000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
140000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
TSG101/ CD40/ integrin beta-1
Not detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
0-1000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220305 2/2 Homo sapiens Cell culture supernatant (d)(U)C Ngo NH 2022 78%

Study summary

Full title
All authors
Ngo NH, Chang YH, Vuong CK, Yamashita T, Obata-Yasuoka M, Hamada H, Osaka M, Hiramatsu Y, Ohneda O
Journal
Front Cell Dev Biol
Abstract
The therapeutic effects of endothelial progenitor cells (EPC) in neovascularization have been sugges (show more...)The therapeutic effects of endothelial progenitor cells (EPC) in neovascularization have been suggested/ however, to date, few studies have been conducted on the ability of EPC-derived extracellular vesicles (EV) to rescue the ischemic tissues. In order to examine the functional sources of EV for cell-free therapy of ischemic diseases, we compared the functions of EPC-EV and those of Wharton's Jelly-derived mesenchymal stem cell (WJ-EV) in the flap mouse model. Our results demonstrated that in the intravenous injection, EPC-EV, but not WJ-EV, were uptaken by the ischemic tissues. However, EPC-EV showed poor abilities to induce neovascularization and the recovery of ischemic tissues. In addition, compared to EPC-EV, WJ-EV showed a higher ability to rescue the ischemic injury when being locally injected into the mice. In order to induce the secretion of high-functional EPC-EV, EPC were internalized with hypoxic pre-treated WJ-EV, which resulted in a transformed hwEPC. In comparison to EPC, hwEPC showed induced proliferation and upregulation of angiogenic genes and miRNAs and promoted angiogenic ability. Interestingly, hwEPC produced a modified EV (hwEPC-EV) that highly expressed miRNAs related to angiogenesis, such as miR-155, miR-183, and miR-296. Moreover, hwEPC-EV significantly induced the neovascularization of the ischemic tissues which were involved in promoting the proliferation, the expression of VEGF and miR-183, and the angiogenic functions of endothelial cells. Of note, hwEPC-EV were highly uptaken by the ischemic tissues and showed a greater effect with regard to inducing recovery from ischemic injury in the intravenous administration, compared to EPC-EV. Therefore, hwEPC-EV can be considered a functional candidate for cell-free therapy to treat the distal ischemic tissues. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
treated with Hypoxia WJ-MSC derived EV
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ CD40/ Integrin beta-1
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
endothelial progenitor cell
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99.9
Cell count
1000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
140000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
140000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
TSG101/ CD40/ integrin beta-1
Not detected contaminants
ApoA1
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
500
RNAse treatment
Yes
RNAse type
RNase A
RNAse concentration
0.01
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
0-1000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190029 1/8 Homo sapiens Cell culture supernatant (d)(U)C Bhome R 2022 78%

Study summary

Full title
All authors
Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE
Journal
J Extracell Vesicles
Abstract
Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
DLD-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
TFT 50.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
20
Wash: time (min)
70
Wash: Rotor Type
TFT 50.38
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101/ Alix/ CD81
Not detected contaminants
cytochrome c/ GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
110
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190029 2/8 Homo sapiens Cell culture supernatant (d)(U)C Bhome R 2022 78%

Study summary

Full title
All authors
Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE
Journal
J Extracell Vesicles
Abstract
Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HCT-116
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
TFT 50.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
20
Wash: time (min)
70
Wash: Rotor Type
TFT 50.38
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD63/ TSG101/ CD81
Not detected contaminants
cytochrome c/ GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
98
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190029 3/8 Homo sapiens Cell culture supernatant (d)(U)C Bhome R 2022 78%

Study summary

Full title
All authors
Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE
Journal
J Extracell Vesicles
Abstract
Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
SW620
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
TFT 50.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
20
Wash: time (min)
70
Wash: Rotor Type
TFT 50.38
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD63/ TSG101/ CD81
Not detected contaminants
cytochrome c/ GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
115
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190029 4/8 Homo sapiens Cell culture supernatant (d)(U)C Bhome R 2022 78%

Study summary

Full title
All authors
Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE
Journal
J Extracell Vesicles
Abstract
Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
SW480
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
TFT 50.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
20
Wash: time (min)
70
Wash: Rotor Type
TFT 50.38
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD63/ TSG101/ CD81
Not detected contaminants
cytochrome c/ GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
124
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190029 5/8 Homo sapiens Cell culture supernatant (d)(U)C Bhome R 2022 78%

Study summary

Full title
All authors
Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE
Journal
J Extracell Vesicles
Abstract
Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
ZEB-1 knock down
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
SW480
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
TFT 50.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
20
Wash: time (min)
70
Wash: Rotor Type
TFT 50.38
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101/ Alix/ CD81
Not detected contaminants
cytochrome c/ GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
105
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210345 1/17 Homo sapiens Cell culture supernatant (d)(U)C Osteikoetxea X 2022 78%

Study summary

Full title
All authors
Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ CD81/ Flotillin­1/ TSG101/ syntenin-1/ B-actin
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Drug delivery
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
95.4
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Concentration Method
Nanodrop
Protein Yield (µg)
number of particles per million cells
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD63/ CD81/ Flotillin­1/ TSG101/ syntenin-1/ B-actin
Detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
120
EV concentration
Yes
Particle yield
number of particles per million cells: 4.00e+4
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210345 3/17 Homo sapiens Cell culture supernatant (d)(U)C Osteikoetxea X 2022 78%

Study summary

Full title
All authors
Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
MysPalm-PHYB-PIF6-Cas9
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ CD81/ Flotillin­1/ TSG101/ B-actin/ syntenin-1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Drug delivery
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
95.4
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Concentration Method
Nanodrop
Protein Yield (µg)
number of particles per million cells
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Not detected EV-associated proteins
Alix/ CD63/ CD81/ Flotillin­1/ TSG101/ B-actin/ syntenin-1/ spCas9
Detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
120
EV concentration
Yes
Particle yield
number of particles per million cells: 2.00e+4
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210345 5/17 Homo sapiens Cell culture supernatant (d)(U)C Osteikoetxea X 2022 78%

Study summary

Full title
All authors
Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
Sample origin
MysPalm-CIBN-CRY2-Cas9
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ CD81/ Flotillin­1/ TSG101/ B-actin/ Syntenin-1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Drug delivery
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
95.4
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Concentration Method
Nanodrop
Protein Yield (µg)
number of particles per million cells
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD63/ CD81/ Flotillin­1/ TSG101/ B-actin/ Syntenin-1/ spCas9
Detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
120
EV concentration
Yes
Particle yield
number of particles per million cells: 4.00e+4
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210199 1/1 Homo sapiens Urine (d)(U)C
Filtration
Correll, Vanessa 2022 78%

Study summary

Full title
All authors
Vanessa L. Correll, Joseph J. Otto, Cristina M. Risi, Brian P. Main, Paul C. Boutros, Thomas Kislinger, Vitold E. Galkin, Julius O. Nyalwidhe, O. John Semmes, Lifang Yang
Journal
J Extracell Vesicles
Abstract
The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient (show more...)The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs. (hide)
EV-METRIC
78% (97th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
prostate cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD9
non-EV: Calnexin/ Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods/Biomarker/New methodological development
Sample
Species
Homo sapiens
Sample Type
Urine
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
175000
Wash: volume per pellet (ml)
12.5
Wash: time (min)
130
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
175000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
per 1E10 particles: 1.16
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Tamm-Horsfall protein
Not detected contaminants
Calnexin
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
145.9
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.20E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210187 1/5 Homo sapiens Cell culture supernatant (d)(U)C Swatler, Julian 2022 78%

Study summary

Full title
All authors
Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka
Journal
Blood advances
Abstract
Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (hide)
EV-METRIC
78% (96th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ Alix/ CD63/ CD81
non-EV: APOA1/ GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
K562
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
98
Cell count
3.00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
60
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ TSG101/ Alix/ CD81
Not detected contaminants
APOA1/ GM130
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
94
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 3.00E+07
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210002 1/3 Mus musculus dissociated tissues (d)(U)C
Delcorte, Ophélie 2022 78%

Study summary

Full title
All authors
Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux
Journal
Biomediscines
Abstract
Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide)
EV-METRIC
78% (83rd 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
dissociated tissues
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
No extra separation steps
Protein markers
EV: Alix/ CD63/ Flotillin1/ CD9/ CD81
non-EV: Calnexin/ PDI
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
dissociated tissues
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Wash: volume per pellet (ml)
7
Wash: time (min)
90
Wash: Rotor Type
Type 80 Ti
Wash: speed (g)
150000
Other
Name other separation method
No extra separation steps
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ Alix/ CD81
Not detected contaminants
Calnexin/ PDI
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNA sequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
Before
RNAse type
RNase A
RNAse concentration
0,01
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
Per dissociated thyroid;Yes, other: 1,50E+09
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV220163 1/3 Homo sapiens Serum qEV70 Lauren A. Newman 2022 75%