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You searched for: EV170014 (EV-TRACK ID)

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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
Experiment number
  • Experiments differ in Isolation, Proteïn analysis, Particle analysis
Experiment number
  • Experiments differ in Isolation, Proteïn analysis, Particle analysis
Experiment number
  • Experiments differ in Isolation, Proteïn analysis, Particle analysis
Experiment number
  • Experiments differ in Isolation, Proteïn analysis, Particle analysis
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV170014 1/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
SEC
Tangential flow filtration (50 nm pore size)
Dionysios C Watson 2018 75%

Study summary

Full title
All authors
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
Journal
J Extracell Vesicles
Abstract
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide)
EV-METRIC
75% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK293
Sample origin
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC
Tangential flow filtration (50 nm pore size)
Protein markers
EV: IL-15/ hetIL-15/lactadherin/ CD63
non-EV: calnexin/ ferritin
Proteomics
yes
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
EV-producing cells
HEK293
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
5
Resin type
Superdex 200
Other
Name other separation method
Tangential flow filtration (50 nm pore size)
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected contaminants
calnexin
Flow cytometry specific beads
Selected surface protein(s)
CD63
Proteomics
Proteomics database
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
Particle yield
480000000000
EM
EM-type
Immune-EM
Proteïns
IL-15
Image type
Close-up, Wide-field
EV170014 2/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Dionysios C Watson 2018 55%

Study summary

Full title
All authors
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
Journal
J Extracell Vesicles
Abstract
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide)
EV-METRIC
55% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK293
Sample origin
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Adj. k-factor
111.1 (pelleting) / 111.1 (washing)
Protein markers
EV: hetIL-15/Lactadherin
non-EV: ferritin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
EV-producing cells
HEK293
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70.1Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
111.1
Wash: time (min)
120
Wash: Rotor Type
Type 70.1Ti
Wash: speed (g)
110000
Wash: adjusted k-factor
111.1
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics
Proteomics database
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
Particle yield
96000000000
EV170014 4/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
SEC
Dionysios C Watson 2018 50%

Study summary

Full title
All authors
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
Journal
J Extracell Vesicles
Abstract
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK293
Sample origin
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC
Protein markers
EV: hetIL-15/Lactadherin
non-EV: ferritin
Proteomics
yes
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
EV-producing cells
HEK293
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Size-exclusion chromatography
Total column volume (mL)
24
Sample volume/column (mL)
0.5
Resin type
Superdex 200
Characterization: Protein analysis
Protein Concentration Method
Bradford
Flow cytometry specific beads
Selected surface protein(s)
CD63
Proteomics
Proteomics database
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
Particle yield
110000000000
EV170014 3/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
SEC
Dionysios C Watson 2018 37%

Study summary

Full title
All authors
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
Journal
J Extracell Vesicles
Abstract
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide)
EV-METRIC
37% (66th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK293
Sample origin
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC
Protein markers
EV: hetIL-15/Lactadherin
non-EV: ferritin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
EV-producing cells
HEK293
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
5
Resin type
Superdex 200
Characterization: Protein analysis
Protein Concentration Method
Bradford
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
Particle yield
240000000000
1 - 4 of 4
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV170014
species
Homo sapiens
sample type
Cell culture
cell type
HEK293
condition
human hetIL-15 stably transfected
human hetIL-15/lactadherin stably transfected
separation protocol
(d)(U)C
Filtration
SEC
Tangential flow filtration (50 nm pore size)
(d)(U)C
Filtration
(d)(U)C
Filtration
SEC
(d)(U)C
Filtration
SEC
Exp. nr.
1
2
4
3
EV-METRIC %
75
55
50
37