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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV240006	4/4	Mus musculus	Neuro-2a (N2a) CCL-131	(d)(U)C Total Exosome Isolation lipid-based affinity capture	Weerakkody, Jonathan S.	2024	50%
Study summary Full title Photosensitive Nanoprobes for Rapid High Purity Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations All authors Jonathan S. Weerakkody, Tiffany Tseng, Mackenzie Topper, Sikha Thoduvayil, Abhijith Radhakrishnan, Frederic Pincet, Themis R. Kyriakides, Roshan W. Gunasekara, Sathish Ramakrishnan Journal Abstract The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as (show more...)The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 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 Total Exosome Isolation lipid-based affinity capture Protein markers EV: CD9/ CD81 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Neuro-2a (N2a) CCL-131 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 12500 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Other Name other separation method lipid-based affinity capture EV-subtype Distinction between multiple subtypes Size Used subtypes 150-350 Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 105 NTA Report type Mean Reported size (nm) 122 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(1) dSTORM single molecule localization microscopy Report type Mean Report size 52 EV-concentration No Extra information This paper was to validate the efficacy of photosensitive lipid nanoprobe for the isolation and size selective enrichment of native extracellular vesicles

	EV230572	2/6	Homo sapiens	HEK293	PEI precipitation	Djeungoue-Petga, Marie-Ange	2024	50%
Study summary Full title A simple scalable extracellular vesicle isolation method using polyethylenimine polymers for use in cellular delivery All authors Marie Ange Djeungoue Petgaa, Catherine Taylora, Alexander Macpherson, Surendar Reddy Dhadi, Thomas Rollin, Jeremy W. Roya, Anirban Ghosh, Stephen M. Lewis, Rodney J. Ouellette Journal Abstract Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular (show more...)Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular delivery of therapeutic cargo. Precipitation-based methods for the isolation of EVs remain popular due to ease of use and lack of requirements for specialized equipment. We describe here a novel charge-based EV isolation method that is simple, scalable, and uses inexpensive polyethylenimine (PEI) polymers. GFP-expressing EVs were isolated from the conditioned cell culture (CCM) media of HEK293-GFP cells using either branched 10 kDa PEI (B-PEI) or linear 25 kDa PEI (L-PEI). Isolated EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy (TEM), and flow cytometry. Western blotting for common EV markers, including CD63, CD9, flotillin-1, and heat shock protein 70 were positive, while GRP94, a marker for cellular contamination, was negative. Isolated EVs had a mean diameter of 146 nm for B-PEI and 175 nm for L- PEI, while TEM revealed a spherical cup-shaped appearance typical of EVs. In addition, we determined that PEI-based EV isolation methods were scalable up to volumes of at least 50 mL. EVs isolated from CCM collected from SUM159 cells that express CD63 fused to a dual EGFP-Renilla-split tag were tested for their ability to reconstitute functional luciferase by delivering the CD63-EGFP-Renilla-split tag to SUM159 recipient cells loaded with a cytopermeable Renilla luciferase substrate. Although EVs isolated using L-PEI behaved similarly to EVs isolated using ultracentrifugation, we observed that EVs isolated using B-PEI produced a more rapid uptake and delivery of active luciferase. In this study we demonstrate that both branched and linear PEI polymers can precipitate EVs from CCM. Furthermore, once eluted from the polymers, the isolated EVs were able to deliver functional protein cargo to recipient cells. Overall, our data support PEI-based isolation of EVs as a simple, rapid method for the recovery of functional EVs. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 PEI precipitation Protein markers EV: CD9/ CD63/ Flotillin-1/ HSP70/ GFP non-EV: GRP94 Proteomics no Show all info Study aim Mechanism of uptake/transfer/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Other Name other separation method PEI precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Flotillin-1/ HSP70 Not detected EV-associated proteins GFP Not detected contaminants GRP94 Flow cytometry Type of Flow cytometry Beckman Coulter Cytoflex Hardware adaptation to ~100nm EV's The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells) Calibration bead size 0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9 Antibody details provided? No Not detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240006	1/4	Mus musculus	Neuro-2a (N2a) CCL-131	(d)(U)C Total Exosome Isolation lipid-based affinity capture	Weerakkody, Jonathan S.	2024	44%
Study summary Full title Photosensitive Nanoprobes for Rapid High Purity Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations All authors Jonathan S. Weerakkody, Tiffany Tseng, Mackenzie Topper, Sikha Thoduvayil, Abhijith Radhakrishnan, Frederic Pincet, Themis R. Kyriakides, Roshan W. Gunasekara, Sathish Ramakrishnan Journal Abstract The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as (show more...)The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. (hide) EV-METRIC 44% (84th 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 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 Total Exosome Isolation lipid-based affinity capture Protein markers EV: CD9/ CD63/ CD81/ Syt1 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Neuro-2a (N2a) CCL-131 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 12500 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Other Name other separation method lipid-based affinity capture EV-subtype Distinction between multiple subtypes Size Used subtypes 30-150 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ Syt1 Detected EV-associated proteins CD9/ CD63/ Syt1 Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 100-150 Extra information This paper was to validate the efficacy of photosensitive lipid nanoprobe for the isolation and size selective enrichment of native extracellular vesicles

	EV240006	2/4	Mus musculus	Primary bone marrow-derived macrophage (BMDM) cells from hind limbs in C57BL/6J wild type mice	(d)(U)C Total Exosome Isolation lipid-based affinity capture	Weerakkody, Jonathan S.	2024	44%
Study summary Full title Photosensitive Nanoprobes for Rapid High Purity Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations All authors Jonathan S. Weerakkody, Tiffany Tseng, Mackenzie Topper, Sikha Thoduvayil, Abhijith Radhakrishnan, Frederic Pincet, Themis R. Kyriakides, Roshan W. Gunasekara, Sathish Ramakrishnan Journal Abstract The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as (show more...)The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. (hide) EV-METRIC 44% (84th 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 Total Exosome Isolation lipid-based affinity capture Protein markers EV: CD9/ CD63/ CD81/ Syt1 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Primary bone marrow-derived macrophage (BMDM) cells from hind limbs in C57BL/6J wild type mice EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 12500 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Other Name other separation method lipid-based affinity capture EV-subtype Distinction between multiple subtypes Size Used subtypes 30-200 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ Syt1 Detected EV-associated proteins CD9/ CD63/ Syt1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 220-350 Extra information This paper was to validate the efficacy of photosensitive lipid nanoprobe for the isolation and size selective enrichment of native extracellular vesicles

	EV230985	1/5	Capra hircus	Milk	(d)(U)C Filtration	Santoro J	2024	44%
Study summary Full title Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application. All authors Santoro J, Nuzzo S, Franzese M, Salvatore M, Grimaldi AM Journal Heliyon Abstract Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is (show more...)Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine. (hide) EV-METRIC 44% (70th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 Milk 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 Filtration Protein markers EV: CD81/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Capra hircus Sample Type Milk 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: rotor type MLA-80 Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Filtration steps Between 0.22 and 0.45 ?m/ 0.2 or 0.22 ?m Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 161.1 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.60E+12

	EV230985	2/5	Capra hircus	Milk	(d)(U)C EDTA pretreatment	Santoro J	2024	44%
Study summary Full title Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application. All authors Santoro J, Nuzzo S, Franzese M, Salvatore M, Grimaldi AM Journal Heliyon Abstract Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is (show more...)Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine. (hide) EV-METRIC 44% (70th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 Milk 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 EDTA pretreatment Protein markers EV: CD81/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Capra hircus Sample Type Milk 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: rotor type MLA-80 Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Filtration steps Between 0.22 and 0.45 ?m/ 0.2 or 0.22 ?m Size-exclusion chromatography Resin type Other Name other separation method EDTA pretreatment Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 179.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.44E+11

	EV230985	3/5	Capra hircus	Milk	(d)(U)C HCl pretreatment	Santoro J	2024	44%
Study summary Full title Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application. All authors Santoro J, Nuzzo S, Franzese M, Salvatore M, Grimaldi AM Journal Heliyon Abstract Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is (show more...)Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine. (hide) EV-METRIC 44% (70th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 Milk 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 HCl pretreatment Protein markers EV: CD81/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Capra hircus Sample Type Milk 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: rotor type MLA-80 Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Filtration steps Between 0.22 and 0.45 ?m/ 0.2 or 0.22 ?m Size-exclusion chromatography Resin type Other Name other separation method HCl pretreatment Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 122.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.20E+10

	EV230372	14/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	43%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 43% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 NSCLC Focus vesicles small EVs 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 Biomarker Sample Species Homo sapiens Sample Type Blood plasma 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 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis None Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 143.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.85E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV240036	1/6	Homo sapiens	Serum	Exoquick	Shinde U	2024	38%
Study summary Full title Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G. All authors Shinde U, Rao A, Bansal V, Das DK, Balasinor NH, Madan T Journal Reproduction Abstract Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic (show more...)Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. (hide) EV-METRIC 38% (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. 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 Serum Sample origin Non pregnant 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 Commercial method Protein markers EV: CD9/ CD63/ PLAP/ Cullin 7 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit Exoquick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ PLAP/ Cullin 7 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240036	3/6	Homo sapiens	Serum	Exoquick IAF	Shinde U	2024	38%
Study summary Full title Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G. All authors Shinde U, Rao A, Bansal V, Das DK, Balasinor NH, Madan T Journal Reproduction Abstract Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic (show more...)Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. (hide) EV-METRIC 38% (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. 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 Serum Sample origin Non pregnant 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 Commercial method Immunoaffinity capture (non-commercial) Protein markers EV: PLAP/ HLA-G non-EV: Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit Exoquick Immunoaffinity capture Selected surface protein(s) PLAP/ HLA-G EV-subtype Distinction between multiple subtypes affinity capture Used subtypes PLAP+ HLA-G+ Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins PLAP/ HLA-G Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230985	4/5	Capra hircus	Milk	(d)(U)C SEC (non-commercial) Filtration EDTA pretreatment	Santoro J	2024	38%
Study summary Full title Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application. All authors Santoro J, Nuzzo S, Franzese M, Salvatore M, Grimaldi AM Journal Heliyon Abstract Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is (show more...)Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine. (hide) EV-METRIC 38% (58th 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 Milk 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 Size-exclusion chromatography (non-commercial) Filtration EDTA pretreatment Protein markers EV: CD81/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Capra hircus Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 ?m/ 0.2 or 0.22 ?m Size-exclusion chromatography Total column volume (mL) 2 Sample volume/column (mL) 2 Other Name other separation method EDTA pretreatment Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 179.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.88E+10

	EV230985	5/5	Capra hircus	Milk	(d)(U)C SEC (non-commercial) Filtration HCl pretreatment	Santoro J	2024	38%
Study summary Full title Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application. All authors Santoro J, Nuzzo S, Franzese M, Salvatore M, Grimaldi AM Journal Heliyon Abstract Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is (show more...)Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine. (hide) EV-METRIC 38% (58th 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 Milk 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 Size-exclusion chromatography (non-commercial) Filtration HCl pretreatment Protein markers EV: CD81/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Capra hircus Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 ?m/ 0.2 or 0.22 ?m Size-exclusion chromatography Total column volume (mL) 2 Sample volume/column (mL) 2 Other Name other separation method HCl pretreatment Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 170.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.60E+10

	EV230994	1/6	Bos taurus	Commercially available FBS	(d)(U)C	Urzì O	2024	29%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 29% (25th 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 Commercially available FBS Sample origin no-HI 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 Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Commercially available FBS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 74680000 EM EM-type Transmission-EM Image type Close-up

	EV230994	3/6	Bos taurus	Commercially available FBS	(d)(U)C	Urzì O	2024	29%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 29% (25th 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 Commercially available FBS Sample origin HI-after EV-depl 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 Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Commercially available FBS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 21600000 EM EM-type Transmission-EM Image type Close-up

	EV230994	5/6	Bos taurus	Commercially available FBS	(d)(U)C	Urzì O	2024	29%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 29% (25th 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 Commercially available FBS Sample origin HI-before EV-depl 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 Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Commercially available FBS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 24400000 EM EM-type Transmission-EM Image type Close-up

	EV240020	1/2	Escherichia coli	REL606	(d)(U)C Filtration	Schaack B	2024	22%
Study summary Full title Rapid Biodistribution of Fluorescent Outer-Membrane Vesicles from the Intestine to Distant Organs via the Blood in Mice. All authors Schaack B, Mercier C, Katby M, Hannani D, Vollaire J, Robert JS, Caffaratti C, Blanquet F, Nicoud O, Josserand V, Laurin D Journal Int J Mol Sci Abstract A cell's ability to secrete extracellular vesicles (EVs) for communication is present in all three d (show more...)A cell's ability to secrete extracellular vesicles (EVs) for communication is present in all three domains of life. Notably, Gram-negative bacteria produce a specific type of EVs called outer membrane vesicles (OMVs). We previously observed the presence of OMVs in human blood, which could represent a means of communication from the microbiota to the host. Here, in order to investigate the possible translocation of OMVs from the intestine to other organs, the mouse was used as an animal model after OMVs administration. To achieve this, we first optimized the signal of OMVs containing the fluorescent protein miRFP713 associated with the outer membrane anchoring peptide OmpA by adding biliverdin, a fluorescence cofactor, to the cultures. The miRFP713-expressing OMVs produced in REL606 strain were then characterized according to their diameter and protein composition. Native- and miRFP713-expressing OMVs were found to produce homogenous populations of vesicles. Finally, in vivo and ex vivo fluorescence imaging was used to monitor the distribution of miRFP713-OMVs in mice in various organs whether by intravenous injection or oral gavage. The relative stability of the fluorescence signals up to 3 days post-injection/gavage paves the way to future studies investigating the OMV-based communication established between the different microbiotas and their host. (hide) EV-METRIC 22% (59th 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 Outer membrane vasicles 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: OmpA non-EV: None Proteomics no Show all info Study aim Mechanism of uptake/transfer Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells REL606 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins OmpA Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 56 EM EM-type Transmission-EM Image type Close-up Report size (nm) 56

	EV240020	2/2	Escherichia coli	REL606	(d)(U)C	Schaack B	2024	22%
Study summary Full title Rapid Biodistribution of Fluorescent Outer-Membrane Vesicles from the Intestine to Distant Organs via the Blood in Mice. All authors Schaack B, Mercier C, Katby M, Hannani D, Vollaire J, Robert JS, Caffaratti C, Blanquet F, Nicoud O, Josserand V, Laurin D Journal Int J Mol Sci Abstract A cell's ability to secrete extracellular vesicles (EVs) for communication is present in all three d (show more...)A cell's ability to secrete extracellular vesicles (EVs) for communication is present in all three domains of life. Notably, Gram-negative bacteria produce a specific type of EVs called outer membrane vesicles (OMVs). We previously observed the presence of OMVs in human blood, which could represent a means of communication from the microbiota to the host. Here, in order to investigate the possible translocation of OMVs from the intestine to other organs, the mouse was used as an animal model after OMVs administration. To achieve this, we first optimized the signal of OMVs containing the fluorescent protein miRFP713 associated with the outer membrane anchoring peptide OmpA by adding biliverdin, a fluorescence cofactor, to the cultures. The miRFP713-expressing OMVs produced in REL606 strain were then characterized according to their diameter and protein composition. Native- and miRFP713-expressing OMVs were found to produce homogenous populations of vesicles. Finally, in vivo and ex vivo fluorescence imaging was used to monitor the distribution of miRFP713-OMVs in mice in various organs whether by intravenous injection or oral gavage. The relative stability of the fluorescence signals up to 3 days post-injection/gavage paves the way to future studies investigating the OMV-based communication established between the different microbiotas and their host. (hide) EV-METRIC 22% (59th 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 miRFP713-expressing Focus vesicles Outer membrane vasicles 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: OmpA non-EV: None Proteomics no Show all info Study aim Mechanism of uptake/transfer Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells REL606 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins OmpA Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 56 EM EM-type Transmission-EM Image type Close-up Report size (nm) 56

	EV230572	5/6	Homo sapiens	SUM159-DSP1-CD63/DSP2	(d)(U)C	Djeungoue-Petga, Marie-Ange	2024	14%
Study summary Full title A simple scalable extracellular vesicle isolation method using polyethylenimine polymers for use in cellular delivery All authors Marie Ange Djeungoue Petgaa, Catherine Taylora, Alexander Macpherson, Surendar Reddy Dhadi, Thomas Rollin, Jeremy W. Roya, Anirban Ghosh, Stephen M. Lewis, Rodney J. Ouellette Journal Abstract Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular (show more...)Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular delivery of therapeutic cargo. Precipitation-based methods for the isolation of EVs remain popular due to ease of use and lack of requirements for specialized equipment. We describe here a novel charge-based EV isolation method that is simple, scalable, and uses inexpensive polyethylenimine (PEI) polymers. GFP-expressing EVs were isolated from the conditioned cell culture (CCM) media of HEK293-GFP cells using either branched 10 kDa PEI (B-PEI) or linear 25 kDa PEI (L-PEI). Isolated EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy (TEM), and flow cytometry. Western blotting for common EV markers, including CD63, CD9, flotillin-1, and heat shock protein 70 were positive, while GRP94, a marker for cellular contamination, was negative. Isolated EVs had a mean diameter of 146 nm for B-PEI and 175 nm for L- PEI, while TEM revealed a spherical cup-shaped appearance typical of EVs. In addition, we determined that PEI-based EV isolation methods were scalable up to volumes of at least 50 mL. EVs isolated from CCM collected from SUM159 cells that express CD63 fused to a dual EGFP-Renilla-split tag were tested for their ability to reconstitute functional luciferase by delivering the CD63-EGFP-Renilla-split tag to SUM159 recipient cells loaded with a cytopermeable Renilla luciferase substrate. Although EVs isolated using L-PEI behaved similarly to EVs isolated using ultracentrifugation, we observed that EVs isolated using B-PEI produced a more rapid uptake and delivery of active luciferase. In this study we demonstrate that both branched and linear PEI polymers can precipitate EVs from CCM. Furthermore, once eluted from the polymers, the isolated EVs were able to deliver functional protein cargo to recipient cells. Overall, our data support PEI-based isolation of EVs as a simple, rapid method for the recovery of functional EVs. (hide) EV-METRIC 14% (44th 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 DSP1-CD63/DSP2 overexpression 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 Mechanism of uptake/transfer/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SUM159-DSP1-CD63/DSP2 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) ~180 EV concentration Yes Particle yield particles per milliliter of starting sample: 5-7E07

	EV230372	12/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	14%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 14% (38th 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 small EVs 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 Biomarker Sample Species Homo sapiens Sample Type Blood plasma 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 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis None Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 137.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.41E+08

	EV230572	6/6	Homo sapiens	SUM159-DSP1-CD63/DSP2	PEI precipitation	Djeungoue-Petga, Marie-Ange	2024	0%
Study summary Full title A simple scalable extracellular vesicle isolation method using polyethylenimine polymers for use in cellular delivery All authors Marie Ange Djeungoue Petgaa, Catherine Taylora, Alexander Macpherson, Surendar Reddy Dhadi, Thomas Rollin, Jeremy W. Roya, Anirban Ghosh, Stephen M. Lewis, Rodney J. Ouellette Journal Abstract Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular (show more...)Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular delivery of therapeutic cargo. Precipitation-based methods for the isolation of EVs remain popular due to ease of use and lack of requirements for specialized equipment. We describe here a novel charge-based EV isolation method that is simple, scalable, and uses inexpensive polyethylenimine (PEI) polymers. GFP-expressing EVs were isolated from the conditioned cell culture (CCM) media of HEK293-GFP cells using either branched 10 kDa PEI (B-PEI) or linear 25 kDa PEI (L-PEI). Isolated EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy (TEM), and flow cytometry. Western blotting for common EV markers, including CD63, CD9, flotillin-1, and heat shock protein 70 were positive, while GRP94, a marker for cellular contamination, was negative. Isolated EVs had a mean diameter of 146 nm for B-PEI and 175 nm for L- PEI, while TEM revealed a spherical cup-shaped appearance typical of EVs. In addition, we determined that PEI-based EV isolation methods were scalable up to volumes of at least 50 mL. EVs isolated from CCM collected from SUM159 cells that express CD63 fused to a dual EGFP-Renilla-split tag were tested for their ability to reconstitute functional luciferase by delivering the CD63-EGFP-Renilla-split tag to SUM159 recipient cells loaded with a cytopermeable Renilla luciferase substrate. Although EVs isolated using L-PEI behaved similarly to EVs isolated using ultracentrifugation, we observed that EVs isolated using B-PEI produced a more rapid uptake and delivery of active luciferase. In this study we demonstrate that both branched and linear PEI polymers can precipitate EVs from CCM. Furthermore, once eluted from the polymers, the isolated EVs were able to deliver functional protein cargo to recipient cells. Overall, our data support PEI-based isolation of EVs as a simple, rapid method for the recovery of functional EVs. (hide) EV-METRIC 0% (median: 14% 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 DSP1-CD63/DSP2 overexpression 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 PEI precipitation Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Mechanism of uptake/transfer/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SUM159-DSP1-CD63/DSP2 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Other Name other separation method PEI precipitation Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 200-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 2-3.5E06

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