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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210144	3/9	Homo sapiens	Saliva	(d)(U)C DC	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63 Not detected EV-associated proteins HSC70 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 205.7 +/- 2.3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.89E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	4/9	Homo sapiens	Saliva	(d)(U)C Chitosan-based	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63 Not detected EV-associated proteins HSC70 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 218.2 +/- 5.4 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8.39E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	5/9	Homo sapiens	Urine	(d)(U)C DC	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: Tamm-Horsfall protein/ CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63 Not detected EV-associated proteins HSC70 Detected contaminants Tamm-Horsfall protein Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 165.2 +/- 0.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 7.40E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	6/9	Homo sapiens	Urine	(d)(U)C Chitosan-based	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: Tamm-Horsfall protein/ CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSC70/ CD9/ CD63 Detected contaminants Tamm-Horsfall protein Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136.4 +/- 5.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.17E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	7/9	Homo sapiens	HEK293	(d)(U)C DC	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development 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 Cell count 7.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSC70/ CD9/ CD63 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 164.8 +/- 1.6 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.08E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	1/9	Homo sapiens	Blood plasma	(d)(U)C DC	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: FCN3/ SAA/ APOB/ APOA1/ TETN Proteomics no Show all info Study aim New methodological development 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 Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSC70 Detected contaminants APOB/ FCN3 Not detected contaminants APOA1/ TETN/ SAA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 222.1 +/- 6.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.18E+08 EM EM-type Transmission-EM Image type Close-up

	EV210144	2/9	Homo sapiens	Blood plasma	(d)(U)C Chitosan-based	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: FCN3/ APOA1/ TETN Proteomics no Show all info Study aim New methodological development 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 Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSC70 Not detected contaminants APOA1/ FCN3/ TETN Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 89.8 +/- 2.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.94E+08 EM EM-type Transmission-EM Image type Close-up

	EV210144	8/9	Homo sapiens	HEK293	(d)(U)C DG	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim New methodological development 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 Cell count 7.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 12 Lowest density fraction 10% Highest density fraction 90% Total gradient volume, incl. sample (mL) 12.1 Sample volume (mL) 0.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 200,000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 90 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210144	9/9	Homo sapiens	HEK293	(d)(U)C Chitosan-based	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development 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 Cell count 7.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSC70/ CD9/ CD63 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133.0 +/- 5.2 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.02E+08 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV210144
species	Homo sapiens
sample type	Saliva	Saliva	Urine	Urine	Cell culture	Blood plasma	Blood plasma	Cell culture	Cell culture
cell type	NA	NA	NA	NA	HEK293	NA	NA	HEK293	HEK293
medium	NA	NA	NA	NA	EV-depleted medium	NA	NA	EV-depleted medium	EV-depleted medium
condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C DC	(d)(U)C Chitosan-based	(d)(U)C DC	(d)(U)C Chitosan-based	(d)(U)C DC	(d)(U)C DC	(d)(U)C Chitosan-based	(d)(U)C DG	(d)(U)C Chitosan-based
Exp. nr.	3	4	5	6	7	1	2	8	9
EV-METRIC %	75	75	75	75	75	63	63	63	63

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