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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210505	1/3	Mus musculus	B16BL6	(d)(U)C Filtration	Charoenviriyakul C	2018	33%
Study summary Full title Preservation of exosomes at room temperature using lyophilization. All authors Charoenviriyakul C, Takahashi Y, Nishikawa M, Takakura Y Journal Int J Pharm Abstract The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by dev (show more...)The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by developing a method to preserve exosomes. Although exosomes are generally stored at -80 °C, this temperature is not suitable for their handling or transportation and, therefore, other storage methods are desirable. Lyophilization is a promising storage method that can be used to preserve various substances at room temperature. In this study, we sought to develop a room temperature preservation method for exosomes using lyophilization and compared the properties of the lyophilized exosomes with ones stored at -80 °C. Lyophilization without cryoprotectant resulted in the aggregation of B16BL6 melanoma-derived exosomes, while the addition of trehalose, a cryoprotectant, prevented aggregation during lyophilization. PAGE analysis revealed that the proteins and RNA of exosomes were protected following lyophilization in the presence of trehalose. Lyophilization had little effect on the pharmacokinetics of Gaussia luciferase (gLuc)-labeled exosomes after an intravenous injection into mice. Moreover, it was found that lyophilized exosomes retained the activity of loaded gLuc and immunostimulatory CpG DNA for approximately 4 weeks even when stored at 25 °C. In conclusion, lyophilization with trehalose is an effective method for the storage of exosomes for various applications. (hide) EV-METRIC 33% (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 Cell culture supernatant Sample origin Control condition Focus vesicles exosome 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: Alix/ HSP70/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16BL6 EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g/ Other preparation Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 60 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins HSP70/ Alix/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210505	2/3	Mus musculus	B16BL6	(d)(U)C Filtration	Charoenviriyakul C	2018	0%
Study summary Full title Preservation of exosomes at room temperature using lyophilization. All authors Charoenviriyakul C, Takahashi Y, Nishikawa M, Takakura Y Journal Int J Pharm Abstract The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by dev (show more...)The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by developing a method to preserve exosomes. Although exosomes are generally stored at -80 °C, this temperature is not suitable for their handling or transportation and, therefore, other storage methods are desirable. Lyophilization is a promising storage method that can be used to preserve various substances at room temperature. In this study, we sought to develop a room temperature preservation method for exosomes using lyophilization and compared the properties of the lyophilized exosomes with ones stored at -80 °C. Lyophilization without cryoprotectant resulted in the aggregation of B16BL6 melanoma-derived exosomes, while the addition of trehalose, a cryoprotectant, prevented aggregation during lyophilization. PAGE analysis revealed that the proteins and RNA of exosomes were protected following lyophilization in the presence of trehalose. Lyophilization had little effect on the pharmacokinetics of Gaussia luciferase (gLuc)-labeled exosomes after an intravenous injection into mice. Moreover, it was found that lyophilized exosomes retained the activity of loaded gLuc and immunostimulatory CpG DNA for approximately 4 weeks even when stored at 25 °C. In conclusion, lyophilization with trehalose is an effective method for the storage of exosomes for various applications. (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 plasmid DNA gLuc-L Focus vesicles exosome 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: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16BL6 EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g/ Other preparation Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 60 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210505	3/3	Mus musculus	B16BL6	(d)(U)C Filtration	Charoenviriyakul C	2018	0%
Study summary Full title Preservation of exosomes at room temperature using lyophilization. All authors Charoenviriyakul C, Takahashi Y, Nishikawa M, Takakura Y Journal Int J Pharm Abstract The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by dev (show more...)The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by developing a method to preserve exosomes. Although exosomes are generally stored at -80 °C, this temperature is not suitable for their handling or transportation and, therefore, other storage methods are desirable. Lyophilization is a promising storage method that can be used to preserve various substances at room temperature. In this study, we sought to develop a room temperature preservation method for exosomes using lyophilization and compared the properties of the lyophilized exosomes with ones stored at -80 °C. Lyophilization without cryoprotectant resulted in the aggregation of B16BL6 melanoma-derived exosomes, while the addition of trehalose, a cryoprotectant, prevented aggregation during lyophilization. PAGE analysis revealed that the proteins and RNA of exosomes were protected following lyophilization in the presence of trehalose. Lyophilization had little effect on the pharmacokinetics of Gaussia luciferase (gLuc)-labeled exosomes after an intravenous injection into mice. Moreover, it was found that lyophilized exosomes retained the activity of loaded gLuc and immunostimulatory CpG DNA for approximately 4 weeks even when stored at 25 °C. In conclusion, lyophilization with trehalose is an effective method for the storage of exosomes for various applications. (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 plasmid DNA streptavidin-LA Focus vesicles exosome 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: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16BL6 EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g/ Other preparation Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 60 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210505
species	Mus musculus
sample type	Cell culture
cell type	B16BL6
condition	Control condition	plasmid DNA gLuc-L	plasmid DNA streptavidin-LA
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2	3
EV-METRIC %	33	0	0