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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV160002	2/3	Homo sapiens	Extruded cells (NIH3T3)	DC Sequential extrusion	Lunavat TR	2016	37%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 37% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Extruded cells (NIH3T3) Sample origin Control condition Focus vesicles Nanovesicles 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 DC Sequential extrusion Protein markers EV: PDGFR/ Flotillin-1/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (NIH3T3) Separation Method Density cushion Density medium Iodixanol Other Name other separation method Sequential extrusion Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins PDGFR, Flotillin-1, CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-200 EM EM-type Transmission-EM Image type Wide-field

	EV160002	3/3	Homo sapiens	Extruded cells (NIH3T3)	DC Sequential extrusion	Lunavat TR	2016	37%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 37% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Extruded cells (NIH3T3) Sample origin cMyc shRNA Focus vesicles Nanovesicles 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 DC Sequential extrusion Protein markers EV: PDGFR/ Flotillin-1/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (NIH3T3) Separation Method Density cushion Density medium Iodixanol Other Name other separation method Sequential extrusion Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins PDGFR, Flotillin-1, CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-200 EM EM-type Transmission-EM Image type Wide-field

	EV160002	1/3	Homo sapiens	Extruded cells (U973)	DC Sequential extrusion	Lunavat TR	2016	0%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 0% (median: 0% 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 Extruded cells (U973) Sample origin Control condition Focus vesicles Nanovesicles 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 DC Sequential extrusion Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (U973) Separation Method Density cushion Density medium Iodixanol Other Name other separation method Sequential extrusion Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus Reported size (nm) 150

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EV-TRACK ID	EV160002
species	Homo sapiens
sample type	Extruded cells (NIH3T3)	Extruded cells (NIH3T3)	Extruded cells (U973)
condition	Control condition	cMyc shRNA	Control condition
separation protocol	DC Sequential extrusion	DC Sequential extrusion	DC Sequential extrusion
Exp. nr.	2	3	1
EV-METRIC %	37	37	0