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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200049	1/2	Homo sapiens	placental tissue culture supernatrant	DG (d)(U)C	Bergamelli, Mathilde	2021	100%
Study summary Full title Human Cytomegalovirus Infection Changes the Pattern of Surface Markers of Small Extracellular Vesicles Isolated From First Trimester Placental Long-Term Histocultures All authors Mathilde Bergamelli, Hélène Martin, Mélinda Bénard, Jérôme Ausseil, Jean-Michel Mansuy, Ilse Hurbain, Maïlys Mouysset, Marion Groussolles, Géraldine Cartron, Yann Tanguy le Gac, Nathalie Moinard, Elsa Suberbielle, Jacques Izopet, Charlotte Tscherning, Graça Raposo, Daniel Gonzalez-Dunia, Gisela D'Angelo, Cécile E Malnou Journal Front Cell Dev Biol Abstract Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of p (show more...)Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking. (hide) EV-METRIC 100% (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 placental tissue culture supernatrant Sample origin Control condition Focus vesicles Other / 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 DG (d)(U)C Protein markers EV: CD29/ CD63/ CD81/ CD44/ CD326/ CD9 non-EV: Calreticulin Proteomics no EV density (g/ml) 1.103 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type placental tissue culture supernatrant 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: time(min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1.7 Fraction processing Centrifugation Pelleting: volume per fraction 25 Pelleting: duration (min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Not detected EV-associated proteins CD81/ CD9 Not detected contaminants Calreticulin Detected EV-associated proteins CD9/ CD29/ CD44/ CD326/ CD81/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 143 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.00E+08 Particle analysis: flow cytometry Flow cytometer type mascquant VYB Hardware adjustment Use of calibration beads FITC positive, of different size (500, 240, 200 and 160 nm) and granulosity that allow bead population separation and calibration of the cytometer , creation of a gate on 200 nm and smaller events Calibration bead size 0.16 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 95

	EV200049	2/2	Homo sapiens	placental tissue culture supernatrant	DG (d)(U)C	Bergamelli, Mathilde	2021	100%
Study summary Full title Human Cytomegalovirus Infection Changes the Pattern of Surface Markers of Small Extracellular Vesicles Isolated From First Trimester Placental Long-Term Histocultures All authors Mathilde Bergamelli, Hélène Martin, Mélinda Bénard, Jérôme Ausseil, Jean-Michel Mansuy, Ilse Hurbain, Maïlys Mouysset, Marion Groussolles, Géraldine Cartron, Yann Tanguy le Gac, Nathalie Moinard, Elsa Suberbielle, Jacques Izopet, Charlotte Tscherning, Graça Raposo, Daniel Gonzalez-Dunia, Gisela D'Angelo, Cécile E Malnou Journal Front Cell Dev Biol Abstract Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of p (show more...)Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking. (hide) EV-METRIC 100% (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 placental tissue culture supernatrant Sample origin in vitro hCMV infected placental tissue Focus vesicles Other / 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 DG (d)(U)C Protein markers EV: CD29/ CD63/ CD81/ CD44/ CD326/ CD9 non-EV: Calreticulin Proteomics no EV density (g/ml) 1.103 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type placental tissue culture supernatrant 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: time(min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1.7 Fraction processing Centrifugation Pelleting: volume per fraction 25 Pelleting: duration (min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Not detected EV-associated proteins CD63/ CD9 Not detected contaminants Calreticulin Detected EV-associated proteins CD9/ CD29/ CD44/ CD326/ CD81/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle analysis: flow cytometry Flow cytometer type mascquant VYB Hardware adjustment Use of calibration beads FITC positive, of different size (500, 240, 200 and 160 nm) and granulosity that allow bead population separation and calibration of the cytometer , creation of a gate on 200 nm and smaller events Calibration bead size 0.16 Report type Mean Reported size (nm) 140 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 100

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EV-TRACK ID	EV200049
species	Homo sapiens
sample type	placental tissue culture supernatrant
condition	Control condition	in vitro hCMV infected placental tissue
separation protocol	DG (d)(U)C	DG (d)(U)C
Exp. nr.	1	2
EV-METRIC %	100	100