Este trabajo contó con el apoyo del Fondo Nacional de Ciencias para Jóvenes Académicos Distinguidos (82025024); el proyecto Key de la Fundación Nacional de Ciencias Naturales de China (82230080); el Programa Nacional Clave de Investigación y Desarrollo de China (2021YFA1300604); la Fundación Nacional de Ciencias Naturales de China (81871735, 82272438 y 82002245); Fondo de Ciencias Naturales de Guangdong para Jóvenes Académicos Distinguidos (2023B1515020058); la Fundación de Ciencias Naturales de la Provincia de Guangdong (2021A1515011639); el Programa Estatal de Desarrollo de la Investigación Básica de la Fundación de Ciencias Naturales de la Provincia de Shandong (ZR2020ZD11); la Fundación de Ciencias Postdoctorales (2022M720059); el Plan de Desarrollo de Jóvenes Sobresalientes del Hospital de Nanfang, Universidad Médica del Sur (2022J001).

Aislamiento mejorado de vesículas extracelulares bacterianas para microbiota multiómica

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+changes+in+stool,+saliva,+serum,+and+urine+before+and+after+anti-TNF-&#945;+therapy+in+patients+with+inflammatory+bowel+diseases.">Microbial changes in stool, saliva, serum, and urine before and after anti-TNF-&#945; therapy in patients with inflammatory bowel diseases.</a> Scientific Reports. 12 (1), 6359 (2022).</li><li>Tulkens, J., De Wever, O., Hendrix, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+bacterial+extracellular+vesicles+in+human+body+fluids+by+orthogonal+biophysical+separation+and+biochemical+characterization.">Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization.</a> Nature Protocols. 15 (1), 40-67 (2020).</li><li>Tulkens, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+levels+of+systemic+LPS-positive+bacterial+extracellular+vesicles+in+patients+with+intestinal+barrier+dysfunction.">Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction.</a> Gut. 69 (1), 191-193 (2020).</li><li>Kang, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+derived+from+gut+microbiota,+especially+Akkermansia+muciniphila,+protect+the+progression+of+dextran+sulfate+sodium-induced+colitis.">Extracellular vesicles derived from gut microbiota, especially Akkermansia muciniphila, protect the progression of dextran sulfate sodium-induced colitis.</a> PloS one. 8 (10), 76520 (2013).</li><li>Simonsen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+we+looking+at?+Extracellular+vesicles,+lipoproteins,+or+both.">What are we looking at? Extracellular vesicles, lipoproteins, or both.</a> Circulation Research. 121 (8), 920-922 (2017).</li><li>Correll, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+small+extracellular+vesicle+isolation+from+expressed+prostatic+secretions+in+urine+for+in-depth+proteomic+analysis.">Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis.</a> Journal of Extracellular Vesicles. 11 (2), 12184 (2022).</li><li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+bacterial+extracellular+vesicles:+important+players+in+regulating+intestinal+microenvironment.">Gut bacterial extracellular vesicles: important players in regulating intestinal microenvironment.</a> Gut Microbes. 14 (1), 2134689 (2022).</li><li>Alberti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+derived+from+gut+microbiota+in+inflammatory+bowel+disease+and+colorectal+cancer.">Extracellular vesicles derived from gut microbiota in inflammatory bowel disease and colorectal cancer.</a> Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czech Republic. 165 (3), 233-240 (2021).</li><li>D&#237;ez-Sainz, E., Milagro, F. I., Riezu-Boj, J. I., Lorente-Cebri&#225;n, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+gut+microbiota-derived+extracellular+vesicles+on+obesity+and+diabetes+and+their+potential+modulation+through+diet.">Effects of gut microbiota-derived extracellular vesicles on obesity and diabetes and their potential modulation through diet.</a> Journal of Physiology and Biochemistry. 78 (2), 485-499 (2022).</li><li>Lajqi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiota-derived+small+extracellular+vesicles+endorse+memory-like+inflammatory+responses+in+murine+neutrophils.">Gut microbiota-derived small extracellular vesicles endorse memory-like inflammatory responses in murine neutrophils.</a> Biomedicines. 10 (2), 442 (2022).</li><li>Lee, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+vesicle+of+gut+microbial+Paenalcaligenes+hominis+is+a+risk+factor+for+vagus+nerve-mediated+cognitive+impairment.">The extracellular vesicle of gut microbial Paenalcaligenes hominis is a risk factor for vagus nerve-mediated cognitive impairment.</a> Microbiome. 8 (1), 107 (2020).</li><li>Villard, A., Boursier, J., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+and+eukaryotic+extracellular+vesicles+and+nonalcoholic+fatty+liver+disease:+new+players+in+the+gut-liver+axis.">Bacterial and eukaryotic extracellular vesicles and nonalcoholic fatty liver disease: new players in the gut-liver axis.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 320 (4), G485-G495 (2021).</li><li>Wei, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+vesicles+enhance+tau+phosphorylation+and+contribute+to+cognitive+impairment.">Outer membrane vesicles enhance tau phosphorylation and contribute to cognitive impairment.</a> Journal of Cellular Physiology. 235 (5), 4843-4855 (2020).</li><li>Bitto, N. 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Los autores declaran no tener conflictos de intereses.

Las vesículas extracelulares bacterianas (BEV) son nanopartículas de bicapa lipídica secretadas por bacterias, portadoras de una gran cantidad de proteínas, lípidos, ácidos nucleicos y otras moléculas bioactivas, que contribuyen a mediar los efectos funcionales de las bacterias20. Se ha comprobado que los BEV derivados del intestino están implicados en el desarrollo de enfermedades, como la enfermedad inflamatoria intestinal, la enfermedad de Crohn y el cáncer colorrectal, y también afec...

El intestino es ampliamente reconocido como el órgano que alberga las comunidades microbianas más abundantes en el cuerpo humano, con más del 90% de las bacterias involucradas en la colonización y multiplicación 1,2. Numerosas pruebas han demostrado que la microbiota intestinal modula el microambiente intestinal y, al mismo tiempo, interactúa con la disfunción en órganos distantes, principalmente a través de una barrera intestinal alterada 3,4. Cada vez hay más pruebas que indican una correlación entre el desequilibrio de la microbiota intestinal y la progresión de la enfermedad inflamatoria intestinal (EII)5,6, así como de los trastornos cognitivos a través del eje intestino-cerebro 5,6,7,8. Las vesículas extracelulares bacterianas (BEV) producidas por bacterias desempeñan un papel importante en estos procesos patológicos.
Los BEV son partículas a nanoescala que encapsulan derivados bacterianos, con diámetros que oscilan entre 20 y 400 nm. Se ha demostrado que facilitan las interacciones entre las bacterias y sus organismos huéspedes 9,10. A pesar de su invisibilidad, estas partículas han atraído cada vez más la atención de los investigadores debido a sus amplias aplicaciones prospectivas como biomarcadores de diagnóstico, dianas terapéuticas y vehículos de administración de fármacos11. Las heces humanas, que a menudo se utilizan como muestras biológicas para estudiar los BEV, procedentes principalmente de bacterias intestinales, contienen una mezcla compleja de agua, bacterias, lípidos, proteínas, residuos de alimentos no digeridos y células epiteliales exfoliadas, entre otros. La intrincada composición fecal plantea desafíos para el aislamiento y la pureza de los BEV, lo que impide un análisis exhaustivo, objetivo y realista de los BEV. Por lo tanto, las estrategias efectivas para minimizar la interferencia de los componentes contaminantes y mejorar el rendimiento de los BEV se han convertido en cuestiones críticas que merecen atención inmediata.
Las estrategias de aislamiento existentes se basan en gran medida en técnicas como la centrifugación de ultra alta velocidad (CU), la centrifugación en gradiente de densidad (DGC) y la cromatografía de exclusión por tamaño (SEC)12,13,14,15,16,17. Actualmente, el DGC es uno de los métodos más aplicados en el campo de la separación de BEV, que abarca dos modos de sedimentación flotante, "Top-down" y "Bottom-up", que están determinados por la posición de carga inicial de la muestra. Estas metodologías diferencian las vesículas extracelulares (VE) de otros componentes en función de las disparidades de tamaño y densidad, lo que produce tasas variables de pureza y recuperación. Investigaciones anteriores han indicado que las estrategias de enfoque único son insuficientes para separar adecuadamente las VE de las proteínas solubles en muestras de fluidos corporales, como la lipoproteína en sangre18 y la proteína Tamm-Horsfall en orina19. Además, la distribución del tamaño de las vesículas extracelulares eucariotas (EEV) a menudo se superpone con la de los BEV, lo que requiere más mejoras metodológicas para optimizar el rendimiento de los BEV. En consecuencia, el avance en el estudio de los BEV depende del desarrollo de metodologías eficaces de separación y purificación. En particular, Tulkens et al 15 emplearon una estrategia biofísica ortogonal para separar los BEV fecales de los EEV, en la que el tiempo de centrifugación de un modo DGC de abajo hacia arriba fue de hasta18 h. Por el contrario, este estudio lo redujo a 7 h, ahorrando en gran medida el tiempo de ultracentrifugación en gradiente y simplificando el proceso.
En el presente estudio, aislamos y purificamos BEV fecales empleando dos modos DGC en condiciones de tampón optimizadas, después de enriquecer BEV con un rango de velocidades de centrifugación diferenciales, de baja a extremadamente alta velocidad. Las evaluaciones basadas en la morfología, el tamaño de partícula y la concentración indicaron un rendimiento encomiable con este método mejorado. Este estudio podría servir como base para futuras investigaciones, extendiendo sus aplicaciones a un dominio más amplio y ofreciendo información sobre la heterogeneidad de los BEV dentro del cuerpo humano. También contribuye a la estandarización de las técnicas de separación y análisis de BEV.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 % (w/v) glutaraldehyde (prepared from 2.5 % stock solution in deionized water)</td>
			<td>ACMEC</td>
			<td>AP1126</td>
			<td>Morphological observation for BEVs using TEM at Step 8.3.3</td>
		</tr>
		<tr>
			<td>1 % (w/v) methylcellulose (prepared from original powder in deionized water)</td>
			<td>Sigma-Aldrich</td>
			<td>M7027</td>
			<td>Morphological observation for BEVs using TEM at Step 8.3.6</td>
		</tr>
		<tr>
			<td>1.5 % (w/v) uranyl acetate (prepared from original powder in deionized water)</td>
			<td>Polysciences</td>
			<td>21447-25</td>
			<td>Morphological observation for BEVs using TEM at Step 8.3.5</td>
		</tr>
		<tr>
			<td>1000 &#956;L, 200 &#956;L, 10 &#956;L Pipette</td>
			<td>KIRGEN</td>
			<td>KG1313, KG1212, KG1011</td>
			<td>Transfer the solution</td>
		</tr>
		<tr>
			<td>5 % (w/v) bovine serum albumin solution (prepared from the original powder in TBST buffer)</td>
			<td>Fdbio science</td>
			<td>FD0030</td>
			<td>Used in western blotting for blocking at Step 8.5.6</td>
		</tr>
		<tr>
			<td>5 &#215; loading buffer</td>
			<td>Fdbio science</td>
			<td>FD006</td>
			<td>Used in western blotting and Coomassie brilliant blue stain at Step 8.5.1</td>
		</tr>
		<tr>
			<td>75 % (v/v) alcohol</td>
			<td>LIRCON</td>
			<td>LIRCON-500 mL</td>
			<td>Surface disinfection</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Rar</td>
			<td>A8096</td>
			<td>Measure the absorbance values&#160;</td>
		</tr>
		<tr>
			<td>Anti-Calnexin antibody</td>
			<td>Abcam</td>
			<td>ab92573</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-CD63 antibody</td>
			<td>Abcam</td>
			<td>ab134045</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-CD9 antibody</td>
			<td>Abcam</td>
			<td>ab236630</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-Flagellin antibody</td>
			<td>Sino Biological</td>
			<td>40067-MM06</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-Integrin beta 1 antibody</td>
			<td>Abcam</td>
			<td>ab30394</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-LPS antibody</td>
			<td>Thermo Fisher</td>
			<td>MA1-83152</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-LTA antibody</td>
			<td>Thermo Fisher&#160;</td>
			<td>MA1-7402</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-OmpA antibody</td>
			<td>CUSABIO</td>
			<td>CSB-PA359226ZA01EOD, https://www.cusabio.com/</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-Syntenin antibody</td>
			<td>Abcam</td>
			<td>ab133267</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Anti-TSG101 antibody</td>
			<td>Abcam</td>
			<td>ab125011</td>
			<td>Western blotting (Primary Antibody)</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>ZEALWAY</td>
			<td>GR110DP</td>
			<td>Sterilization for supplies and mediums used in the experiment</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Mettler Toledo</td>
			<td>AL104</td>
			<td>Balance the tube sample-loaded with PBS</td>
		</tr>
		<tr>
			<td>Bicinchoninic acid assay&#160;</td>
			<td>Fdbio science</td>
			<td>FD2001</td>
			<td>Measure protein content of BEVs at Step 8.2</td>
		</tr>
		<tr>
			<td>BioRender</td>
			<td>BioRender</td>
			<td>https://app.biorender.com</td>
			<td>Make the schematic workflow of BEVs isolation and purification showed in Figure 1</td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Haier</td>
			<td>HR1200- II B2</td>
			<td>Peform the procedures about feces sample handling</td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R; Rotor F-34-6-38</td>
			<td>Eppendorf</td>
			<td>5805000092; 5804727002, adapter: 5804774000</td>
			<td>Preprocess for BEVs (Step 3)</td>
		</tr>
		<tr>
			<td>Chemiluminescence Apparatus</td>
			<td>BIO-OI</td>
			<td>OI600SE-MF</td>
			<td>Used in western blotting for signal detection at Step 8.5.12</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td>F01</td>
			<td>Microplate detector for measuring the absorbance (Step 8.1) and fluorescence (Figure 6) values&#160;</td>
		</tr>
		<tr>
			<td>Dil-labled low density lipoprotein</td>
			<td>ACMEC</td>
			<td>AC12038</td>
			<td>Definition of distribution of interfering components&#160;</td>
		</tr>
		<tr>
			<td>Electrophoresis equipment</td>
			<td>Bio-rad</td>
			<td>1658033</td>
			<td>Used in western blotting for protein separation and transfer at Step 8.5.2, 8.5.3, 8.5.5</td>
		</tr>
		<tr>
			<td>Enhanced Chemiluminescence kit HRP&#160;</td>
			<td>Fdbio science</td>
			<td>FD8020</td>
			<td>Used in western blotting for signal detection at Step 8.5.12</td>
		</tr>
		<tr>
			<td>Escherichia coli&#160;</td>
			<td>American Type Culture Collection</td>
			<td>ATCC8739</td>
			<td>Isolate BEVs as a positive control. Protocol: Dissolve 25 g of the LB powder in 1 L deionized water, and autoclave. Transfer the 800 &#956;L of preserved Escherichia coli into the medium. Cultivate at 37 &#176;C in the incubator shaker. Then centrifuge at 3, 000 &#215; g for 20 min at 4 &#176;C, 12, 000 &#215; g for 30 min at 4 &#176;C, filter the supernatant through 0.22 &#956;m membrane, and perform ultra-speed centrifugation at 160, 000 &#215; g for 70 min at 4 &#176;C. Pellet defined as crude BEVs from Escherichia coli was suspended in 1.2 mL PBS (Step 3, 4).&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Falcon tubes 50 mL</td>
			<td>KIRGEN</td>
			<td>KG2811</td>
			<td>Preprocess for BEVs (Step 3)</td>
		</tr>
		<tr>
			<td>Feto Protein Staining Buffer</td>
			<td>Absci</td>
			<td>ab.001.50</td>
			<td>Coomassie brilliant blue staining at Step 8.5.4</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Biosharp</td>
			<td>BS-TFP-070B</td>
			<td>Morphological observation for BEVs using TEM at Step 8.3 (Blotting the solution)</td>
		</tr>
		<tr>
			<td>Formvar/Carbon supported copper grids&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>TEM-FCF200CU50</td>
			<td>Morphological observation for BEVs using TEM at Step 8.3</td>
		</tr>
		<tr>
			<td>HEPES powder</td>
			<td>Meilunbio</td>
			<td>MB6078</td>
			<td>Prepare iodixanol buffers with different concentrations for density gradient centrifugation</td>
		</tr>
		<tr>
			<td>HRP AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td>Fdbio science</td>
			<td>FDM007</td>
			<td>Western blotting (Secondary Antibody)</td>
		</tr>
		<tr>
			<td>HRP AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Fdbio science</td>
			<td>FDR007</td>
			<td>Western blotting (Secondary Antibody)</td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>Qiangwen</td>
			<td>DHZ-L</td>
			<td>Cultivate Escherichia coli&#160;</td>
		</tr>
		<tr>
			<td>Kimwipes&#8482; Delicate Task Wipes</td>
			<td>Kimtech Science</td>
			<td>34155</td>
			<td>Wipe the inner wall of the ultracentrifuge tube at Step 4.15</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Hopebio</td>
			<td>HB0128</td>
			<td>Cultivate Escherichia coli&#160;</td>
		</tr>
		<tr>
			<td>Low temperature freezer (-80 &#176;C)</td>
			<td>Haier</td>
			<td>DW-86L338J</td>
			<td>Store the samples</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Alalddin</td>
			<td>M116118</td>
			<td>Used in western blotting for activating PVDF membrane at Step 8.5.5</td>
		</tr>
		<tr>
			<td>Micro tubes 1.5 mL</td>
			<td>KIRGEN</td>
			<td>KG2211</td>
			<td>Recover fractions after density gradient centrifugation</td>
		</tr>
		<tr>
			<td>Micro tubes 2 mL</td>
			<td>KIRGEN</td>
			<td>KG2911</td>
			<td>Recover fractions after density gradient centrifugation</td>
		</tr>
		<tr>
			<td>Micro tubes 5 mL</td>
			<td>BBI</td>
			<td>F610888-0001</td>
			<td>Recover fractions after density gradient centrifugation</td>
		</tr>
		<tr>
			<td>Microplate reader&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Multiskan MK3</td>
			<td>Measure protein content of BEVs at Step 8.2</td>
		</tr>
		<tr>
			<td>Millipore filter 0.22 &#956;m</td>
			<td>Merck millipore</td>
			<td>SLGP033RB</td>
			<td>Filtration sterilization; Material: polyethersulfone, PES</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>GHTECH</td>
			<td>1.01307.040</td>
			<td>Density gradient centrifugation solution</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>GHTECH</td>
			<td>1.01394.068</td>
			<td>Density gradient centrifugation solution (pH adjustment)</td>
		</tr>
		<tr>
			<td>Optima&#8482; XPN-100</td>
			<td>Beckman Coulter</td>
			<td>A94469</td>
			<td>Ultracentrifugation for BEVs isolation at Step 4, 7</td>
		</tr>
		<tr>
			<td>OptiPrep&#8482;</td>
			<td>Serumwerk Bernburg AG</td>
			<td>1893</td>
			<td>Density gradient centrifugation stock solution</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Youning</td>
			<td>CS-100</td>
			<td>Dissolve feces at Step 2</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Procell</td>
			<td>PB180327</td>
			<td>Dissolve feces at Step 2</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Eppendorf</td>
			<td>3120000267, 3120000259</td>
			<td>Transfer the solution</td>
		</tr>
		<tr>
			<td>Plastic pasteur pipette</td>
			<td>ABCbio</td>
			<td>ABC217003-4</td>
			<td>Remove supernatant in preprocessing at Step 3.4</td>
		</tr>
		<tr>
			<td>Polyvinylidene difluoride (PVDF) membranes</td>
			<td>Millipore</td>
			<td>ISEQ00010, IPVH00010</td>
			<td>Used in western blotting for protein transfer at Step 8.5.5</td>
		</tr>
		<tr>
			<td>Prefabricated polyacrylamide gel, 4&#8211;20% 15 Wells</td>
			<td>ACE</td>
			<td>F15420Gel</td>
			<td>Used in western blotting for protein separation at Step 8.5.2, 8.5.3</td>
		</tr>
		<tr>
			<td>Primary antibody diluent</td>
			<td>Fdbio science</td>
			<td>FD0040</td>
			<td>Used in western blotting at Step 8.5.8</td>
		</tr>
		<tr>
			<td>Protein ladder</td>
			<td>Fdbio science</td>
			<td>FD0672</td>
			<td>Used in western blotting and Coomassie brilliant blue stain at Step 8.5</td>
		</tr>
		<tr>
			<td>Rapid protein blotting solution</td>
			<td>UBIO</td>
			<td>UW0500</td>
			<td>Used in western blotting for protein transfer at Step 8.5.5</td>
		</tr>
		<tr>
			<td>Rotor SW 32 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>369650</td>
			<td>Ultracentrifugation for BEVs isolation at Step 4, 7</td>
		</tr>
		<tr>
			<td>Syringe 20 mL, 50 mL&#160;</td>
			<td>Jetway</td>
			<td>ZSQ-20ML, YCXWJZSQ-50 mL</td>
			<td>Transfer buffers amd remove supernatant in preprocessing</td>
		</tr>
		<tr>
			<td>TBS powder</td>
			<td>Fdbio science</td>
			<td>FD1021</td>
			<td>Used in western blotting at Step 8.5</td>
		</tr>
		<tr>
			<td>Transmission electron microscope (TEM)</td>
			<td>Hitachi&#160;</td>
			<td>H-7650</td>
			<td>Morphological observation for BEVs at Step 8.3</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fdbio science</td>
			<td>FD0020</td>
			<td>Used in western blotting at Step 8.5</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tube</td>
			<td>Beckman</td>
			<td>326823, 355642</td>
			<td>Ultracentrifugation for BEVs isolation at Step 4, 7</td>
		</tr>
		<tr>
			<td>Ultra-clean bench</td>
			<td>AIRTECH</td>
			<td>SW-CJ-2FD</td>
			<td>Peform the procedures about liquid handling</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Bluepard</td>
			<td>CU600</td>
			<td>Used for measuring protein content of BEVs at Step 8.2.5</td>
		</tr>
		<tr>
			<td>ZetaView</td>
			<td>Particle Metrix</td>
			<td>S/N 21-734, Software ZetaView (version 8.05.14 SP7)</td>
			<td>Nanoparticle tracking analysis (NTA) for measuring the particle size and concentrarion of BEVs at Step 8.4</td>
		</tr>
	</tbody>
</table>

isolation and purification of bacterial extracellular vesicles from human feces using density gradient centrifugation

Las vesículas extracelulares bacterianas (BEV) son nanovesículas derivadas de bacterias que desempeñan un papel activo en la comunicación bacteria-bacteria y bacteria-huésped, transfiriendo moléculas bioactivas como proteínas, lípidos y ácidos nucleicos heredados de la bacteria parental. Los BEV derivados de la microbiota intestinal tienen efectos en el tracto gastrointestinal y pueden llegar a órganos distantes, lo que tiene importantes implicaciones para la fisiología y la patología. Las investigaciones teóricas que exploran los tipos, las cantidades y las funciones de los BEV derivados de las heces humanas son cruciales para comprender la secreción y la función de los BEV de la microbiota intestinal. Estas investigaciones también requieren una mejora en la estrategia actual para aislar y purificar los BEV.
Este estudio optimizó el proceso de aislamiento y purificación de BEV mediante el establecimiento de dos modos de centrifugación en gradiente de densidad (DGC): Top-down y Bottom-up. La distribución enriquecida de los BEV se determinó en las fracciones 6 a 8 (F6-F8). La efectividad del enfoque se evaluó en función de la morfología de las partículas, el tamaño, la concentración y el contenido de proteínas. Se calcularon las tasas de recuperación de partículas y proteínas, y se analizó la presencia de marcadores específicos para comparar la recuperación y pureza de los dos modos de DGC. Los resultados indicaron que el modo de centrifugación de arriba hacia abajo tenía niveles de contaminación más bajos y lograba una tasa de recuperación y pureza similar a la del modo de abajo hacia arriba. Un tiempo de centrifugación de 7 h fue suficiente para alcanzar una concentración fecal de BEV de 108/mg.
Además de las heces, este método podría aplicarse a otros tipos de fluidos corporales con la modificación adecuada de acuerdo con las diferencias en los componentes y la viscosidad. En conclusión, este protocolo detallado y fiable facilitaría el aislamiento y la purificación estandarizados de los BEV y, por lo tanto, sentaría las bases para el posterior análisis multiómico y experimentos funcionales.

The gut is widely recognized as the organ harboring the most abundant microbial communities in the human body, with over 90% of bacteria involved in colonization and multiplication1,2. Extensive evidence has demonstrated that the gut microbiota modulates the gut microenvironment and simultaneously interacts with dysfunction in distant organs, primarily through an impaired intestinal barrier3,4. Mounting evidence indicates a correlation between the imbalance of gut microbiota and the progression of inflammatory bowel disease (IBD)5,6, as well as cognitive disorders through the gut-brain axis5,6,7,8. Bacterial extracellular vesicles (BEVs) produced by bacteria play significant roles in these pathological processes.
BEVs are nanoscale particles encapsulating bacterial derivatives, with diameters ranging from 20 to 400 nm. They have been demonstrated to facilitate interactions between bacteria and their host organisms9,10. Despite their invisibility, these particles have garnered increasing attention from researchers due to their prospective broad applications as diagnostic biomarkers, therapeutic targets, and drug delivery vehicles11. Human feces, often used as biospecimens for studying BEVs, predominantly sourced from gut bacteria, contain a complex mixture of water, bacteria, lipids, proteins, undigested food residue, and exfoliated epithelial cells among others. The intricate fecal composition poses challenges to the isolation and purity of BEVs, thereby impeding a comprehensive, objective, and realistic analysis of BEVs. Hence, effective strategies to minimize interference from contaminating components and enhance the yield of BEVs have emerged as critical issues warranting immediate attention.
Existing isolation strategies largely rely on techniques such as ultra-high-speed centrifugation (UC), density gradient centrifugation (DGC), and size exclusion chromatography (SEC)12,13,14,15,16,17. Currently, DGC is one of the most widely applied methods in the field of BEV separation, encompassing two sedimentation-floating modes, &#34;Top-down&#34; and &#34;Bottom-up&#34;, which are determined by the initial loading position of the sample. These methodologies differentiate extracellular vesicles (EVs) from other components based on size and density disparities, yielding variable purity and recovery rates. Prior research has indicated that single-approach strategies are insufficient for adequately separating EVs from soluble proteins in body fluid samples, such as lipoprotein in blood18 and Tamm-Horsfall protein in urine19. Additionally, the size distribution of eukaryotic extracellular vesicles (EEVs) often overlaps with that of BEVs, thereby necessitating further methodological enhancements to optimize BEV yield. Consequently, advancing the study of BEVs hinges on the development of effective separation and purification methodologies. Notably, Tulkens et al15 employed an orthogonal biophysical strategy to separate fecal BEVs from EEVs, in which the centrifugation time of a Bottom-up DGC mode was up to 18 h. In contrast, this study reduced it to 7 h, greatly saving the gradient-ultracentrifugation time and simplifying the process.
In the present study, we isolated and purified fecal BEVs employing two DGC modes under optimized buffer conditions, after enriching BEVs with a range of differential centrifugation speeds, from low to extremely high velocity. Evaluations based on morphology, particle size, and concentration indicated a commendable performance by this enhanced method. This study could serve as the foundation for future research, extending its applications to a broader domain, and offering insights into the heterogeneity of BEVs within the human body. It also contributes to the standardization of BEV separation and analysis techniques.

Autor destacado: Aceleración de la investigación sobre la separación y heterogeneidad de vesículas extracelulares bacterianas 

Aislamiento y purificación de vesículas extracelulares bacterianas a partir de heces humanas mediante centrifugación en gradiente de densidad

Our study isolates, purifies, and characterize bacterial extracellular vesicles from physicists using density gradient centrifugation. We aim to address the separation of BEVs from contaminants and provide a reliable method for researchers. Our protocol reduces centrifugation time from 18 to 7 hours.Top down mode improves BEV separation and reduces contamination from liver proteins. Our method simplifies the separation of BEVs, setting a standard for future studies. It can be applied to other body fluids with appropriate modification, enhancing our understanding and facilitating the study of BEVs revealing their inherent heterogeneity within the human body.

Determinar la distribución de las fracciones enriquecidas con BEV Para determinar la distribución de las fracciones enriquecidas con vesículas extracelulares bacterianas (BEV), se estableció un control en blanco para medir los valores de absorbancia a DO 340 nm, y se calculó la densidad de cada fracción en función de las mediciones y las directrices de yodixanol (Paso 8.1). En la Tabla 2 se presentan los resultados de densidad, demostrando que las fracciones F4 a F9 exhibieron ...

Watch this Scientific Journal Video about Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity at JoVE.com

Este estudio describe un método para aislar y purificar vesículas extracelulares bacterianas (BEV) enriquecidas a partir de heces humanas mediante centrifugación en gradiente de densidad (DGC), identifica las características físicas de los BEV a partir de la morfología, el tamaño de partícula y la concentración, y discute las posibles aplicaciones del enfoque DGC en la investigación clínica y científica.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

Nuestro estudio aísla, purifica y caracteriza vesículas extracelulares bacterianas de físicos mediante centrifugación en gradiente de densidad. Nuestro objetivo es abordar la separación de los BEV de los contaminantes y proporcionar un método fiable para los investigadores. Nuestro protocolo reduce el tiempo de centrifugación de 18 a 7 horas.El modo descendente mejora la separación de BEV y reduce la contaminación por proteínas hepáticas. Nuestro método simplifica la separación de BEV, estableciendo un estándar para futuros estudios. Se puede aplicar a otros fluidos corporales con la modificación adecuada, mejorando nuestra comprensión y facilitando el estudio de los BEV revelando su heterogeneidad inherente dentro del cuerpo humano.

isolation-purification-bacterial-extracellular-vesicles-from-human

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JoVE Journal

Biology

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

1.6K vistas.  Southern Medical University. Nuestro estudio aísla, purifica y caracteriza vesículas extracelulares bacterianas de físicos mediante centrifugación en gradiente de densidad. Nuestro objetivo es abordar la separación de los BEV de los contaminantes y proporcionar un método fiable para los investigadores. Nuestro protocolo reduce el tiempo de centrifugación de 18 a 7 horas.El modo descendente mejora la separación de BEV y reduce la contaminación por proteínas hepáticas. Nuestro método simplifica la separa...