Name: В центре внимания автора: Ускорение исследований по разделению и гетерогенности бактериальных внеклеточных везикул
Uploaded: 2023-09-01T14:30:00
Description: Watch this Scientific Journal Video about Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity at JoVE.com

Работа выполнена при поддержке Национального научного фонда для выдающихся молодых ученых (82025024); Ключевой проект Национального фонда естественных наук Китая (82230080); Национальная программа ключевых исследований и разработок Китая (2021YFA1300604); Национальный фонд естественных наук Китая (81871735, 82272438 и 82002245); Гуандунский фонд естественных наук для выдающихся молодых ученых (2023B1515020058); Фонд естественных наук провинции Гуандун (2021A1515011639); Крупная государственная программа развития фундаментальных исследований Фонда естественных наук провинции Шаньдун в Китае (ZR2020ZD11); Научный фонд постдокторантуры (2022M720059); Программа развития выдающейся молодежи больницы Наньфан Южного медицинского университета (2022J001).

Комитет по этике больницы Наньфан Южного медицинского университета санкционировал это исследование, которое проводилось с информированного согласия участников. Все методы, используемые в настоящем документе, соответствуют стандартным операционным рекомендациям, предоставленным М?...

Улучшенная изоляция бактериальных внеклеточных везикул для мультиомики микробиоты

<ol>
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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. 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Бактериальные внеклеточные везикулы (БЭВ) представляют собой липидно-бислойные наночастицы, секретируемые бактериями, несущие множество белков, липидов, нуклеиновых кислот и других биологически активных молекул, способствующих опосредованию функциональных эффектов бактерий<sup class="x...

Кишечник широко признан органом, содержащим самые многочисленные микробные сообщества в организме человека, причем более 90% бактерий участвуют в колонизации и размножении 1,2. Обширные данные свидетельствуют о том, что микробиота кишечника модулирует микроокружение кишечника и одновременно взаимодействует с дисфункцией отдаленных органов, в первую очередь через нарушение кишечного барьера 3,4. Появляется все больше данных, указывающих на корреляцию между дисбалансом микробиоты кишечника и прогрессированием воспалительных заболеваний кишечника (ВЗК)5,6, а также когнитивных нарушений через ось кишечник-мозг 5,6,7,8. Бактериальные внеклеточные везикулы (БЭВ), продуцируемые бактериями, играют значительную роль в этих патологических процессах.
БЭВ представляют собой наноразмерные частицы, инкапсулирующие производные бактерий, диаметром от 20 до 400 нм. Было продемонстрировано, что они облегчают взаимодействие между бактериями и их организмами-хозяевами 9,10. Несмотря на свою невидимость, эти частицы привлекают все большее внимание исследователей из-за их предполагаемого широкого применения в качестве диагностических биомаркеров, терапевтических мишеней и средств доставки лекарств11. Человеческие фекалии, часто используемые в качестве биологических образцов для изучения БЭВ, в основном полученные из кишечных бактерий, содержат сложную смесь воды, бактерий, липидов, белков, непереваренных остатков пищи и отслоившихся эпителиальных клеток. Сложный состав фекалий создает проблемы для изоляции и чистоты БЭВ, тем самым препятствуя всестороннему, объективному и реалистичному анализу БЭВ. Таким образом, эффективные стратегии минимизации помех от загрязняющих компонентов и повышения производительности электромобилей стали критически важными проблемами, требующими немедленного внимания.
Существующие стратегии изоляции в значительной степени основаны на таких методах, как сверхвысокоскоростное центрифугирование (UC), центрифугирование с градиентом плотности (DGC) и эксклюзионная хроматография (SEC)12,13,14,15,16,17. В настоящее время ДГК является одним из наиболее широко применяемых методов в области разделения БЭВ, охватывающим два седиментационно-плавающих режима, «сверху-вниз» и «снизу-вверх», которые определяются начальным положением загрузки образца. Эти методы позволяют дифференцировать внеклеточные везикулы (ВВ) от других компонентов на основе различий в размерах и плотности, обеспечивая различную чистоту и скорость восстановления. Предыдущие исследования показали, что стратегии однократного подхода недостаточны для адекватного отделения ВВ от растворимых белков в образцах биологических жидкостей, таких как липопротеин в крови18 и белок Тамма-Хорсфолла в моче19. Кроме того, распределение по размерам эукариотических внеклеточных везикул (ВЭВ) часто пересекается с распределением БЭВ, что обуславливает необходимость дальнейших методологических усовершенствований для оптимизации выхода БЭВ. Следовательно, дальнейшее изучение БЭВ зависит от разработки эффективных методологий сепарации и очистки. Примечательно, что Tulkens et al.15 использовали ортогональную биофизическую стратегию для отделения фекальных БЭВ от ВЭЭ, в которой время центрифугирования в режиме ДГК «снизу вверх» составляло до 18 ч. В отличие от этого, это исследование сократило его до 7 ч, что значительно сэкономило время градиентно-ультрацентрифугирования и упростило процесс.
В настоящем исследовании мы выделили и очистили фекальные БЭВ с использованием двух режимов ДГК в оптимизированных буферных условиях после обогащения БЭВ диапазоном дифференциальных скоростей центрифугирования, от низкой до чрезвычайно высокой. Оценки, основанные на морфологии, размере частиц и концентрации, показали похвальную эффективность этого усовершенствованного метода. Это исследование может послужить основой для будущих исследований, расширяя его применение в более широкой области и предлагая понимание гетерогенности БЭВ в организме человека. Это также способствует стандартизации методов разделения и анализа электромобилей.

<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

Бактериальные внеклеточные везикулы (БЭВ) — это нановезикулы, полученные из бактерий, которые играют активную роль в коммуникации бактерии-бактерии и бактерии-хозяина, перенося биологически активные молекулы, такие как белки, липиды и нуклеиновые кислоты, унаследованные от родительских бактерий. БЭВ, полученные из микробиоты кишечника, оказывают воздействие на желудочно-кишечный тракт и могут достигать отдаленных органов, что приводит к значительным последствиям для физиологии и патологии. Теоретические исследования, изучающие типы, количества и роль БЭВ, полученных из фекалий человека, имеют решающее значение для понимания секреции и функции БЭВ из микробиоты кишечника. Эти исследования также требуют совершенствования существующей стратегии по изоляции и очистке БЭВ.
В этом исследовании оптимизирован процесс изоляции и очистки электромобилей путем установления двух режимов центрифугирования с градиентом плотности (DGC): сверху вниз и снизу вверх. Обогащенное распределение БЭВ определяли во фракциях от 6 до 8 (F6-F8). Эффективность подхода оценивалась на основе морфологии частиц, размера, концентрации и содержания белка. Были рассчитаны скорости извлечения частиц и белков, а также проанализировано наличие специфических маркеров для сравнения восстановления и чистоты двух режимов ДГК. Результаты показали, что режим центрифугирования «Сверху вниз» имеет более низкие уровни загрязнения и обеспечивает скорость восстановления и чистоту, аналогичные режиму «Снизу вверх». Время центрифугирования 7 ч было достаточным для достижения концентрации фекальных БЭВ 108/мг.
Помимо фекалий, этот метод может быть применен и к другим типам жидкостей организма с соответствующей модификацией в соответствии с различиями в компонентах и вязкости. В заключение, этот подробный и надежный протокол облегчит стандартизированную изоляцию и очистку БЭВ и, таким образом, заложит основу для последующего мультиомиксного анализа и функциональных экспериментов.

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.

В центре внимания автора: Ускорение исследований по разделению и гетерогенности бактериальных внеклеточных везикул 

Выделение и очистка бактериальных внеклеточных везикул из фекалий человека с помощью центрифугирования с градиентом плотности

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.

Определение распределения фракций, обогащенных BEV Для определения распределения фракций, обогащенных внеклеточными везикулами бактерий (БЭВ), был установлен холостой контроль для измерения значений абсорбции при ОД 340 нм, а плотность каждой фракции рассчитывалась на основе...

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

В этом исследовании описывается метод выделения и очистки бактериальных внеклеточных везикул (БЭВ), обогащенных из фекалий человека с помощью центрифугирования с градиентом плотности (ДГК), определяются физические характеристики БЭВ с точки зрения морфологии, размера частиц и концентрации, а также обсуждаются потенциальные применения подхода ДГК в клинических и научных исследованиях.

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

Наше исследование выделяет, очищает и характеризует внеклеточные пузырьки бактерий у физиков с помощью центрифугирования с градиентом плотности. Мы стремимся решить проблему отделения электромобилей от загрязняющих веществ и предоставить исследователям надежный метод. Наш протокол сокращает время центрифугирования с 18 до 7 часов.Нисходящий режим улучшает отделение БЭВ и снижает загрязнение белков печени. Наш метод упрощает разделение электромобилей, устанавливая стандарт для будущих исследований. Он может быть применен к другим жидкостям организма с соответствующей модификацией, расширяя наше понимание и облегчая изучение БЭВ, выявляя присущую им гетерогенность в организме человека.

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

Research

JoVE Journal

Biology

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

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

Bacterial Extracellular Vesicles (BEVs) Enrichment from Human Faeces Using Differential Centrifugation Speeds

Обогащение бактериальных внеклеточных везикул (БЭВ) из фекалий человека с использованием дифференциальных скоростей центрифугирования  

To begin, take the prepared human fecal samples. Cool the high speed refrigerated centrifuge to 4 degrees Celsius. Use PBS to adjust the weight of the sample tubes to 0.1 grams, and centrifuge them at 3, 000 G for 20 minutes at 4 degrees Celsius.Using a plastic pipette, transfer the supernatant into two 50 milliliter tubes, leaving 1 milliliter behind. Then, centrifuge the supernatant at 12, 000 G for 30 minutes at 4 degrees Celsius. Using a 20 milliliter syringe, aspirate the supernatant, and remove the needle from the syringe.Filter the liquid through 0.22 micrometer filters into 50 milliliter tubes. Next, wipe the bucket with 75%alcohol to eliminate any remaining contamination. Transfer approximately 30 milliliters of the filtered fecal supernatant into the ultracentrifuge tube.Fill the tube with approximately 8 milliliters of PBS, leaving a 3 millimeter gap from the tube's opening. Place the two ultracentrifuge tubes in opposing ultracentrifugation buckets, and equalize the weight of the two buckets using PBS. Load all buckets onto the rotor, irrespective of whether they are loaded with tubes.Place the samples in the centrifuge, initiate the vacuum and spin them at 160, 000 G for 70 minutes at 4 degrees Celsius. As soon as the centrifugation completes, release the chamber vacuum and open the door of the ultra centrifuge. Then, promptly remove the rotor.Transfer the buckets from the rotor to the rack, and pick up the tubes using a nipper. Discard the supernatant, and save the visible brown pellets. Add 1 milliliter of pre-chilled PBS to resuspend the pellets completely.Fill the tube with approximately 37 milliliters of PBS, leaving a 3 millimeter gap from the opening. After performing another round of ultracentrifugation, discard the supernatant and leave the tube inverted for 5 minutes. Remove any remaining solution on the inner wall with wipers.Using a 1, 000 microliter pipette, add 1.2 milliliters of pre-chilled PBS to resuspend pellets, and mix them by pipetting. Transfer 1.2 milliliters of the PBS BEV solution from one ultracentrifuge tube to a clean 1.5 milliliter micro tube.

Обогащение бактериальных внеклеточных везикул (БЭВ) из фекалий человека с использованием дифференциальных скоростей центрифугирования

Establishment of a Density Gradient Centrifugation System to Isolate and Purify Bacterial Extracellular Vesicles (BEVs) from Human Faeces

Создание системы центрифугирования с градиентом плотности для выделения и очистки бактериальных внеклеточных везикул (BEV) из фекалий человека  

To begin, take 500 microliters of the prepared PBS/BEV solution. Combine it with three milliliters of PBS and mix gently using a 1, 000 microliter pipette. Place a 31 milliliter ultracentrifuge tube on a foam plate with holes, and label the tube with a downward arrow.Then hold a 1, 000 microliter pipette vertically and add three milliliters of the 50%iodixanol solution to the bottom of the tube. Place a tube holder slightly above the level of the foam plate below the tube opening, and tilt the tube to an angle of 70 degrees. Using a 1, 000 microliter pipette, add three milliliters of 40%iodixanol solution on top of the 50%iodixanol solution.Then layer three milliliters of 20%iodixanol solution on the 40%iodixanol solution. Using a 1, 000 microliter pipette, add three milliliters of a 10%iodixanol solution on top of the prepared 20%iodixanol solution. Next, add 3.5 milliliters of the PBS/BEV solution on top of the 10%iodixanol solution, and gently return the tubes to an upright position.Now, position a 31 milliliter ultracentrifuge tube on a foam plate with holes, and label it with an upward arrow. Using a 1, 000 microliter pipette, vertically add 2.5 milliliters of the 60%iodixanol stock solution into the bottom of the tube, and combine it with 500 microliters of PBS/BEV solution to get three milliliters of 50%iodixanol BEV solution. Mix the solution thoroughly by pipetting.Then after layering 40, 20 and 10%iodixanol solution in sequence, use a 1, 000 microliter pipette to add 3.5 milliliters of PBS on top of the 10%iodixanol solution. Adjust the weight of the two tubes with PBS to a total weight within plus minus 0.005 grams. Next, position the tubes in the buckets and centrifuge them at 160, 000 G for seven hours at four degrees Celsius.Using a pipetter, collect the fractions obtained from a gradient ultracentrifugation mode sequentially from top to bottom against the sidewall into 10 separate 38.5 milliliter ultracentrifuge tubes. Then ultracentrifuge the fractions at 160, 000 G for 70 minutes at four degrees Celsius. Remove the supernatant, and invert the ultracentrifuge tubes for five minutes.Next, resuspend the pellets in 200 microliters of pre-chilled PBS using a 200 microliter pipette. Transfer the PBS/BEV solution into a clean 1.5 milliliter microtube. Label the fractions from F1 to F10 as obtained from one gradient ultracentrifugation mode, and proceed for analysis.