Name: Autor em destaque: acelerando a pesquisa sobre separação e heterogeneidade de vesículas extracelulares bacterianas
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

Este trabalho foi apoiado pelo National Science Fund for Distinguished Young Scholars (82025024); o projeto-chave da Fundação Nacional de Ciências Naturais da China (82230080); o Programa Nacional de P&D da China (2021YFA1300604); a Fundação Nacional de Ciências Naturais da China (81871735, 82272438 e 82002245); Fundo de Ciências Naturais de Guangdong para Jovens Académicos Ilustres (2023B1515020058); Fundação de Ciências Naturais da Província de Guangdong (2021A1515011639); o Programa Estadual de Desenvolvimento de Pesquisa Básica da Fundação de Ciências Naturais da Província de Shandong, na China (ZR2020ZD11); a Fundação de Ciência Pós-doutoral (2022M720059); o Esquema de Desenvolvimento de Jovens Excepcionais do Hospital Nanfang, Southern Medical University (2022J001).

O Comitê de Ética do Hospital Nanfang, Southern Medical University, sancionou este estudo, que foi conduzido com o consentimento informado dos participantes. Todos os métodos aqui empregados seguiram as diretrizes operacionais padrão fornecidas pelas Normas Internacionais de Microbioma Humano (IHMS: http://www.microbiome-standards.org/). Todos os procedimentos subsequentes de manuseio de líquidos foram obrigados a serem realizados dentro de um gabinete de biossegurança ou uma bancada ultralimpa.
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Isolamento Aprimorado de Vesículas Extracelulares Bacterianas para Microbiota Multi-Ômica

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As vesículas extracelulares bacterianas (VEB) são nanopartículas de bicamada lipídica secretadas por bactérias, carregando uma riqueza de proteínas, lipídios, ácidos nucléicos e outras moléculas bioativas, contribuindo para mediar os efeitos funcionais das bactérias20. Verificou-se que os BEVs derivados do intestino estão envolvidos no desenvolvimento de doenças, como doença inflamatória intestinal, doença de Crohn e câncer colorretal, além de afetar o metabolismo geral e mediar ...

O intestino é amplamente reconhecido como o órgão que abriga as comunidades microbianas mais abundantes no corpo humano, com mais de 90% das bactérias envolvidas na colonização e multiplicação 1,2. Extensas evidências têm demonstrado que a microbiota intestinal modula o microambiente intestinal e, simultaneamente, interage com disfunção em órgãos distantes, principalmente através de uma barreira intestinal prejudicada 3,4. Evidências crescentes indicam uma correlação entre o desequilíbrio da microbiota intestinal e a progressão da doença inflamatória intestinal (DII)5,6, bem como distúrbios cognitivos através do eixo intestino-cérebro5,6,7,8. As vesículas extracelulares bacterianas (BEVs) produzidas por bactérias desempenham papéis significativos nesses processos patológicos.
BEVs são partículas em nanoescala que encapsulam derivados bacterianos, com diâmetros variando de 20 a 400 nm. Eles têm sido demonstrados para facilitar as interações entre bactérias e seus organismos hospedeiros 9,10. Apesar de sua invisibilidade, essas partículas têm recebido cada vez mais atenção dos pesquisadores devido às suas amplas aplicações prospectivas como biomarcadores diagnósticos, alvos terapêuticos e veículos de liberação de fármacos11. As fezes humanas, frequentemente usadas como bioespécimes para estudar BEVs, predominantemente provenientes de bactérias intestinais, contêm uma mistura complexa de água, bactérias, lipídios, proteínas, resíduos alimentares não digeridos e células epiteliais esfoliadas, entre outros. A intrincada composição fecal coloca desafios ao isolamento e pureza dos BEVs, impedindo uma análise abrangente, objetiva e realista dos BEVs. Assim, estratégias efetivas para minimizar a interferência de componentes contaminantes e aumentar o rendimento dos BEVs têm emergido como questões críticas que merecem atenção imediata.
As estratégias de isolamento existentes dependem em grande parte de técnicas como centrifugação em ultra-alta velocidade (UC), centrifugação por gradiente de densidade (DGC) e cromatografia de exclusão de tamanho (SEC)12,13,14,15,16,17. Atualmente, a DGC é um dos métodos mais amplamente aplicados no campo da separação de BEV, englobando dois modos de sedimentação-flutuação, "Top-down" e "Bottom-up", que são determinados pela posição de carregamento inicial da amostra. Essas metodologias diferenciam as vesículas extracelulares (EVs) de outros componentes com base nas disparidades de tamanho e densidade, produzindo taxas variáveis de pureza e recuperação. Pesquisas anteriores indicaram que estratégias de abordagem única são insuficientes para separar adequadamente os EVs das proteínas solúveis em amostras de fluido corporal, como a lipoproteína no sangue18 e a proteína Tamm-Horsfall na urina19. Além disso, a distribuição de tamanho das vesículas extracelulares (EEVs) eucarióticas frequentemente se sobrepõe à dos BEVs, necessitando de maiores aprimoramentos metodológicos para otimizar o rendimento do BEV. Consequentemente, o avanço do estudo dos BEVs depende do desenvolvimento de metodologias eficazes de separação e purificação. Notavelmente, Tulkens e cols.15 empregaram uma estratégia biofísica ortogonal para separar BEVs fecais de EEVs, na qual o tempo de centrifugação de um modo Bottom-up DGC foi de até18 h. Em contrapartida, este estudo reduziu para 7 h, economizando muito o tempo de gradiente-ultracentrifugação e simplificando o processo.
No presente estudo, isolamos e purificamos BEVs fecais empregando dois modos de DGC sob condições de tampão otimizadas, após enriquecer BEVs com uma faixa de velocidades de centrifugação diferencial, de baixa a extremamente alta velocidade. Avaliações baseadas em morfologia, tamanho de partícula e concentração indicaram um desempenho louvável por este método aprimorado. Este estudo poderia servir como base para pesquisas futuras, estendendo suas aplicações para um domínio mais amplo e oferecendo insights sobre a heterogeneidade dos BEVs dentro do corpo humano. Também contribui para a padronização das técnicas de separação e análise 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

As vesículas extracelulares bacterianas (BEVs) são nanovesículas derivadas de bactérias que desempenham um papel ativo na comunicação bactéria-bactéria e bactéria-hospedeiro, transferindo moléculas bioativas como proteínas, lipídios e ácidos nucléicos herdados das bactérias mãe. Os BEVs derivados da microbiota intestinal têm efeitos dentro do trato gastrointestinal e podem atingir órgãos distantes, resultando em implicações significativas para a fisiologia e patologia. Investigações teóricas que explorem os tipos, quantidades e papéis de BEVs derivados de fezes humanas são cruciais para entender a secreção e a função de BEVs da microbiota intestinal. Essas investigações também requerem um aprimoramento na estratégia atual de isolamento e purificação de BEVs.
Este estudo otimizou o processo de isolamento e purificação de BEVs estabelecendo dois modos de centrifugação por gradiente de densidade (DGC): Top-down e Bottom-up. A distribuição enriquecida dos BEVs foi determinada nas frações 6 a 8 (F6-F8). A eficácia da abordagem foi avaliada com base na morfologia das partículas, tamanho, concentração e teor de proteínas. As taxas de recuperação de partículas e proteínas foram calculadas, e a presença de marcadores específicos foi analisada para comparar a recuperação e pureza dos dois modos de DGC. Os resultados indicaram que o modo de centrifugação Top-down apresentou menores níveis de contaminação e alcançou uma taxa de recuperação e pureza semelhante à do modo Bottom-up. Um tempo de centrifugação de 7 h foi suficiente para atingir uma concentração de BEV fecal de 108/mg.
Além das fezes, este método pode ser aplicado a outros tipos de fluidos corporais com modificação adequada de acordo com as diferenças de componentes e viscosidade. Em conclusão, este protocolo detalhado e confiável facilitaria o isolamento e purificação padronizados de BEVs e, assim, estabeleceria uma base para análises multi-ômicas subsequentes e experimentos funcionais.

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 em destaque: acelerando a pesquisa sobre separação e heterogeneidade de vesículas extracelulares bacterianas 

Isolamento e Purificação de Vesículas Extracelulares Bacterianas de Fezes Humanas Utilizando Centrifugação por Gradiente de Densidade

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 a distribuição das frações enriquecidas com BEV Para determinar a distribuição das frações enriquecidas com vesículas extracelulares bacterianas (BEVs), um controle em branco foi estabelecido para medir os valores de absorbância em OD 340 nm, e a densidade de cada fração foi calculada com base nas medidas e diretrizes de iodixanol (Passo 8.1). A Tabela 2 apresenta os resultados de densidade, demonstrando que as frações F4 a F9 exibiram densidades dentro da fai...

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

Este estudo descreve um método para isolar e purificar vesículas extracelulares bacterianas (BEVs) enriquecidas de fezes humanas via centrifugação por gradiente de densidade (DGC), identifica as características físicas dos BEVs a partir da morfologia, tamanho de partícula e concentração, e discute as potenciais aplicações da abordagem DGC em pesquisa clínica e científica.

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

Nosso estudo isola, purifica e caracteriza vesículas extracelulares bacterianas de físicos usando centrifugação por gradiente de densidade. Nosso objetivo é abordar a separação de BEVs de contaminantes e fornecer um método confiável para pesquisadores. Nosso protocolo reduz o tempo de centrifugação de 18 para 7 horas.O modo de cima para baixo melhora a separação do BEV e reduz a contaminação das proteínas do fígado. Nosso método simplifica a separação dos VEBs, estabelecendo um padrão para estudos futuros. Pode ser aplicado a outros fluidos corporais com modificação apropriada, aumentando nossa compreensão e facilitando o estudo dos BEVs revelando sua heterogeneidade inerente dentro do corpo humano.

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.

Biologia

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

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.

Enriquecimento de vesículas extracelulares bacterianas (BEVs) de fezes humanas usando velocidades de centrifugação diferencial

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

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.

Estabelecimento de um Sistema de Centrifugação por Gradiente de Densidade para Isolar e Purificar Vesículas Extracelulares Bacterianas (BEVs) de Fezes Humanas