The research described above was supported by NIH TL1 TR002549-03 training grant. We thank Drs. John C. Tilton and Zachary Troyer (Case Western Reserve University) for facilitating access to the particle size analyzer instrument; Lew Brown (Spectradyne) for technical assistance with analysis of the particle size distribution data; Dr. David Putnam at Cornell University for providing pClyA-GFP plasmid14; and Dr. Mark Goulian at the University of Pennsylvania for providing us with the E. coli MP113.

<ol>
	<li>Yanez-Mo, M., 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=Biological+properties+of+extracellular+vesicles+and+their+physiological+functions.">Biological properties of extracellular vesicles and their physiological functions.</a> Journal of Extracellular Vesicles. 4, 27066 (2015).</li><li>Chatterjee, S. N., Das, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+observations+on+the+excretion+of+cell-wall+material+by+Vibrio+cholerae.">Electron microscopic observations on the excretion of cell-wall material by Vibrio cholerae.</a> Journal of General Microbiology. 49 (1), 1-11 (1967).</li><li>Ciofu, O., Beveridge, T. J., Kadurugamuwa, J., Walther-Rasmussen, J., Hoiby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+beta-lactamase+is+packaged+into+membrane+vesicles+and+secreted+from+Pseudomonas+aeruginosa.">Chromosomal beta-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa.</a> Journal of Antimicrobial Chemotherapy. 45 (1), 9-13 (2000).</li><li>Yonezawa, H., 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+of+Helicobacter+pylori+TK1402+are+involved+in+biofilm+formation.">Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation.</a> BMC Microbiology. 9, 197 (2009).</li><li>Mashburn, L. M., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+traffic+signals+and+facilitate+group+activities+in+a+prokaryote.">Membrane vesicles traffic signals and facilitate group activities in a prokaryote.</a> Nature. 437 (7057), 422-425 (2005).</li><li>Kato, S., Kowashi, Y., Demuth, D. R. <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-like+vesicles+secreted+by+Actinobacillus+actinomycetemcomitans+are+enriched+in+leukotoxin.">Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin.</a> Microbial Pathogenesis. 32 (1), 1-13 (2002).</li><li>Petousis-Harris, H., 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=Effectiveness+of+a+group+B+outer+membrane+vesicle+meningococcal+vaccine+against+gonorrhoea+in+New+Zealand:+a+retrospective+case-control+study.">Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study.</a> Lancet. 390 (10102), 1603-1610 (2017).</li><li>Kim, O. Y., 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=Bacterial+outer+membrane+vesicles+suppress+tumor+by+interferon-gamma-mediated+antitumor+response.">Bacterial outer membrane vesicles suppress tumor by interferon-gamma-mediated antitumor response.</a> Nature Communications. 8 (1), 626 (2017).</li><li>Thery, C., 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=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Consortium, E. -. 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=EV-TRACK:+transparent+reporting+and+centralizing+knowledge+in+extracellular+vesicle+research.">EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.</a> Nature Methods. 14 (3), 228-232 (2017).</li><li>Watson, D. C., 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=Efficient+production+and+enhanced+tumor+delivery+of+engineered+extracellular+vesicles.">Efficient production and enhanced tumor delivery of engineered extracellular vesicles.</a> Biomaterials. 105, 195-205 (2016).</li><li>Watson, D. C., 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=Scalable,+cGMP-compatible+purification+of+extracellular+vesicles+carrying+bioactive+human+heterodimeric+IL-15/lactadherin+complexes.">Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes.</a> Journal of Extracellular Vesicles. 7 (1), 1442088 (2018).</li><li>Lasaro, M., 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=Escherichia+coli+isolate+for+studying+colonization+of+the+mouse+intestine+and+its+application+to+two-component+signaling+knockouts.">Escherichia coli isolate for studying colonization of the mouse intestine and its application to two-component signaling knockouts.</a> Journal of Bacteriology. 196 (9), 1723-1732 (2014).</li><li>Kim, J. Y., 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=Engineered+bacterial+outer+membrane+vesicles+with+enhanced+functionality.">Engineered bacterial outer membrane vesicles with enhanced functionality.</a> Journal of Molecular Biology. 380 (1), 51-66 (2008).</li><li>Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+gram-negative+cell+walls+and+their+derived+membrane+vesicles.">Structures of gram-negative cell walls and their derived membrane vesicles.</a> Journal of Bacteriology. 181 (16), 4725-4733 (1999).</li><li>Reimer, S. 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=Comparative+analysis+of+outer+membrane+vesicle+isolation+methods+with+an+Escherichia+coli+tolA+mutant+reveals+a+hypervesiculating+phenotype+with+outer-inner+membrane+vesicle+content.">Comparative analysis of outer membrane vesicle isolation methods with an Escherichia coli tolA mutant reveals a hypervesiculating phenotype with outer-inner membrane vesicle content.</a> Frontiers in Microbiology. 12, 628801 (2021).</li></ol>

The authors have no conflicts of interest to declare.

In the protocol above, a method is described that is scalable and reliably isolates EVs from various gram-negative/positive and aerobic/anaerobic bacteria. It has several potential stopping points throughout the procedure, although it is better to avoid taking longer than 48 h to isolate EVs from conditioned bacterial culture media.
First, it consists of culturing bacteria to generate conditioned bacterial culture medium. It was found that increasing the culture time to at least 48 h and using...

Extracellular vesicles (EVs) are nanometer-sized, liposome-like structures comprised of lipids, proteins, glycans, and nucleic acids, secreted by both prokaryotic and eukaryotic cells1. Since the early studies visualizing the release of EVs from gram-negative bacteria2, the number of biological functions attributed to bacterial EVs (20-300 nm in diameter) has constantly been growing in the past decades. Their functions include transferring antibiotic resistance3, biofilm formation4, quorum sensing5, and toxin delivery6. There is also growing interest in the use of bacterial EVs as therapeutics, especially in vaccinology7 and cancer therapy8.
Despite the growing interest in EV research, there are still technical challenges regarding methods of isolation. Specifically, there is a need for isolation methods that are reproducible, scalable, and compatible with diverse EV-producing organisms. To create a unified set of principles for planning and reporting EV isolation and research methods, the International Society for Extracellular Vesicles publishes and updates the MISEV position paper9. Moreover, the EV-TRACK consortium provides an open platform for reporting detailed methodologies for EV isolation used in published manuscripts to enhance transparency10.
In this protocol, previous methodologies used for the isolation of EVs from mammalian cell culture were adapted11,12 to enable the isolation of EVs from bacterial cell culture. We sought to employ methods that enable EV isolation from a variety of microbes, which can be scalable, and balance EV purity and yield (as discussed in the MISEV position paper9). After removing bacterial cells and debris by centrifugation and filtration, the culture medium is concentrated either by centrifugal device ultrafiltration (for a volume of up to ~100 mL) or pump-driven TFF (for larger volumes). EVs are then isolated by SEC using columns optimized for the purification of small EVs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63155/63155fig01.jpg" /> 
Figure 1: Bacterial EV isolation workflow schematic overview. Abbreviations: EV = extracellular vesicle; TFF = tangential flow filtration; SEC = size exclusion chromatography; MWCO = molecular weight cut-off. <a href="https://www.jove.com/files/ftp_upload/63155/63155fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A mouse-commensal strain of Escherichia coli (i.e., E. coli MP113) was used as a model organism and modified to express EV-associated nanoluciferase by fusion to cytolysin A, as previously reported14. The methods used here can process at least up to several liters of bacterial cultures and effectively separate EV-associated from non-EV-associated proteins. Finally, this method can also be used for other gram-positive and gram-negative bacterial species. All relevant data of the reported experiments were submitted to the EV-TRACK knowledgebase (EV-TRACK ID: EV210211)10.

<table><tbody><tr><td>0.5 mL flat cap, thin-walled PCR tubes</td><td>Thermo Scientific</td><td>3430</td><td>it is important to use thin-walled PCR tubes to obtain accurate readings with Qubit</td></tr><tr><td>16% Paraformaldehyde (formaldehyde) aqueous solution</td><td>Electron microscopy sciences</td><td>15700</td><td></td></tr><tr><td>250 mL Fiberlite polypropylene centrifuge bottles</td><td>ThermoFisher</td><td>010-1495</td><td></td></tr><tr><td>500 mL Fiberlite polypropylene centrifuge bottles</td><td>ThermoFisher</td><td>010-1493</td><td></td></tr><tr><td>65 mm Polypropylene Round-Bottom/Conical Bottle Adapter</td><td>Beckman Coulter</td><td>392077</td><td>Allows Vivacell to fit in rotor</td></tr><tr><td>&lt;em&gt;Akkermansia mucinophila&lt;/em&gt;</td><td>ATCC</td><td>BAA-835</td><td></td></tr><tr><td>Amicon-15 (100 kDa MWCO)</td><td>MilliporeSigma</td><td>UFC910024</td><td></td></tr><tr><td>Avanti J-20 XPI centrifuge</td><td>Beckman Coulter</td><td></td><td>No longer sold by Beckman. Avanti J-26XP is closest contemporary model.</td></tr><tr><td>&lt;em&gt;Bacteroides thetaiotaomicron &lt;/em&gt;VPI 5482</td><td>ATCC</td><td>29148</td><td></td></tr><tr><td>&lt;em&gt;Bifidobacterium breve&lt;/em&gt;</td><td>NCIMB</td><td>B8807</td><td></td></tr><tr><td>&lt;em&gt;Bifidobacterium dentium&lt;/em&gt;</td><td>ATCC</td><td>27678</td><td></td></tr><tr><td>Brain Heart infusion (BHI) broth</td><td>Himedia</td><td>M2101</td><td>After autoclaving, Both BHI broth and agar were introduced into the anaerobic chamber, supplemented with Menadione (1 &amp;micro;g/L), hematin (1.2 &amp;micro;g/L), and L-Cysteine Hydrochloride (0.05%). They were then incubated for at least 24 h under anaerobic conditions before inoculation with the anaerobic bacterial strains.</td></tr><tr><td>C-300 microfluidics cartridge</td><td>Spectradyne</td><td></td><td></td></tr><tr><td>Chloramphenicol</td><td>MP Biomedicals</td><td>ICN19032105</td><td></td></tr><tr><td>Electron microscope</td><td>FEI company</td><td>Tecnai G2 SpiritBT</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; HST08 (Steller competent cells)</td><td>Takara</td><td>636763</td><td></td></tr><tr><td>Escherichia coli MP1</td><td>Dr. Mark Goulian (gift)</td><td></td><td>commensal bacteria derived from mouse gut</td></tr><tr><td>Fiberlite 500 mL to 250 mL adapter</td><td>ThermoFisher</td><td>010-0151-05</td><td>used with Fiberlite rotor to enable 250 mL bottles to be used for smaller size of starting bacterial culture</td></tr><tr><td>Fiberlite fixed-angle centrifuge rotor</td><td>ThermoFisher</td><td>F12-6x500-LEX</td><td>fits 6 x 500 mL bottles</td></tr><tr><td>Formvar Carbon Film 400 Mesh, Copper</td><td>Electron microscopy sciences</td><td>FCF-400-CU</td><td></td></tr><tr><td>Glutaraldehyde (EM-grade, 10% aqeous solution)</td><td>Electron microscopy sciences</td><td>16100</td><td></td></tr><tr><td>Hematin</td><td>ChemCruz</td><td>207729B</td><td>Stock solution was made in 0.2 M L-histidine solution as&amp;nbsp; 1.2 mg/mL</td></tr><tr><td>Infinite M Nano+ Microplate reader</td><td>Tecan</td><td></td><td>This equibment was used to measure the mCherry fluorescence</td></tr><tr><td>In-Fusion&amp;nbsp; HD Cloning Plus</td><td>Takara</td><td>638909</td><td>For cloning of the PCR fragements into the PCR-lineraized vectors</td></tr><tr><td>JS-5.3 AllSpin Swinging-Bucket Rotor</td><td>Beckman Coulter</td><td>368690</td><td></td></tr><tr><td>Lauria Bertani (LB) broth, Miller</td><td>Difco</td><td>244620</td><td></td></tr><tr><td>L-Cysteine Hydrochloride</td><td>J.T. Baker</td><td>2071-05</td><td>It should be weighed and added directly to the autoclaved BHI media inside the anaerobic chamber</td></tr><tr><td>Masterflex Fitting, Polypropylene, Straight, Female Luer to Hose Barb Adapter, 1/8&quot; ID; 25/PK</td><td>cole-parmer - special</td><td>HV-30800-08</td><td>connection adapters for filtration tubing circuit</td></tr><tr><td>Masterflex Fitting, Polypropylene, Straight, Male Luer to Hose Barb Adapter, 1/8&quot; ID; 25/PK</td><td>cole-parmer - special</td><td>HV-30800-24</td><td>connection adapters for filtration tubing circuit</td></tr><tr><td>Masterflex L/S Analog Variable-Speed Console Drive, 20 to 600 rpm</td><td>Masterflex</td><td>HV-07555-00</td><td></td></tr><tr><td>Masterflex L/S Easy-Load Head for Precision Tubing, 4-Roller, PARA Housing, SS Rotor</td><td>Masterflex</td><td>EW-07514-10</td><td></td></tr><tr><td>Masterflex L/S Precision Pump Tubing, PharmaPure, L/S 16; 25 ft</td><td>Cole Palmer</td><td>EW-06435-16</td><td>low-binding/low-leaching tubing</td></tr><tr><td>Menadione (Vitamin K3)</td><td>MP</td><td>102259</td><td>Stock solution was made in ethanol as 1 mg/mL</td></tr><tr><td>MIDIKROS 41.5CM 100K MPES 0.5MM FLL X FLL 1/PK</td><td>Repligen</td><td>D04-E100-05-N</td><td>TFF device we have used to filter up to 2 L of E. coli culture supernatant</td></tr><tr><td>Nano-Glo Luciferase Assay System</td><td>Promega</td><td>N1110</td><td>This assay kit was used to measure the luminescence of the nluc reporter protein</td></tr><tr><td>NanoLuc (Nluc) Luciferase Antibody, clone 965808</td><td>R&amp;amp;D Systems</td><td>MAB10026</td><td></td></tr><tr><td>nCS1 microfluidics resistive pulse sensing instrument</td><td>Spectradyne</td><td></td><td></td></tr><tr><td>nCS1 Viewer</td><td>Spectradyne</td><td></td><td>Analysis software for particle size distribution</td></tr><tr><td>OneTaq 2x Master Mix with Standard Buffer</td><td>NEB</td><td>M0482</td><td>DNA polymerase master mix used to perform the routine PCR reactions for colony checking</td></tr><tr><td>Protein LoBind, 2.0 mL, PCR clean tubes</td><td>Eppendorf</td><td>30108450</td><td></td></tr><tr><td>Q5 High-Fidelity 2x Master Mix</td><td>NEB</td><td>M0492</td><td>DNA polymerase master mix used to perform the PCR reactions needed for cloning</td></tr><tr><td>qEV original, 35 nm</td><td>Izon</td><td></td><td>maximal loading volume of 0.5 mL</td></tr><tr><td>qEV rack</td><td>Izon</td><td></td><td>for use with the qEV-original SEC columns</td></tr><tr><td>qEV-2, 35 nm</td><td>Izon</td><td></td><td>maximal loading volume of 2 mL</td></tr><tr><td>Qubit fluorometer</td><td>ThermoFisher</td><td></td><td>Item no longer available. Closest available product is Qubit 4.0 Fluorometer (cat. No. Q33238)</td></tr><tr><td>Qubit protein assay kit</td><td>ThermoFisher</td><td>Q33211</td><td>Store kit at room temperature. Standards are stored at 4 &amp;deg;C.</td></tr><tr><td>Sorvall Lynx 4000 centrifuge</td><td>ThermoFisher</td><td>75006580</td><td></td></tr><tr><td>SpectraMax i3x Microplate reader</td><td>Molecular Devices</td><td></td><td>This equipment was used to measure the nanoluciferase bioluminescence</td></tr><tr><td>Stericup Quick-release-GP Sterile Vacuum Filtration system (150, 250, or 500 mL)</td><td>MilliporeSigma</td><td>S2GPU01RE&lt;br/&gt; S2GPU02RE&lt;br/&gt; S2GPU05RE</td><td>One or multiple filters can be used to accommodate working volumes. In our experience, you can filter twice the volume listed on the product size.</td></tr><tr><td>Uranyl acetate</td><td>Electron microscopy sciences</td><td>22400</td><td></td></tr><tr><td>Vinyl anaerobic chamber</td><td>Coy Lab</td><td></td><td></td></tr><tr><td>Vivacell 100, 100,000 MWCO PES</td><td>Sartorius</td><td>VC1042</td><td></td></tr><tr><td>Whatman Anotop 10 Plus syringe filters (0.02 micron)</td><td>MilliporeSigma</td><td>WHA68093002</td><td>to filter MRPS diluent</td></tr></tbody></table>

scalable isolation and purification of extracellular vesicles from escherichia coli and other bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

To assess which SEC chromatography fractions were enriched for EVs, the SEC column was loaded with 2 mL of E. coli MP1-conditioned culture medium that had been concentrated 1,000-fold by TFF, and sequential fractions were collected. Using MRPS, it was found that Fractions 1-6 contained the most EVs (Figure 2A). Subsequent fractions contained very few EVs, comprising instead of EV-free proteins (Figure 2B). EVs were primarily &#60;100 nm in diameter (<st...

Watch this Scientific Journal Video about Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria at JoVE.com

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

scalable-isolation-purification-extracellular-vesicles-from

Research

JoVE Journal

Biology

3.7K Views.  Cleveland Clinic. Dit protocol is belangrijk omdat het een manier biedt om extracellulaire blaasjes te isoleren van een verscheidenheid aan bacteriën op een zeer reproduceerbare manier. Het mooie van deze techniek is dat het kan worden gebruikt om EV's te isoleren uit vrij grote omgevingen van bacterieculturen, wat in vivo studies mogelijk maakt. Hoewel de gegevens die we tonen preklinische toepassingen weerspiegelen, is het te verwachten dat dit protocol kan worden aangepast aan de productie van bacteriële ...

Video: Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Biochemistry

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of &#34;large&#34; (350 nm) OMVs and &#34;small&#34; (&#60;150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout ...

Bioengineering

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Immunology and Infection

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Neuroscience

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Production and Imaging of Recombinant Protein-Filled Vesicles from E. coli

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Author Spotlight: The Production of Recombinant Proteins 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an ...

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

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.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

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) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Source: Ryan, J. M., et al. <a href="https://app.jove.com/v/63895">Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography.</a> J. Vis. Exp. (2022).
This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.

1. Preparation of crude Mtb EV concentrate
NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

Source: Urwitz, S. N. et al. <a href="https://www.jove.com/v/59143/extraction-of-extracellular-vesicles-from-whole-tissue">Extraction of Extracellular Vesicles from Whole Tissue.</a> J. Vis. Exp. (2019)
This video demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

Source: Urwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video demonstrates the density gradient ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

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