We thank Emanuele Canonico for technical support. Part of this work was carried out in ALEMBIC, an advanced microscopy laboratory established by IRCCS Ospedale San Raffaele and Universit&#224; Vita-Salute San Raffaele. The work of Enrico Ragni and Laura de&#160;Girolamo was supported by the Italian Ministry of Health, &#34;Ricerca Corrente&#34;.

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

<ol>
	<li>Welsh, J. A., 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+(misev2023):+From+basic+to+advanced+approaches.">Minimal information for studies of extracellular vesicles (misev2023): From basic to advanced approaches.</a> J Extracell Vesicles. 13 (2), e12404 (2024).</li><li>Xu, R., Greening, D. W., Zhu, H. J., Takahashi, N., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+isolation+and+characterization:+Toward+clinical+application.">Extracellular vesicle isolation and characterization: Toward clinical application.</a> J Clin Invest. 126 (4), 1152-1162 (2016).</li><li>Dixson, A. C., Dawson, T. R., Di Vizio, D., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-specific+regulation+of+extracellular+vesicle+biogenesis+and+cargo+selection.">Context-specific regulation of extracellular vesicle biogenesis and cargo selection.</a> Nat Rev Mol Cell Biol. 24 (7), 454-476 (2023).</li><li>Witwer, K. W., Wolfram, J. <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+versus+synthetic+nanoparticles+for+drug+delivery.">Extracellular vesicles versus synthetic nanoparticles for drug delivery.</a> Nat Rev Mater. 6 (2), 103-106 (2021).</li><li>Cheng, L., Hill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutically+harnessing+extracellular+vesicles.">Therapeutically harnessing extracellular vesicles.</a> Nat Rev Drug Discov. 21 (5), 379-399 (2022).</li><li>Du, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+A+rising+star+for+therapeutics+and+drug+delivery.">Extracellular vesicles: A rising star for therapeutics and drug delivery.</a> J Nanobiotechnology. 21 (1), 231 (2023).</li><li>Keshtkar, S., Azarpira, N., Ghahremani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-derived+extracellular+vesicles:+Novel+frontiers+in+regenerative+medicine.">Mesenchymal stem cell-derived extracellular vesicles: Novel frontiers in regenerative medicine.</a> Stem Cell Res Ther. 9 (1), 63 (2018).</li><li>Orbay, H., Tobita, M., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+isolated+from+adipose+and+other+tissues:+Basic+biological+properties+and+clinical+applications.">Mesenchymal stem cells isolated from adipose and other tissues: Basic biological properties and clinical applications.</a> Stem Cells Int. 2012, 461718 (2012).</li><li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+trophic+mediators.">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li><li>Rani, S., Ryan, A. E., Griffin, M. D., Ritter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-derived+extracellular+vesicles:+Toward+cell-free+therapeutic+applications.">Mesenchymal stem cell-derived extracellular vesicles: Toward cell-free therapeutic applications.</a> Mol Ther. 23 (5), 812-823 (2015).</li><li>Galipeau, J., Sens&#233;b&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+Clinical+challenges+and+therapeutic+opportunities.">Mesenchymal stromal cells: Clinical challenges and therapeutic opportunities.</a> Cell Stem Cell. 22 (6), 824-833 (2018).</li><li>Jia, 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=Small+extracellular+vesicles+isolation+and+separation:+Current+techniques,+pending+questions+and+clinical+applications.">Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications.</a> Theranostics. 12 (15), 6548-6575 (2022).</li><li>Ohnuma, K., Yomo, T., Asashima, M., Kaneko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+of+cells+of+the+same+size,+shape,+and+cell+cycle+stage+for+a+single+cell+level+assay+without+staining.">Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining.</a> BMC Cell Biol. 7, 25 (2006).</li><li>Morales-Kastresana, A., 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=High-fidelity+detection+and+sorting+of+nanoscale+vesicles+in+viral+disease+and+cancer.">High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer.</a> J Extracell Vesicles. 8 (1), 1597603 (2019).</li><li>Mortati, 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=In+vitro+study+of+extracellular+vesicles+migration+in+cartilage-derived+osteoarthritis+samples+using+real-time+quantitative+multimodal+nonlinear+optics+imaging.">In vitro study of extracellular vesicles migration in cartilage-derived osteoarthritis samples using real-time quantitative multimodal nonlinear optics imaging.</a> Pharmaceutics. 12 (8), 734 (2020).</li><li>Andreu, Z., Yanez-Mo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraspanins+in+extracellular+vesicle+formation+and+function.">Tetraspanins in extracellular vesicle formation and function.</a> Front Immunol. 5, 442 (2014).</li><li>Mildmay-White, A., Khan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+markers+on+adipose-derived+stem+cells:+A+systematic+review.">Cell surface markers on adipose-derived stem cells: A systematic review.</a> Curr Stem Cell Res Ther. 12 (6), 484-492 (2017).</li><li>Welsh, J. A., Tang, V. A., Van Der Pol, E., Gorgens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIFlowCyt-EV:+The+next+chapter+in+the+reporting+and+reliability+of+single+extracellular+vesicle+flow+cytometry+experiments.">MIFlowCyt-EV: The next chapter in the reporting and reliability of single extracellular vesicle flow cytometry experiments.</a> Cytometry A. 99 (4), 365-368 (2021).</li><li>Maia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Employing+flow+cytometry+to+extracellular+vesicles+sample+microvolume+analysis+and+quality+control.">Employing flow cytometry to extracellular vesicles sample microvolume analysis and quality control.</a> Front Cell Dev Biol. 8, 593750 (2020).</li></ol>

The authors have nothing to disclose.

Analyzing and sorting EVs is challenging due to their small size and the fact that they are near the detection limit of most flow cytometers. Our objective was to develop a protocol for isolating EVs derived from AMSCs labeled with CFSE. CFSE was selected as the staining method due to its reported high EVs labeling efficiency (&#8805;90%), without the formation of unwanted particles such as protein aggregates given by antibodies. Nevertheless, it is possible that few EVs without esterase could be missed and future studie...

Extracellular Vesicles (EVs) are a heterogeneous group of membrane-structured vesicles released by almost all cells, delimited by a lipid bilayer, unable to replicate on their own1. They can be found in several biofluids such as blood plasma, serum, saliva, breast milk, urine, bronchial lavage fluid, amniotic fluid, cerebrospinal fluid, and malignant ascites2. One of the main functions of EVs is to transport various molecules, including nucleic acids, proteins, lipids, and carbohydrates, between a donor and a recipient cell. This can occur through various mechanisms, such as direct membrane fusion, receptor-ligand interaction, endocytosis, and phagocytosis3,4. For this reason, they have been demonstrated to play an important role in a lot of physiological and pathological processes, and they show considerable promise as novel biomarkers of disease, as drug delivery vehicles, and as therapeutic agents5,6.
Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from many tissues, including adipose tissue, dental pulp, umbilical cord blood, placenta, amniotic fluid, Wharton&#39;s jelly, and even the brain, lung, thymus, pancreas, spleen, liver, and kidney. In recent years, they have attracted considerable interest in regenerative medicine7. Adipose-derived mesenchymal stem cells (ASCs) can be harvested from fat tissue through a less invasive procedure compared to other sources like bone marrow, resulting in lower risks of severe complications and avoiding ethical issues8.
Additionally, adipose tissue contains a significantly higher concentration of MSCs than bone marrow (1% versus &#62;0.01%) and other sources such as the dermis, dental pulp, umbilical cord, and placenta. MSCs are crucial in the regeneration of injured tissues and cells due to their differentiation ability and their secretion of a broad repertoire of growth factors, chemokines, and cytokines; these therapeutic benefits are attributable to their differentiation ability but also to the fact that they secrete a broad repertoire of growth factors, chemokines, and cytokines. A striking example is given by MSCs&#39; therapeutic potential for orthopedic conditions, with the term &#34;Musculoskeletal Diseases&#34; having the higher number of registered clinical studies under clinicaltrials.gov (accessed 13th May 2024).
Moreover, MSCs can also secrete EVs that take part in tissue regeneration via transferring information to damaged cells or tissue and exert biological activity similar to the mother cells9,10. For this reason, MSC-EVs may be a valuable substitute for cell therapy to achieve a cell-free approach11, with two clinical studies involving MSC-EVs for orthopedic conditions (NCT05261360 and NCT04998058). However, several challenges still exist for the clinical applications of EVs. For example, there are some concerns about EV isolation techniques: most of them do not guarantee vesicle purity or integrity. Moreover, some isolation techniques are complex, time-consuming, and have low repeatability, making them unsuitable for clinical use12.
Cell sorting, on the other hand, is a commonly used method that allows for the isolation of single cells from heterogeneous cell suspensions by using specific fluorescent markers13. It can be used for many applications and adapted to different sample types. However, although cell sorting is a well-established and widely used technology, EV sorting is still very challenging because most EVs are below the minimum detection threshold for even the most sensitive flow cytometers. There are some features that make a sorter more suitable for this purpose. First of all, using a Jet-in-air system in which the stream suspending the particles is interrogated by lasers in air, rather than within a flow cell; this system preserves the sample by decreasing the stress to which it is subjected. A second important point is the presence of an &#34;obscuration&#34; bar between the stream and the collection lens that decrease the background optical noise of the instrument. Although it is low, the background noise is not completely eliminated and constitutes a reference that provides a partial window into the events that fall under the threshold: it is very important for the analysis of events that are close to the &#34;limit of detection&#34; of the instrument14. Finally, the sorter features a dual-path Forward Scatter (FSC) with two different masks that allow for improved discrimination between small and large particles in the sample.
Based on this, we developed a protocol aimed to separate carboxyfluorescein succinimidyl ester (CFSE) labeled MSC-EVs by using a high-sensitivity cell sorter. To minimize the manipulation of EVs and preserve their integrity and quantity, we avoided ultracentrifugation steps during the sample preparation. Furthermore, sorting conditions were adjusted to minimize stress on the vesicles, including further optimization of our instrument by reducing the sorting pressure associated with the nozzle size (70 &#956;m nozzle for a pressure of 35 psi).

<table><tbody><tr><td>5(6)-Carboxyfluorescein diacetate&amp;nbsp;N-succinimidyl ester</td><td>Merck</td><td>150347-59-4</td><td></td></tr><tr><td>Adipose Mesenchymal Stromal Cells</td><td>Wepredic, Parc d&#039;affaires, 35760 Saint-Gr&amp;eacute;goire, France</td><td></td><td>Cells used in this study</td></tr><tr><td>Alexa 488 anti-Caveolin</td><td>R&amp;amp;D Systems</td><td>IC5736G</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD44</td><td>BioLegend</td><td>338805</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD63</td><td>BioLegend</td><td>353007</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD81 (TAPA-1)</td><td>BioLegend</td><td>349509</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD9</td><td>BioLegend</td><td>312107</td><td>Flow cytometry antibody</td></tr><tr><td>BC CytoFLEX S</td><td>Beckman Coulter</td><td></td><td>BC CytoFLEX S equipped with 3 lasers, Blue, Red and Violet</td></tr><tr><td>Flow-Check Pro Fluorospheres</td><td>Beckman Coulter</td><td>A63493</td><td>Fluorescent control beads for MoFLO Astrios EQ</td></tr><tr><td>FlowJo software (version 10.8.1)</td><td>BD</td><td>version 10.8.1</td><td>Analysis software</td></tr><tr><td>IntraSure kit</td><td>BD Biosciences</td><td>641776</td><td>Fixation and permeabilization for intracellular staining</td></tr><tr><td>Megamix-Plus FSC</td><td>BioCytex</td><td>7802</td><td>FSC polystyrene beads&amp;nbsp;</td></tr><tr><td>MoFLO Astrios EQ</td><td>Beckman Coulter</td><td></td><td>MoFLO Astrios EQ equipped with 4 lasers, Blue, Yellow - Green, Violet and Red</td></tr><tr><td>Mouse anti-FLOT1 antibody</td><td>BD Transduction Laboratories</td><td>610820</td><td>Western Blot antibody</td></tr><tr><td>NanoSight NS300</td><td>Malvern</td><td>NS300</td><td></td></tr><tr><td>Rabbit anti-Calnexin antibody</td><td>Origene</td><td>TA336279</td><td>Western Blot antibody</td></tr><tr><td>Rabbit anti-CD9 and CD81 antibody (ExoAb antibody kit)</td><td>System Biosciences</td><td>EXOAB-KIT-1</td><td>Western Blot antibodies</td></tr><tr><td>Rabbit anti-TSG101 antibody&amp;nbsp;</td><td>Merck</td><td>HPA006161</td><td>Western Blot antibody</td></tr><tr><td>Triton X-100</td><td>Merck</td><td>9036-19-5</td><td></td></tr><tr><td>Ultra Rainbow Fluorescent Particles</td><td>Spherotech</td><td>URFP-30-2</td><td></td></tr><tr><td>Ultracel 100 kDa MWCO</td><td>Merck</td><td>UFC910024</td><td></td></tr><tr><td>VER01 - Verity Shells</td><td>Exometry</td><td></td><td>Organo silica beads for scatter calibration</td></tr></tbody></table>

setting a successful sorting for extracellular vesicle isolation

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications.
First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. 
In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique.
Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

The FSC polystyrene beads have been sorted to validate the instrument setup and sorting conditions. FSC polystyrene beads are a mix of fluorescent beads ranging from 100 nm, 300 nm, 500 nm, and 900 nm and are visible on the FITC channel. Figure 7A shows the SSC log scale versus the FITC log scale dot plot with the four populations of beads before sorting. The fluorescent populations of 100 nm, 300 nm, and 500 nm were gated and sorted. Sorted beads were analyzed for purity and enrichment as a...

Setting a Successful Sorting for Extracellular Vesicle Isolation

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. ...

setting-a-successful-sorting-for-extracellular-vesicle-isolation

Research

JoVE Journal

Biology

924 Views.  IRCCS Ospedale San Raffaele. De belangrijkste focus van ons onderzoek is dus het bestuderen van mesenchymale stromale cellen, extracellulair blaasje voor weefselregeneratie. Een kwestie en een doel van ons onderzoek is om de celvrije benadering te vertalen naar klinische toepassing naast celtherapieën. Karakterisering is cruciaal en tegenwoordig is het erg moeilijk om een zuiver blaasje te hebben voor een diepe karakterisering omdat er geen protocollen zijn om een hoge zuiverheid te ber...

Video: Een succesvolle sortering instellen voor extracellulaire isolatie van blaasjes

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Biochemistry

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 ...

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. ...

Bioengineering

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, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

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 ...

Characterizing Extracellular Vesicles from Biological Fluids

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from one cell to another. EVs have been shown to travel short and far distances in the body and are tissue-specific. EVs are not only found in tissues, but they can also be found in practically all bodily fluids, such as tears, saliva, cerebral spinal fluid, blood, etc. Even though EVs can be collected non-invasively from tears and saliva, only small volumes can be collected at a time, which can cause issues in obtaining enough EVs to analyze proteins. The scanner discussed in this paper is a nanoparticle analyzer that provides a solution to&#160;this problem, allowing us to characterize and study the phenotype, size, and total particle count of EVs from as little as 1 &#181;L of biological fluid. This protocol will expand the knowledge of EVs from small volumes of samples that are difficult to extract from patients. This could enhance patient comfort and potentially identify new therapeutic targets for a range of diseases and disorders.

Here, we demonstrate the characterization method for extracellular vesicles (EVs) collected from biological fluids, such as tears and saliva, of human subjects. The scanner used in this method is capable of detecting the phenotype, size, and total particle count of EVs from 1 &#181;L of the sample.

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from ...

Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation

Source: Hurwitz, 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 describes the technique of extracting extracellular vesicles from fresh or frozen tissues. The isolated extracellular vesicles can be used for further analysis to uncover their significant roles in disease pathogenesis.

Source: Hurwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video describes the technique of extracting ...

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vitro studies

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 ...

Setting a Successful Sorting for Extracellular Vesicle Isolation

Samenvatting

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