This work was supported by the National Science Centre, Poland, grant number 2019/33/B/NZ5/00647. We would like to thank Professor Tomasz Gosiewski and Agnieszka Krawczyk from the Department of Molecular Medical Microbiology, Jagiellonian University Medical College for their invaluable help in the detection of bacterial DNA in EVs.

1. Preparation of ultracentrifuge tubes
<ol>
	<li>Use sterile, single-use tubes. If this is not possible, reuse the ultracentrifuge tubes after washing them with a detergent using a sterile Pasteur pipette or other single-use applicators. Remember that ultracentrifuge tubes should be dedicated to one type of centrifuged material (cell culture supernatant/serum/plasma) and species (human/mouse/etc.).</li>
	<li>After a detergent wash, rinse the ultracentrifuge tubes with deionized, LPS-free water 3x. 
	NOTE: Do not use low-quality water.</li>
	<li>Dry the ultracentrifuge tubes, and then fill them up with 70% ethanol. Leave the tubes with 70% ethanol for overnight disinfection. Remove the ethanol and dry the tubes again.</li>
	<li>Place the tubes and caps in sterilization packaging and close them tightly. Use the plasma or gas (ethylene oxide) sterilization method18,19. Store the ultracentrifuge tubes enclosed in the sterilization packaging in a dry place, and use them before the expiration date. 
	&#8203;NOTE: Perform monthly monitoring of the LPS level in phosphate buffered saline (PBS) and the water used for EVs isolation. Monitoring should include the tubes as well (e.g., by testing the water [wash control] which has been stored in the ultracentrifuge tubes overnight at room temperature [RT]).</li>
</ol>
2. Preparation of EV-depleted low-endotoxin fetal bovine serum (EE-FBS)
<ol>
	<li>Ensure to use ultra-low endotoxin FBS (commercially available; see Table of Materials; &#60;0.1 EU/mL).</li>
	<li>Place a bottle of ultra-low endotoxin FBS in a water bath and incubate at 56 &#176;C for 30 min. This step is required to inactivate the complement system.</li>
	<li>Pipette the inactivated FBS to ultracentrifuge tubes and centrifuge it at 100,000 x g for 4 h at 4 &#176;C. Collect the supernatant into sterile 50 mL tubes, ensuring not to exceed 45 mL per tube to avoid cap contamination and wetting the ring of the tube.</li>
	<li>Store the EE-FBS serum prepared in this manner at -20 &#176;C. Check the concentration of LPS in EE-FBS (step 6). The LPS concentration should be at the same level as before ultracentrifugation.</li>
</ol>
3. Cell culture
<ol>
	<li>For this study, culture SW480 and SW620 cell lines in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 4.5 g/L glucose, L- glutamine, sodium pyruvate, and gentamicin (50 &#181;L/mL) supplemented with 10% EE-FBS accordingly in 75 cm2 culture flasks using aseptic technique. Seed nearly 4.5 x 106 cells&#160;and 6.5 x 106 cells per flask for SW480 and SW620, respectively.</li>
	<li>Collect the supernatants twice a week (~10 mL per flask) when the cells reach full confluency (~13.5 x 106 and 20 x 106 cells per flask for SW480 and SW620, respectively), and split. Ensure that the viability of cells is not less than 99% and that the cells are cultured at 37 &#176;C in a 5% CO2 atmosphere.</li>
	<li>Collect supernatants from the cell culture into appropriately labeled 15 mL tubes. Centrifuge the collected supernatants at 500 x g for 5 min at RT to remove cell debris.</li>
	<li>Collect the supernatants carefully, avoid aspirating debris, place in labeled tubes, and centrifuge again at 3,200 x g for 12 min at 4 &#176;C.</li>
	<li>Collect supernatants from the second centrifugation step into sterile, labeled 50 mL tubes. Avoid wetting the edges of tubes. Ensure that the volume of supernatant does not exceed 45 mL and that the tubes are kept vertically.</li>
	<li>Wrap the tube&#39;s cap with a sterile transparent film and store supernatants in the vertical position at -80 &#176;C. 
	&#8203;NOTE: Perform monthly control of Mycoplasma spp. and LPS contamination in fresh culture supernatants (step 6). If the level of endotoxin in the supernatants exceeds 0.05 EU/mL, verify at which stage contamination might have occurred and restart the process from the beginning. If contamination of the cell culture by Mycoplasma spp. is detected, isolation of the EVs from such supernatants should be discontinued.</li>
</ol>
4. Isolation of EVs from cell culture supernatants
<ol>
	<li>Prepare 0.22 &#181;m syringe filters and syringes of proper volume. Prepare two tubes with culture supernatants (90 mL).</li>
	<li>Fill the syringe with the supernatant and attach the filter to the needle adapter. Place the filled syringe with the filter over a 50 mL tube. Push on the plunger flange and collect ~90 mL of filtrate.</li>
	<li>Pipette the filtrate (7 mL) to the ultracentrifuge tubes (ensuring that the same volume is pipetted to each tube for proper balance) and centrifuge at 100,000 x g for 2 h at 4 &#176;C. 
	NOTE: The efficiency of EVs pelleting depends on many factors (e.g., rotor type, its k-factor, migration path length, the viscosity of the medium, etc.), which need to be optimized for better recovery of EVs20.</li>
	<li>Discard the supernatant using a sterile Pasteur pipette. Collect the remaining pellets using long, filtered pipette tips and pool them into two ultracentrifuge tubes. Fill them up to 7 mL with filtered endotoxin-free PBS to rinse the EVs.</li>
	<li>Centrifuge the PBS-resuspended pellets at 100,000 x g for 2 h at 4 &#176;C. Completely remove the supernatant using a sterile Pasteur pipette, and add 200 &#181;L of filtered, endotoxin-free PBS to both tubes to resuspend the EVs. Gently pipette the EVs pellet to collect all of the EVs.</li>
	<li>Transfer the EVs suspension into a sterile 1.5 mL test tube (if possible, use a low protein binding tube). Retain 10 &#181;L of the EV suspension for preparing a dilution for nanoparticle tracking analysis (NTA) and for other purposes (e.g., protein concentration measurement).</li>
	<li>Dilute the EVs with filtered PBS (1:1,000) for measurements by NTA, according to the manufacturer&#39;s instructions. Secure the EV tubes by wrapping the cap with a sterile transparent film. Store the EVs at -80 &#176;C.</li>
</ol>
5. Specific markers detection by western blotting
<ol>
	<li>Determine the protein level in the samples (e.g., by Bradford assay), and prepare the samples (20 &#181;g) with loading buffer. Prepare the loading buffer by mixing 5 &#181;L of sample buffer (4x) and 2 &#181;L of sample reducing agent (10x) to a total volume of 20 &#181;L. Incubate the samples at 70 &#176;C for 10 min.</li>
	<li>Prepare polyacrylamide gels with SDS (10%-14%) and load the 20 &#181;g of EVs to each well.</li>
	<li>Run the electrophoresis with running buffer (30 g of Tris, 144 g of glycine, 10% SDS per 1 L) for 45 min at 150 V.</li>
	<li>Perform semi-dry transfer of proteins from the gel onto the polyvinylidene difluoride (PVDF) membrane in a transfer machine with Towbin buffer (1.51 g of Tris, 7.2 g of glycine, 10% methanol per 0.5 L) at 25 V for 1 h.</li>
	<li>Place the membrane in TBST buffer (1 mL of Tween, 100 mL of Tris-buffered saline (TBS 10x) per 1 L) for blocking with 1% bovine serum albumin (BSA) in TBST buffer, and incubate for 1 h on the rocker at RT.</li>
	<li>Add diluted (1:1,000) antibodies, anti-CD91 (clone#D8O1A) or anti-Alix1 (clone#3A9), in 1% BSA and incubate with the membrane overnight at 4&#160;&#176;C on the rocker.&#160;Remove antibodies and wash membrane 3x with 10 mL of 1x TBST for 10 minutes.</li>
	<li>Add goat anti-rabbit or goat anti-mouse (dilution: 1:2,000) secondary antibody conjugated with horseradish peroxidase, depending on the primary antibody on the membrane, and incubate for 1 h on the rocker at RT.&#160;Remove antibodies and wash membrane 3x with 10 mL of 1x TBST for 10 minutes.</li>
	<li>Mix the substrate and luminol at a 1:1 ratio to obtain a 1 mL solution. Pour the solution on the membrane. Place the membrane immediately in the measuring chamber of the imaging system, choose the chemiluminescence module, and visualize protein bands on the screen.</li>
</ol>
6. Measurement of endotoxin level by Limulus Amebocyte Lysate test (LAL)
<ol>
	<li>Perform chromogenic LAL measurement of the endotoxin level, according to the manufacturer&#39;s recommendation. Briefly, use the method based on the interaction of the endotoxin with the limulus amebocyte lysate (LAL). The detection limit of this method is 0.005 EU/mL. 
	NOTE: LAL assay, despite its limitations, currently remains the standard method for detecting and quantifying endotoxin contamination in different kinds of solutions and other products used in science or medicine8. The limiting factor of this kit is the stability of the reconstituted amebocyte lysate solution, which is stable for only 1 week at -20 &#176;C if frozen immediately after reconstitution.</li>
	<li>Use a tenfold dilution of EVs and other samples and a 50-fold dilution of serum. For further experiments, use only low-endotoxin reagents such as EE-FBS, DMEM, PBS, RPMI (&#60;0.005 EU/mL), and LPS-free culture supernatants.</li>
</ol>
7. Detection of prokaryotic 16S rRNA gene in EV samples
<ol>
	<li>Isolate DNA from the EV samples. Try to keep the reagents and the site of isolation aseptic. Measure the DNA concentration and quality (e.g., using a spectrophotometer).</li>
	<li>Perform polymerase chain reaction (PCR), as described previously21. Prepare 1.5% agarose gel with ethidium bromide or SYBR green to visualize the PCR products in ultraviolet light (UV).</li>
	<li>Run electrophoresis of the sample and weight marker with TRIS-acetate-EDTA buffer at 75 V for 45 min. Visualize DNA bands using the imaging system.</li>
</ol>
8. Determination of effective LPS concentration for stimulation in human monocyte model
<ol>
	<li>Prepare 2 x 106 monocytes/mL of monocyte suspension in RPMI 1640 supplemented with L-glutamine, glucose, and 2% EE-FBS. Add 50 &#181;L of the suspension per well of a 96-well tissue culture plate22.</li>
	<li>Add a proper volume of LPS or culture medium to obtain the following final concentrations: 0 pg/mL (control), 10 pg/mL, 50 pg/mL, 100 pg/mL, 1 ng/mL, and 100 ng/mL, in 100 &#181;L total volume of cell suspension. Make triplicates of each LPS concentration.</li>
	<li>Culture the cells for 18 h at 37 &#176;C, 5% CO2. Collect the supernatant.</li>
	<li>Spin down the supernatant at 2,000 x g for 5 min. Transfer the supernatants into new tubes. Measure TNF8,23 and IL-1023 concentration in collected supernatants with the cytometric beads array (CBA) human cytokine kit, according to the manufacturer&#39;s procedure, with a flow cytometer.</li>
</ol>

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

In the last few years, methods for proper EVs isolation have become increasingly important, enabling their further reliable analyses, for example, in the context of obtaining reliable omics and functional data24. Based on previous research experience, it seems that not only the type of isolation method, but also other conditions during this procedure may be important. The use of EV-depleted FBS is widely recognized as a necessity25,26; how...

Extracellular vesicles (EVs), according to the MISEV 2018 nomenclature, are a collective term describing various subtypes of cell-secreted membranous vesicles that play crucial roles in numerous physiological and pathological processes1,2. Moreover, EVs show promise as novel biomarkers for various diseases, as well as therapeutic agents and drug delivery vehicles. However, realizing their full potential is difficult as there are still many technical obstacles related to their acquisition3. One such challenge is the isolation of endotoxin-free EVs, which has been neglected in many cases. One of the most common endotoxins is lipopolysaccharide (LPS), which is a major component of gram-negative bacterial cell walls and can cause an acute inflammatory response, owing to the release of a large number of inflammatory cytokines by various cells4,5. LPS induces a response by binding to LPS binding protein, followed by interaction with the CD14/TLR4/MD2 complex on myeloid cells. This interaction leads to the activation of MyD88- and TRIF-dependent signaling pathways, which in turn triggers the nuclear factor kappa B (NFkB). Translocation of NFkB to the nucleus initiates the production of cytokines6. Monocytes and macrophages are highly sensitive to LPS, and their exposure to LPS results in a release of inflammatory cytokines and chemokines (e.g., IL-6, IL-12, CXCL8, and TNF-&#945;)7,8. The CD14 structure enables the binding of different LPS species with similar affinity and serves as a co-receptor for other toll-like receptors (TLRs) (TLR1, 2, 3, 4, 6, 7, and 9)6. The number of studies being conducted on the effects of EVs on monocytes/macrophages is still increasing9,10,11. Especially from the perspective of studying the functions of monocytes, their subpopulations, and other immune cells, the presence of endotoxin and even their masked presence in EVs is of great importance12. The overlooked contamination of EVs with endotoxins may lead to misleading conclusions and hide their true biological activity. In other words, working with monocytic cells requires confidence in the absence of endotoxin contamination13. Potential sources of endotoxins can be water, commercially obtained media and sera, media components and additives, laboratory glassware, and plasticware5,14,15.
Therefore, this study aimed to develop a protocol for the isolation of low endotoxin-containing EVs. The protocol provides simple hints on how to avoid endotoxin contamination during EVs isolation instead of removing endotoxins from EVs. Previously, many protocols have been presented on how to remove endotoxins from, for example, engineered nanoparticles used in nanomedicine; however, none of them are useful for biological structures such as EVs. The effective depyrogenation of nanoparticles can be carried out by ethanol or acetic acid rinsing, heating at 175 &#176;C for 3 h, &#947; irradiation, or triton X-100 treatment; however, these procedures lead to the destruction of EVs16,17.
The presented protocol is a pioneering study focused on avoiding endotoxin impurities in EVs, unlike previous studies on the effect of EVs on monocytes9. Applying proposed principles to laboratory practice may help to obtain reliable research results, which can be crucial when considering the potential use of EVs as therapeutic agents in the clinic12.

<table><tbody><tr><td>&amp;nbsp;Alix (3A9) Mouse mAb&amp;nbsp;</td><td>Cell Signaling Technology</td><td>2171</td><td></td></tr><tr><td>1250ul Filter Universal Pipette Tips, Clear, Polypropylene, Non-Pyrogenic</td><td>GoogLab Scientific</td><td>GBFT1250-R-NS</td><td></td></tr><tr><td>BD FACSCanto II Flow Cytometr</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>CBA Human Th1/Th2 Cytokine Kit II</td><td>BD Biosciences</td><td>551809</td><td></td></tr><tr><td>CD9 (D8O1A) Rabbit mAb</td><td>Cell Signaling Technology</td><td>13174</td><td></td></tr><tr><td>ChemiDoc Imaging System</td><td>Bio-Rad Laboratories, Inc.&amp;nbsp;</td><td>17001401</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium)&amp;nbsp;</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>ELX800NB, Universal Microplate Reader</td><td>BIO-TEK INSTRUMENTS, INC</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000044</td><td></td></tr><tr><td>Fetal Bovine Serum South America Ultra Low Endotoxin</td><td>&amp;nbsp;Biowest</td><td>&amp;nbsp;S1860-500</td><td></td></tr><tr><td>Gentamicin, 50 mg/mL</td><td>&amp;nbsp;PAN &amp;ndash; Biotech</td><td>P06-13100</td><td></td></tr><tr><td>Goat anti-Mouse IgG- HRP</td><td>Santa Cruz Biotechnology</td><td>sc-2004</td><td></td></tr><tr><td>Goat anti-Rabbit IgG- HRP</td><td>Santa Cruz Biotechnology</td><td>sc-2005</td><td></td></tr><tr><td>Immun-Blot PVDF Membrane</td><td>Bio-Rad Laboratories, Inc.&amp;nbsp;</td><td>1620177</td><td></td></tr><tr><td>LPS from Salmonella abortus equi S-form (TLRGRADE)</td><td>&amp;nbsp;Enzo Life Sciences, Inc.</td><td>ALX-581-009-L002</td><td></td></tr><tr><td>Mini Trans-Blot Electrophoretic Transfer Cell</td><td>Bio-Rad Laboratories, Inc.&amp;nbsp;</td><td>1703930</td><td></td></tr><tr><td>Nanoparticle Tracking Analysis&amp;nbsp;</td><td>Malvern Instruments Ltd</td><td></td><td></td></tr><tr><td>NuPAGE LDS Sample Buffer (4X)</td><td>&amp;nbsp;Invitrogen</td><td>&amp;nbsp;NP0007</td><td></td></tr><tr><td>NuPAGE Sample Reducing Agent (10x)</td><td>&amp;nbsp;Invitrogen</td><td>NP0004</td><td></td></tr><tr><td>Parafilm</td><td>Sigma Aldrich</td><td>P7793</td><td>transparent film</td></tr><tr><td>Perfect 100-1000 bp DNA Ladder</td><td>EURx</td><td>E3141-01&amp;nbsp;</td><td></td></tr><tr><td>PierceTM Chromogenic Endotoxin Quant Kit</td><td>Thermo Scientific</td><td>A39552</td><td></td></tr><tr><td>PP Oak Ridge Tube with sealing caps</td><td>Thermo Scientific</td><td>3929, 03613</td><td></td></tr><tr><td>RPMI 1640</td><td>RPMI-1640 (Gibco)</td><td>11875093</td><td></td></tr><tr><td>SimpliAmp Thermal Cycler</td><td>Applied Biosystem</td><td>A24811</td><td></td></tr><tr><td>Sorvall wX+ ULTRA SERIES Centrifuge with T-1270 rotor</td><td>Thermo Scientific</td><td>75000100</td><td></td></tr><tr><td>Sub-Cell GT Horizontal Electrophoresis System</td><td>Bio-Rad Laboratories, Inc.&amp;nbsp;</td><td>1704401</td><td></td></tr><tr><td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34577</td><td></td></tr><tr><td>SW480 cell line</td><td>American Type Culture Collection(ATCC)</td><td></td><td></td></tr><tr><td>SW480 cell line</td><td>American Type Culture Collection (ATCC)</td><td></td><td></td></tr><tr><td>Syringe filter 0.22 um</td><td>TPP</td><td>99722</td><td></td></tr><tr><td>Trans-Blot SD Semi-Dry Transfer Cell</td><td>Bio-Rad Laboratories, Inc.&amp;nbsp;</td><td>1703940</td><td>Transfer machine</td></tr><tr><td>Transfer pipette, 3.5 mL</td><td>SARSTEDT</td><td>86.1171.001</td><td></td></tr></tbody></table>

isolation of low endotoxin content extracellular vesicles derived from cancer cell lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

A prerequisite or obligatory step for this protocol is the exclusion of possible endotoxin contamination from reagents. All the reagents being used, such as FBS, DMEM, RPMI, PBS, and even ultracentrifuge tubes, must be endotoxin-free (&#60;0.005 EU/mL). Maintaining the regime of no endotoxin contamination is not easy as, for example, the regular/standard serum for cell culture can be its rich source (0.364 EU/mL; see Table 1).
Although this protocol was developed to isolate EV...

Watch this Scientific Journal Video about Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines at JoVE.com

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

isolation-low-endotoxin-content-extracellular-vesicles-derived-from

Research

JoVE Journal

Immunology and Infection

1.0K Views.  Jagiellonian University Medical College. Dit protocol biedt richtlijnen voor het voorkomen van besmetting met endotoxine tijdens de isolatie van extracellulaire blaasjes uit celkweeksupernatanten en hoe deze op de juiste manier kunnen worden geëvalueerd. Het belangrijkste voordeel van dit protocol is dat elk laboratorium dat zich bezighoudt met EV's endotoxinebesmetting kan beperken door een aseptische procedure te volgen en eenvoudige en algemeen beschikbare apparatuur te gebruiken. Iedereen die deze techniek voor de eerste keer p...

Video: Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Cancer Research

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Biochemistry

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

Biomanufacturing and Characterization of NK-Extracellular Vesicles

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.

Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release.

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic ...

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 ...

Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. In contrast, traditional extraction methods such as ultracentrifugation and size-exclusion chromatography are limited by their time intensity, cost, and scalability. To overcome these limitations, we propose a method integrating a hemodialyzer and ultracentrifugation. This approach utilizes a hemodialysis device with a 100 kDa molecular weight cut-off (MWCO) membrane, which selectively concentrates sEVs while filtering out a plethora of proteins, thereby enhancing the yield and purity of sEVs. This initial purification step is followed by ultracentrifugation to further refine the sEV preparation. The integration of these two technologies not only significantly reduced the time spent on the entire process but also ensured compliance with good manufacturing practice (GMP) standards. The method here demonstrates high efficiency in isolating sEVs from a large volume of samples, offering a significant advancement over traditional methods. This protocol holds promise for accelerating the translation of EV-based therapies into clinical practice by providing a scalable, cost-effective, and GMP-compliant solution.

Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. This study provides a protocol combining hemodialysis with ultracentrifugation, significantly reducing the time spent on the entire process and ensuring compliance with good manufacturing practice (GMP) standards.

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal ...

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 and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Biology

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.

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.

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

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

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