Name: Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines (Video) | JoVE Journal
Uploaded: 2023-02-17T13:30:00.000+00:00
Duration: 6 min 28 s
Description: Learn to isolate low-endotoxin extracellular vesicles from cancer cell lines using ultracentrifugation and aseptic techniques. This protocol ensures reliable EV research with minimal contamination for accurate biological studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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>

<ol><li>Théry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimal+information+for+studies+of+extracellular+vesicles+2018+%28MISEV2018%29%3A+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">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>Yáñez-Mó, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+properties+of+extracellular+vesicles+and+their+physiological+functions%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">Biological properties of extracellular vesicles and their physiological functions.</a> Journal of Extracellular Vesicles. 4, 27066(2015).</li>
<li>van Niel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+and+directions+in+studying+cell-cell+communication+by+extracellular+vesicles%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Challenges and directions in studying cell-cell communication by extracellular vesicles.</a> Nature Reviews Molecular Cell Biology. 23 (5), 369-382 (2022).</li>
<li>Ngkelo, A., Meja, K., Yeadon, M., Adcock, I., Kirkham, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LPS+induced+inflammatory+responses+in+human+peripheral+blood+mononuclear+cells+is+mediated+through+NOX4+and+Gi%CE%B1+dependent+PI-3+kinase+signalling%5BTitle%5D)+AND+%22Journal+of+Inflammation%22%5BJournal%5D)">LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Giα dependent PI-3 kinase signalling.</a> Journal of Inflammation. 9 (1), (2012).</li>
<li>Schwarz, H., Schmittner, M., Duschl, A., Horejs-Hoeck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Residual+endotoxin+contaminations+in+recombinant+proteins+are+sufficient+to+activate+human+CD1c%2B+dendritic+cells%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.</a> PLOS ONE. 9 (12), 113840(2014).</li>
<li>Zanoni, I., Granucci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+CD14+in+host+protection+against+infections+and+in+metabolism+regulation%5BTitle%5D)+AND+%22Frontiers+in+Cellular+and+Infection+Microbiology%22%5BJournal%5D)">Role of CD14 in host protection against infections and in metabolism regulation.</a> Frontiers in Cellular and Infection Microbiology. 3, 32(2013).</li>
<li>Takashiba, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+monocytes+to+macrophages+primes+cells+for+lipopolysaccharide+stimulation+via+accumulation+of+cytoplasmic+nuclear+factor+kB%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kB.</a> Infection and Immunity. 67 (11), 5573-5578 (1999).</li>
<li>Schwarz, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+activity+of+masked+endotoxin%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Biological activity of masked endotoxin.</a> Scientific Reports. 7, 44750(2017).</li>
<li>Danesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Granulocyte-derived+extracellular+vesicles+activate+monocytes+and+are+associated+with+mortality+in+intensive+care+unit+patients%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Granulocyte-derived extracellular vesicles activate monocytes and are associated with mortality in intensive care unit patients.</a> Frontiers in Immunology. 9, 956(2018).</li>
<li>Barry, O. P., Pratico, D., Savani, R. C., FitzGerald, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+monocyte-endothelial+cell+interactions+by+platelet+microparticles%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">Modulation of monocyte-endothelial cell interactions by platelet microparticles.</a> The Journal of Clinical Investigation. 102 (1), 136-144 (1998).</li>
<li>Sadallah, S., Eken, C., Martin, P. J., Schifferli, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microparticles+%28ectosomes%29+shed+by+stored+human+platelets+downregulate+macrophages+and+modify+the+development+of+dendritic+cells%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells.</a> Journal of Immunology. 186 (11), 6543-6552 (2011).</li>
<li>Soekmadji, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+future+of+Extracellular+Vesicles+as+Theranostics+-+an+ISEV+meeting+report%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">The future of Extracellular Vesicles as Theranostics - an ISEV meeting report.</a> Journal of Extracellular Vesicles. 9 (1), 1809766(2020).</li>
<li>Gioannini, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+an+endotoxin-MD-2+complex+that+produces+Toll-like+receptor+4-dependent+cell+activation+at+picomolar+concentrations%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations.</a> Proceedings of the National Academy of Sciences. 101 (12), 4186-4191 (2004).</li>
<li>Roslansky, P. F., Dawson, M. E., Novitsky, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plastics%2C+endotoxins%2C+and+the+Limulus+amebocyte+lysate+test%5BTitle%5D)+AND+%22Journal+of+Parenteral+Science+and+Technology%22%5BJournal%5D)">Plastics, endotoxins, and the Limulus amebocyte lysate test.</a> Journal of Parenteral Science and Technology. 45 (2), 83-87 (1991).</li>
<li>Fishel, S., Jackson, P., Webster, J., Faratian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endotoxins+in+culture+medium+for+human+in+vitro+fertilization%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Endotoxins in culture medium for human in vitro fertilization.</a> Fertility and Sterility. 49 (1), 108-111 (1988).</li>
<li>Schulz, E., Karagianni, A., Koch, M., Fuhrmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hot+EVs+-+How+temperature+affects+extracellular+vesicles%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutics+and+Biopharmaceutics%22%5BJournal%5D)">Hot EVs - How temperature affects extracellular vesicles.</a> European Journal of Pharmaceutics and Biopharmaceutics. 5 (146), 55-63 (2020).</li>
<li>Osteikoetxea, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+detergent+sensitivity+of+extracellular+vesicle+subpopulations%5BTitle%5D)+AND+%22Organic+%26+Biomolecular+Chemistry%22%5BJournal%5D)">Differential detergent sensitivity of extracellular vesicle subpopulations.</a> Organic & Biomolecular Chemistry. 13 (38), 9775-9782 (2015).</li>
<li>Sakudo, A., Yagyu, Y., Onodera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disinfection+and+sterilization+using+plasma+technology%3A+fundamentals+and+future+perspectives+for+biological+applications%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Disinfection and sterilization using plasma technology: fundamentals and future perspectives for biological applications.</a> International Journal of Molecular Sciences. 20 (20), 5216(2019).</li>
<li>Shintani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethylene+oxide+gas+sterilization+of+medical+devices%5BTitle%5D)+AND+%22Biocontrol+Science%22%5BJournal%5D)">Ethylene oxide gas sterilization of medical devices.</a> Biocontrol Science. 22 (1), 1-16 (2017).</li>
<li>Cvjetkovic, A., Lötvall, J., Lässer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+rotor+type+and+centrifugation+time+on+the+yield+and+purity+of+extracellular+vesicles%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles.</a> Journal of Extracellular Vesicles. 3, (2014).</li>
<li>Salamon, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+iSeq+and+MiSeq+as+the+two+platforms+for+16S+rRNA+sequencing+in+the+study+of+the+gut+of+rat+microbiome%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">Comparison of iSeq and MiSeq as the two platforms for 16S rRNA sequencing in the study of the gut of rat microbiome.</a> Applied Microbiology and Biotechnology. 106 (22), 7671-7681 (2022).</li>
<li>Baj-Krzyworzeka, M., Szatanek, R., Weglarczyk, K., Baran, J., Zembala, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumour-derived+microvesicles+modulate+biological+activity+of+human+monocytes%5BTitle%5D)+AND+%22Immunology+Letters%22%5BJournal%5D)">Tumour-derived microvesicles modulate biological activity of human monocytes.</a> Immunology Letters. 113 (2), 76-82 (2007).</li>
<li>Chaiwut, R., Kasinrerk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Very+low+concentration+of+lipopolysaccharide+can+induce+the+production+of+various+cytokines+and+chemokines+in+human+primary+monocytes%5BTitle%5D)+AND+%22BMC+Research+Notes%22%5BJournal%5D)">Very low concentration of lipopolysaccharide can induce the production of various cytokines and chemokines in human primary monocytes.</a> BMC Research Notes. 15 (1), 42(2022).</li>
<li>Van Deun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+disparate+isolation+methods+for+extracellular+vesicles+on+downstream+RNA+profiling%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.</a> Journal of Extracellular Vesicles. 3, 24858(2014).</li>
<li>Lehrich, B. M., Liang, Y., Fiandaca, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foetal+bovine+serum+influence+on+in+vitro+extracellular+vesicle+analyses%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">Foetal bovine serum influence on in vitro extracellular vesicle analyses.</a> Journal of Extracellular Vesicles. 10 (3), 12061(2021).</li>
<li>Pham, C. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bovine+extracellular+vesicles+contaminate+human+extracellular+vesicles+produced+in+cell+culture+conditioned+medium+when+%27exosome-depleted+serum%27+is+utilised%5BTitle%5D)+AND+%22Archives+of+Biochemistry+and+Biophysics%22%5BJournal%5D)">Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when 'exosome-depleted serum' is utilised.</a> Archives of Biochemistry and Biophysics. 708, 108963(2021).</li>
<li>Li, Y., Boraschi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endotoxin+contamination%3A+a+key+element+in+the+interpretation+of+nanosafety+studies%5BTitle%5D)+AND+%22Nanomedicine%22%5BJournal%5D)">Endotoxin contamination: a key element in the interpretation of nanosafety studies.</a> Nanomedicine. 11 (3), 269-287 (2016).</li>
<li>Álvarez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+system+dynamics+model+to+predict+the+human+monocyte+response+to+endotoxins%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">A system dynamics model to predict the human monocyte response to endotoxins.</a> Frontiers in Immunology. 8, 915(2017).</li>
<li>Busatto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tangential+flow+filtration+for+highly+efficient+concentration+of+extracellular+vesicles+from+large+volumes+of+fluid%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid.</a> Cells. 7 (12), 273(2018).</li>
<li>Gałuszka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transition+metal+containing+particulate+matter+promotes+Th1+and+Th17+inflammatory+response+by+monocyte+activation+in+organic+and+inorganic+compounds+dependent+manner%5BTitle%5D)+AND+%22International+Journal+of+Environmental+Research+and+Public+Health%22%5BJournal%5D)">Transition metal containing particulate matter promotes Th1 and Th17 inflammatory response by monocyte activation in organic and inorganic compounds dependent manner.</a> International Journal of Environmental Research and Public Health. 17 (4), 1227(2020).</li>
<li>Falagas, M. E., Kasiakou, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxicity+of+polymyxins%3A+a+systematic+review+of+the+evidence+from+old+and+recent+studies%5BTitle%5D)+AND+%22Critical+Care%22%5BJournal%5D)">Toxicity of polymyxins: a systematic review of the evidence from old and recent studies.</a> Critical Care. 10 (1), 27(2006).</li>
<li>Petsch, D., Anspach, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endotoxin+removal+from+protein+solutions%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">Endotoxin removal from protein solutions.</a> Journal of Biotechnology. 76 (2-3), 97-119 (2000).</li>
</ol>

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

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 (&lt;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 EVs...

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

Source: Watson, D. C., et.al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation and concentration of extracellular vesicles (EVs) derived from the outer membrane of genetically engineered bacteria. The process involves culturing the bacteria to release EVs, followed by sequential centrifugation and filtration steps to remove cells and debris. Finally, ultrafiltration is used to...

Isolation and Enrichment of Outer Membrane-Derived Extracellular Vesicles from Bacteria

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.

Isolation of Tissue Extracellular Vesicles from the Liver

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.

Extraction of Extracellular Vesicles from Whole Tissue

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

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.

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

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

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

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

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

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

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

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.

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

This protocol is designed to expedite and allow for the effective isolation of extracellular vesicles from human breast milk with high purity.

Rapid Isolation of Human Breast Milk-Derived Extracellular Vesicles

Learn to isolate low-endotoxin extracellular vesicles from cancer cell lines using ultracentrifugation and aseptic techniques. This protocol ensures reliable EV research with minimal contamination for accurate biological studies.

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

Explore More Videos