Name: labeling of extracellular vesicles for monitoring migration and uptake in cartilage explants
Uploaded: 2021-10-04T14:00:00
Description: Watch this Scientific Journal Video about Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants at JoVE.com

This research was funded by Instituto de Salud Carlos III, Ministerio de Econom&#237;a y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124); by the PROGRAMA JUNIOR del proyecto TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands; by the Direcci&#243; General d&#39;Investigaci&#243;, Conselleria d&#39;Investigaci&#243;, Govern Balear (FPI/2046/2017); by the FOLIUM postdoctoral program (FOLIUM 17/01) within the FUTURMed, financed at 50% by the sustainable tourism tax of the Balearic Islands and at 50% by the ESF; and by the Comissio de Docencia i Investigacio de la Fundacio Banc de Sang i Teixits de les Illes Balears (CDI21/03).

NOTE: Cartilage explants were obtained from the IdISBa Biobank (IB 1995/12 BIO) in compliance with institutional guidelines after ethical approval of the project by the CEI-IB (IB 3656118 PI).
1. Column preparation
<ol>
	<li>Equilibrate columns following the manufacturer&#39;s instructions or as follows:
	<ol>
		<li>Remove the column cap and equilibrate the column. Remove the storage buffer by elution.</li>
		<li>Wash the column 3 times with 2.5 mL of phosphate-buffered saline (PBS). During ...

<ol>
	<li>Sutton, 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=The+contribution+of+the+synovium,+synovial+derived+inflammatory+cytokines+and+neuropeptides+to+the+pathogenesis+of+osteoarthritis.">The contribution of the synovium, synovial derived inflammatory cytokines and neuropeptides to the pathogenesis of osteoarthritis.</a> The Veterinary Journal. 179 (1), 10-24 (2009).</li><li>Zyli&#324;ska, B., Silmanowicz, P., Sobczy&#324;ska-Rak, A., Jarosz, &. #. 3. 2. 1. ;., Szponder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+articular+cartilage+defects:+Focus+on+tissue+engineering.">Treatment of articular cartilage defects: Focus on tissue engineering.</a> In Vivo. 32 (6), 1289-1300 (2018).</li><li>Mobasheri, A., Kalamegam, G., Musumeci, G., Batt, M. 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E., 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=Neutrophil-derived+microvesicles+enter+cartilage+and+protect+the+joint+in+inflammatory+arthritis.">Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis.</a> Science Translational Medicine. 7 (315), 1-13 (2015).</li><li>Liu, X., 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=Exosomes+derived+from+platelet-rich+plasma+present+a+novel+potential+in+alleviating+knee+osteoarthritis+by+promoting+proliferation+and+inhibiting+apoptosis+of+chondrocyte+via+Wnt/&#946;-catenin+signaling+pathway.">Exosomes derived from platelet-rich plasma present a novel potential in alleviating knee osteoarthritis by promoting proliferation and inhibiting apoptosis of chondrocyte via Wnt/&#946;-catenin signaling pathway.</a> Journal of Orthopaedic Surgery and Research. 14 (1), 470 (2019).</li><li>Otahal, 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=Characterization+and+chondroprotective+effects+of+extracellular+vesicles+from+plasma-+and+serum-based+autologous+blood-derived+products+for+osteoarthritis+therapy.">Characterization and chondroprotective effects of extracellular vesicles from plasma- and serum-based autologous blood-derived products for osteoarthritis therapy.</a> Frontiers in Bioengineering and Biotechnology. 8 (1), 584050 (2020).</li><li>Penfornis, P., Vallabhaneni, K. C., Whitt, J., Pochampally, R. <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+as+carriers+of+microRNA,+proteins+and+lipids+in+tumor+microenvironment.">Extracellular vesicles as carriers of microRNA, proteins and lipids in tumor microenvironment.</a> International Journal of Cancer. 138 (1), 14-21 (2016).</li><li>Ortega, F. G., 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=Interfering+with+endolysosomal+trafficking+enhances+release+of+bioactive+exosomes.">Interfering with endolysosomal trafficking enhances release of bioactive exosomes.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 20, 102014 (2019).</li><li>de Miguel P&#233;rez, D., 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+vesicle-miRNAs+as+liquid+biopsy+biomarkers+for+disease+identification+and+prognosis+in+metastatic+colorectal+cancer+patients.">Extracellular vesicle-miRNAs as liquid biopsy biomarkers for disease identification and prognosis in metastatic colorectal cancer patients.</a> Scientific Reports. 10 (1), 1-13 (2020).</li><li>Morelli, A. E., 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=Endocytosis,+intracellular+sorting,+and+processing+of+exosomes+by+dendritic+cells.">Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells.</a> Blood. 104 (10), 3257-3266 (2004).</li><li>Feng, D., 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=Cellular+internalization+of+exosomes+occurs+through+phagocytosis.">Cellular internalization of exosomes occurs through phagocytosis.</a> Traffic. 11 (5), 675-687 (2010).</li><li>Chuo, S. T. Y., Chien, J. C. Y., Lai, C. P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+extracellular+vesicles:+Current+and+emerging+methods.">Imaging extracellular vesicles: Current and emerging methods.</a> Journal of Biomedical Science. 25, 91 (2018).</li><li>Rice, B. W., Cable, M. D., Nelson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+light-emitting+probes.">In vivo imaging of light-emitting probes.</a> Journal of Biomedical Optics. 6 (4), 432 (2001).</li><li>Lai, C. P., 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=Visualization+and+tracking+of+tumour+extracellular+vesicle+delivery+and+RNA+translation+using+multiplexed+reporters.">Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.</a> Nature Communications. 6, 7029 (2015).</li><li>Takahashi, 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=Visualization+and+in+vivo+tracking+of+the+exosomes+of+murine+melanoma+B16-BL6+cells+in+mice+after+intravenous+injection.">Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection.</a> Journal of Biotechnology. 165 (2), 77-84 (2013).</li><li>Askenasy, N., Farkas, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+PKH-labeled+hematopoietic+cells+in+recipient+bone+marrow+in+vivo.">Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo.</a> Stem Cells. 20 (6), 501-513 (2002).</li><li>Tamura, R., Uemoto, S., Tabata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppressive+effect+of+mesenchymal+stem+cell-derived+exosomes+on+a+concanavalin+A-induced+liver+injury+model.">Immunosuppressive effect of mesenchymal stem cell-derived exosomes on a concanavalin A-induced liver injury model.</a> Inflammation and Regeneration. 36, 26 (2016).</li><li>Deddens, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+extracellular+vesicles+contain+miRNAs+and+are+released+as+early+biomarkers+for+cardiac+injury.">Circulating extracellular vesicles contain miRNAs and are released as early biomarkers for cardiac injury.</a> Journal of Cardiovascular Translational Research. 9 (4), 291-301 (2016).</li><li>Skardelly, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+benefit+of+human+fetal+neuronal+progenitor+cell+transplantation+in+a+clinically+adapted+model+after+traumatic+brain+injury.">Long-term benefit of human fetal neuronal progenitor cell transplantation in a clinically adapted model after traumatic brain injury.</a> Journal of Neurotrauma. 28 (3), 401-414 (2011).</li><li>Protocol guide: Exosome labeling using PKH lipophilic membrane dyes. Sigma-Aldrich Available from: <a target="_blank" href="https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-culture/exosome-labeling-pkh.html">https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-culture/exosome-labeling-pkh.html</a> (2021)</li><li>Dehghani, M., Gulvin, S. M., Flax, J., Gaborski, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+PKH+labelling+on+extracellular+vesicle+size+by+nanoparticle+tracking+analysis.">Systematic evaluation of PKH labelling on extracellular vesicle size by nanoparticle tracking analysis.</a> Scientific Reports. 10 (1), 1-10 (2020).</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=Labeling+extracellular+vesicles+for+nanoscale+flow+cytometry.">Labeling extracellular vesicles for nanoscale flow cytometry.</a> Scientific Reports. 7 (1), 1-10 (2017).</li><li>Takov, K., Yellon, D. M., Davidson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confounding+factors+in+vesicle+uptake+studies+using+fluorescent+lipophilic+membrane+dyes.">Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes.</a> Journal of Extracellular Vesicles. 6 (1), 1388731 (2017).</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), 1-18 (2020).</li></ol>

EV imaging helps to understand EV properties, such as their release and uptake mechanisms. Their imaging allows the monitoring of their biodistribution and the characterization of their pharmacokinetic properties as drug vehicles. However, EV imaging and tracking may be difficult due to their small sizes, although many imaging devices and labeling techniques have been developed to help researchers monitor EVs under in vitro and in vivo conditions23,24...

Osteoarthritis (OA) is an articular degenerative disease that implies a progressive and irreversible inflammation and destruction of the extracellular matrix of the articular cartilage1. Although various forms of arthritis have numerous treatments2,3,4, these are restricted by their side effects and limited efficacy. Tissue engineering techniques using autologous chondrocyte implantation are routinely applied for the regenerative treatment of injured cartilage in early OA cartilage lesions4. Cell-based therapies are restricted mainly due to the limited number of phenotypically stable chondrocytes or chondroprogenitors capable of effectively repairing the cartilage3. Therefore, the development of new therapeutic strategies to prevent disease progression and regenerate large cartilage lesions is of paramount importance.
Extracellular vesicles (EVs) have been suggested as a treatment for OA by different authors5,6. EVs are membranous bodies secreted by the majority of cell types, are involved in intercellular signaling, and have been shown to exert stem cells&#39; therapeutic effects7,8,9, due to which they have recently elicited interest in regenerative medicine10. EVs derived from mesenchymal stromal cells (MSCs) are the main therapeutic EVs investigated for OA, although other joint-related cells have been used as EV sources, e.g., chondroprogenitors or immune cells11,12.
Platelet concentrates, such as platelet lysates (PLs), are used to enhance wound healing in different injuries, such as corneal ulcers13,14,15 or in tendon tissue regeneration16, because of the hypothesis that the EV component of platelet concentrates may be responsible for these effects17. Some studies related to joint-related diseases use platelet-derived EVs (PL-EVs) as a treatment to ameliorate osteoarthritic conditions. PL-EVs improve chondrocyte proliferation and cell migration by activating the Wnt/&#946;-catenin pathway18, promoting the expression of chondrogenic markers in osteoarthritic chondrocytes19, or showing higher levels of chondrogenic proteins and fewer tissular abnormalities in osteoarthritic rabbits treated with PL-EVs18.
EVs contain proteins, lipids, and nucleic acids that are liberated to the target cell, establishing cell-to-cell communication, which is the main feature related to their therapeutic applications20. The effects of EVs depend on their reaching cells and subsequent cargo release. This effect can be confirmed indirectly by changes caused in cells, such as metabolic activity or gene expression modification. However, these methods do not allow the visualization of how EVs reach cells to exert their function. Thus, this paper presents a method to label these PL-derived EVs to be used as a treatment for inflammation-driven OA cartilage explants. Confocal microscopy was used to monitor EV uptake and progression to the chondrocytes present in the explants in a time-lapse of 5 h.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Centrifuge tube</td>
			<td>SPL life sciences</td>
			<td>PLC60015</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe BD Plastipak</td>
			<td>BD</td>
			<td>303174</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol (Isopropanol)</td>
			<td>Panreac AppliChem</td>
			<td>1.310.901.211</td>
			<td>Prepared at 20% with Milli-Q water</td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>SPL life sciences</td>
			<td>PLC30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol Pharmpur</td>
			<td>Scharlab</td>
			<td>ET0006005P</td>
			<td>Used to prepare 96% and 75% ethanol with Milli-Q water</td>
		</tr>
		<tr>
			<td>Biopsy Punch with plunger 3 mm</td>
			<td>Scandidact</td>
			<td>MTP-33-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum&#160;Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td>Prepared at 5% with PBS</td>
		</tr>
		<tr>
			<td>Cartilage explants</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrating tube 15 mL Nanosep 100 kD Omega</td>
			<td>Pall</td>
			<td>MCP100C41</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrating tube 500 &#181;L Nanosep 100 kD Omega</td>
			<td>Pall</td>
			<td>OD003C33</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass 24 x 60 mm</td>
			<td>Deltalab</td>
			<td>D102460</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 -GlutaMAX medium</td>
			<td>Biowest</td>
			<td>L0092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (1x)</td>
			<td>Capricorn Scientific</td>
			<td>PBS-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedded paraffin tissue blocks</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>Exo-spin mini-HD columns</td>
			<td>Cell guidance systems</td>
			<td>EX05</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather S35 Microtome Blade</td>
			<td>Feather</td>
			<td>43037</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtropur S 0.2 &#181;m syringe filter</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>F-6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Oncostatin M Human</td>
			<td>Sigma-Aldrich</td>
			<td>O9635-10UG</td>
			<td>Prepare a stock solution to a final concentration of 0.1 &#181;g/&#181;L diluten in PBS-0.1% BSA</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>8.18715.1000</td>
			<td>Prepared at 4% with PBS and stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100x</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td>Sigma-Aldrich</td>
			<td>MINI26</td>
			<td>PKH26 and Dliuent C included</td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>Scharlab</td>
			<td>SO019911000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>R&#38;D systems</td>
			<td>210-TA-005</td>
			<td>Prepare a stock solution to a final concentration of 0.01 &#181;g/&#181;L diluted in PBS-0.1% BSA</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used to prepare a 0.1% Triton-0.1% sodium citrate solution with Milli-Q water</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Scharlab</td>
			<td>XI0050005P</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td>5428000210</td>
			<td>F-45-48-11 rotor</td>
		</tr>
		<tr>
			<td>NanoSight NS300</td>
			<td>Malvern</td>
			<td>NS300</td>
			<td>Device with embedded laser at &#955;= 532 nm and camera sCMOS</td>
		</tr>
		<tr>
			<td>Shandon Finesse 325 Manual Microtome</td>
			<td>Thermo Scientific&#8482;</td>
			<td>A78100101</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS-SPE confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>5200000271</td>
			<td></td>
		</tr>
	</tbody>
</table>

labeling of extracellular vesicles for monitoring migration and uptake in cartilage explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

A schematic overview for EV labeling and uptake monitoring is displayed in Figure 1. The particle concentration and EV size detected by NTA in Table 1 show that the EV concentration decreases during the process due to the purification step performed twice after labeling with the column. However, the amount obtained is in the optimal range of the number of particles to use for treatment. This particle concentration is used to calculate the volume of PKH-PL-EV and control that...

Watch this Scientific Journal Video about Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants at JoVE.com

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

This simple protocol allows one to label EVs and monitor them via confocal microscopy. For instance, EV imaging might help in evaluating EV distribution when use it in therapeutics. This technique allows one to monitor EV migration and uptake in cartilage explants easily, with confocal microscopy without any special function.The same protocol can be used for EVs for in vitro or in vivo experiments. Make sure that the columns are prepared beforehand, and that the initial volume of EVs is enough to reach the desired final particle concentration, considering that around 10%of the particles will be lost during the purification step. Begin by concentrating the extracellular vesicle, or EV, sample and the control.Place the samples in a 15 milliliter or 500 microliters concentrating tube, depending on the starting volume of the EV sample. Centrifuge the tubes according to the manufacturer's instructions until an almost dry sample is obtained. Re-suspend the concentrated EV samples with 200 microliters, and the control group with 100 microliters of diluent C, and transfer them to new 1.5-milliliter centrifuge tubes.Separate the EV sample into two aliquots of 100 microliters each. Mark one of the aliquots with dye and use it as treatment. Leave the other unmarked, but process it like the EV sample, and use it to quantify the EV concentration by nanoparticle tracking analysis.Prepare 2x dye solution such that the resulting concentration of PKH26 solution in diluent C is eight micromolar. Mix one microliter of one-millimolar PKH26 linker per 125 microliters of diluent C in the sample. Prepare the volume required to add to the samples in a 1:1 ratio.Add 2x dye solution to PKH platelet lysate-derived EVs and control samples in a 1:1 ratio, to achieve 1x dye concentration, and four micromolar PKH26 concentration. Add the same volume of PBS to the NTA platelet lysate-derived EV sample. Incubate for five minutes at room temperature.Add 5%bovine serum albumin-PBS solution to the samples in a 1:1 ratio, and ensure that the final volume is approximately 400 microliters. Proceed to separate the labeled EVs from the unbound dye and non-specific dye interactions with the column. Remove the cap from the column, add 400 microliters of PKH platelet lysate-derived EVs, NTA platelet lysate-derived EVs, or control and discard all eluted liquid.Wait for the sample to enter the column completely before proceeding to the next step. Add 600 microliters of PBS and discard all eluted liquid. Wait for the PBS to enter the column completely before proceeding to the next step.Add 600 microliters of PBS and collect a fraction of 600 microliters in a 1.5-milliliter centrifuge tube. For a new column, repeat the steps involving the concentration of the samples mentioned at the beginning of the protocol. Add 600 microliters of previously eluted EVs to the column and discard the eluted volume.Then add 400 microliters of PBS and discard all eluted volumes. Add 600 microliters of PBS to the column and collect a fraction of 600 microliters in a 1.5-milliliter centrifuge tube. Wash the cartilage two times with PBS and excise it using a three-millimeter diameter biopsy punch under sterile conditions.Place the explants in 96-well culture plates with DMEM-F12 medium, supplemented with 1%penicillin streptomycin at 37 degrees Celsius, 5%CO2, and 80%humidity. To establish an inflammation-driven OA model, supplement the cell culture medium with 10 nanograms per milliliter of oncostatin M and two nanograms per milliliter of tumor necrosis factor-alpha. Treat the explants with 1 times 10 to the 9th particles per well of PKH platelet lysate-derived EVs, or control, in cell culture medium supplemented with oncostatin M and tumor necrosis factor-alpha.Remove the medium from the 96-well cell culture plates containing cartilage explants. Add 200 microliters of the cell culture medium to each well as described in the manuscript. Stop the in vitro assay every hour from zero to five hours.Wash the cell culture wells containing the cartilage explants two times with 200 microliters of PBS. Add 100 microliters of 4%paraformaldehyde to the tissue to fix it for three hours at four degrees Celsius. Remove the PFA, add 100 microliters of PBS, store the fixed tissue at four degrees Celsius, and process the samples within 48 hours.Images taken at different time points show how EVs enter the tissue until they reach the chondrocytes, and enter the chondrocytes over time. The labeled EVs were already localized around chondrocytes after one hour of incubation. Ensure that the initial volume of EVs is enough to reach the desired final particle concentration.Another important thing to remember is to use the labeled EVs within the next 24 hours. If not use it within 24 hours, a decrease in fluorescence may occur. This technique will help researchers to improve EV imaging, and to study EVs in biology and therapeutics.

Research

JoVE Journal

Bioengineering

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Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

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