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 ...

Research

JoVE Journal

Bioengineering

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