Name: labeling of extracellular vesicles for monitoring migration and uptake in cartilage explants
Uploaded: 2021-10-04T14:00:00.000+00:00
Duration: 7 min 58 s
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 each wash, wait for the column to absorb the whole volume. 
		&#8203;NOTE: Do not let the column dry.</li>
		<li>Cover the column with the cap after the last wash and until sample preparation.</li>
	</ol>
	</li>
</ol>
2. EV labeling
NOTE: This EV labeling protocol uses a PL-EV sample previously isolated by size exclusion chromatography (SEC) with previously described conditions21,22. However, any EV sample from any source may be used with this protocol.
<ol>
	<li>Concentrate the EV sample and the control (PBS) using a concentrating tube.
	<ol>
		<li>Place the samples in a 15 mL or 500 &#181;L concentrating tube, depending on the starting volume of the EV sample starting. Centrifuge the tubes according to the manufacturer&#39;s instructions until an almost-dry sample is obtained. 
		NOTE: The control sample is necessary to check for any dye background. Although this method does not require any specific initial volume, it should be considered that around 10% of particles will be lost during purification steps.&#160;</li>
	</ol>
	</li>
	<li>Resuspend the concentrated samples with diluent C. Resuspend EV samples with 200 &#181;L and the control group with 100 &#181;L and transfer them to new 1.5 mL centrifuge tubes.</li>
	<li>Separate the EV sample into two aliquots of 100 &#181;L. Mark one with dye and use it as treatment (PKH-PL-EV); leave the other unmarked but process it (NTA-PL-EV) like the EV sample and use it to quantify the EV concentration by NTA.</li>
	<li>Prepare 2x dye solution, resulting in 8 &#181;M PKH26 solution in diluent C.</li>
	<li>Mix 1 &#181;L of 1 mM PKH26 linker per 125 &#181;L of diluent C in the sample. Prepare a volume required to add to the samples in a 1:1 ratio.</li>
	<li>Add 2x dye solution to PKH-PL-EV and control samples in a 1:1 ratio to achieve 1x dye concentration and 4 &#181;M PKH26 concentration. Add the same volume of PBS to the NTA-PL-EV sample. Incubate for 5 min at room temperature.</li>
	<li>Add 5% bovine serum albumin-PBS solution to the samples in a 1:1 ratio and ensure that the final volume is ~400 &#181;L. 
	NOTE: This step allows the removal of nonspecific dye interactions or unbound dye.</li>
	<li>Proceed to separate the labeled EVs from the unbound dye and nonspecific interactions of the dye with the column.</li>
</ol>
3. Labeled-EV isolation
<ol>
	<li>Remove the cap from the column, add 400 &#181;L of the sample (PKH-PL-EV, NTA-PL-EV, or control), and discard all eluted liquid.</li>
	<li>Wait for the sample to enter the column completely before proceeding to the next step. Add 600 &#181;L of PBS and discard all eluted liquid.</li>
	<li>Wait for the PBS to enter the column completely before proceeding to the next step. Add 600 &#181;L of PBS and collect a fraction of 600 &#181;L in a 1.5. mL centrifuge tube (EVs or control). 
	NOTE: These steps are needed to remove the excess dye from the samples. Another separation by column is needed to obtain purer EVs. Thus, the following steps should be performed in a new equilibrated column (step 4.1.) or the same column after an initial washing step (step 4.2).</li>
	<li>Prepare the column for a new EV separation step to obtain purer EVs. If it is a new column, repeat steps 2.1. and 2.2. If it is the same column, wash the column with 2.5 mL of 20% isopropanol and then repeat steps 2.1 and 2.2.</li>
	<li>Add 600 &#181;L of previously eluted EVs obtained in step 2.5 to the column and discard the eluted volume. Wait for the liquid to enter the column completely before proceeding to the next step.</li>
	<li>Add 400 &#181;L of PBS and discard all eluted volume. Wait for the liquid to enter the column completely before proceeding to the next step.</li>
	<li>Add 600 &#181;L of PBS and collect a fraction of 600 &#181;L in a 1.5 mL centrifuge tube. Use the EVs and control samples for further analyses or store them overnight at 4 &#7506;C.</li>
	<li>Store the used columns for future use.
	<ol>
		<li>Wash the column with 25 mL of 20% isopropanol and discard the eluted volume. Wash the column 3 times with 2.5 mL of PBS.</li>
		<li>Add the storage buffer removed in step 1.1.1 and wait for the buffer to enter the column. Cover the column with the cap and store at 4 &#7506;C until subsequent use.</li>
	</ol>
	</li>
</ol>
4. EV quantification
<ol>
	<li>Prepare 1:1,000 dilutions of the NTA-PL-EV sample and the initial PL-EV sample as described by the following two steps.
	<ol>
		<li>Prepare 1 mL of 1:10 diluted NTA-PL-EV and 1 mL of 1:10 diluted initial PL-EV with PBS filtered through a 0.2 &#181;m filter.</li>
		<li>Prepare 1 mL of a 1:100 dilution of the previous diluted samples with PBS filtered through a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Inject the 1:1,000 diluted NTA-PL-EV sample or the initial PL-EV sample using a sterile syringe into the NTA pump. Follow the manufacturer&#39;s instructions and recommendations for particle concentration and size distribution determination. 
	&#8203;NOTE: As EV concentration depends on the sample starter volume, it may be necessary to read intermediate dilutions and make adjustments to obtain a correct NTA determination.</li>
</ol>
5. EVs used as a treatment for inflammation-driven OA
<ol>
	<li>Wash the cartilage twice with PBS and excise it using a 3 mm diameter biopsy punch under sterile conditions. 
	NOTE: Perform the procedure from steps 5.1 to 5.6 in a cell culture hood.</li>
	<li>Place the explants in 96-well culture plates with DMEM-F12 medium supplemented with 1% penicillin-streptomycin at 37 &#7506;C, 5% CO2, and 80% humidity.
	<ol>
		<li>To establish an inflammation-driven OA model, supplement the cell culture medium with 10 ng/mL oncostatin M and 2 ng/mL tumor necrosis factor-alpha (TNF&#945;).</li>
	</ol>
	</li>
	<li>Treat the explants with 1 &#215; 109 particles/well of labeled EVs (PKH-PL-EV) or control in cell culture medium supplemented with oncostatin M and TNF&#945;. 
	NOTE: Measure the volume of the sample containing 1 &#215; 109 particles/well and use the same volume for the control.</li>
	<li>Remove the medium from the 96-well cell culture plates containing cartilage explants. Add 200 &#181;L of the cell culture medium described in step 5.3 to each well. 
	NOTE: If the 96-well plates have been in contact with fetal bovine serum (FBS), wash each well three times with 200 &#181;L of PBS to remove any EVs from the FBS.</li>
	<li>Stop the in vitro assay at different times: 0, 1, 2, 3, 4, and 5 h.</li>
	<li>Wash the cell culture wells containing the cartilage explants twice with 200 &#181;L of PBS.</li>
	<li>Add 100 &#181;L of 4% paraformaldehyde (PFA) to the tissue to fix it for 3 h at 4 &#7506;C. 
	&#8203;NOTE: Steps involving PFA should be performed in a fume hood following the Safety Data Sheet recommendations.</li>
	<li>Remove the PFA, add 100 &#181;L of PBS, store the fixed tissue at 4 &#176;C, and process the samples within 48 h.</li>
</ol>
6. Microscopy preparation and visualization
NOTE: This histological procedure consists of dehydration, paraffin embedding, and rehydration steps. These steps may reduce overall dye fluorescence (a limitation mentioned in the datasheet for PKH26). Therefore, other procedures, such as frozen sectioning, may be more suitable for EV visualization by confocal microscopy.
<ol>
	<li>Embed the fixed tissues in paraffin blocks. Cut the tissue into 6 &#181;m-thick sections.</li>
	<li>Deparaffinize the tissue sections. 
	NOTE: All steps using xylene should be performed in a fume hood.
	<ol>
		<li>Immerse the tissue sections in xylene for 30 min, 100% ethanol for 2 min, 96% ethanol for 2 min, 75% ethanol for 1 min, and finally in distilled water for 30 s.</li>
	</ol>
	</li>
	<li>Permeabilize the tissue sections.
	<ol>
		<li>Prepare a 0.1% Triton-0.1% sodium citrate solution to permeabilize the tissue. Add a 20 &#181;L drop to each tissue section and incubate for 10 min at room temperature. Wash each section twice with 20 &#181;L of PBS.</li>
	</ol>
	</li>
	<li>On a microscopic slide, add a drop of mounting medium containing 4&#8242;,6-diamidino-2-phenylindole (DAPI) with an aqueous mounting medium for preserving fluorescence. Cover the slide containing 3 tissue sections from step 6.3.1.</li>
	<li>Incubate the slides overnight at room temperature, protected from light.</li>
	<li>Store at 4 &#176;C, protected from light until confocal microscopy.</li>
</ol>

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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>

The authors have no conflicts of interest to disclose.

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 conditions23,24,25.
Two possibilities exist when tracking EVs by optical microscopy: bioluminescence and fluorescence imaging. Both are used to detect EVs within the visible light spectrum (390-700 nm). Bioluminescence is a type of chemiluminescence produced after a luciferase catalyzes the oxidation of its substrate. Although this signal requires an ultrasensitive charge-coupled device (CCD) camera for detection, it has a high signal-to-noise ratio as the signal does not require an external light source26.
Fluorescence imaging uses proteins or organic dyes that emit signals after excitation from an external light source. Compared to bioluminescence, fluorescence is easier to detect by a CCD camera. Moreover, in bioluminescence, substrate toxicity and half-life of the substrate&#39;s bioluminescence should be considered for real-time EV tracking27,28.
In contrast, fluorescent protein- and organic dye-based labeling have been used with excellent resolution in optical microscopy. Although the fluorescence intensity depends on EV protein expression levels, the efficiency of EV labeling at membrane domains, and the excitation light source, fluorescence dyes provide stable and strong signals for EV imaging25. Most organic fluorescent dyes were initially used for cell membrane imaging. These dyes generally combine fluorophores that label the lipid bilayer or proteins of interest on EVs via different functional groups29.
One organic fluorescent dye family is the lipophilic PKH dye family consisting of fluorophores with a lipophilic carbocyanine that anchors into the lipid bilayer for fluorescence imaging30,31. PKH dyes have been used for in vitro and in vivo studies as their in vivo half-life ranges from 5 to &#62;100 days. Thus, the persistence of the dye in vivo may lead to misleading results in studies of shorter duration than the half-life of the dye. However, PKH dyes are useful as tracers to show EV migration32.
PKH26 is a member of this PKH lipophilic fluorophore family, found in the red spectrum with a peak of excitation at 551 nm and emission at 567 nm. This makes it compatible with other detection channels, such as rhodamine, phycoerythrin, or DAPI33, allowing the detection of, in this case, the migration of EVs marked with PKH26 toward chondrocytes marked with DAPI. It is important to note that although PL was used as an EV source here, this protocol can be used with EVs from other sources and for other purposes, for example, to track labeled the in vivo distribution of EVs.
This protocol has some limitations; for instance, there are some concerns that PKH26 increases EV size, which may affect their biodistribution and cellular uptake. However, in such cases where EV size was increased by PKH26, the labeling procedure was different from that described in this protocol34. These researchers did not include the washing and purification steps, thus leading to higher levels of free dye, which could cause the larger EV size. Moreover, the present protocol overcomes this problem by performing a parallel EV purification with and without PKH26. This allows the characterization of an unlabeled EV sample, which was processed identically as the labeled one. Thus, a misleading quantification due to confounding nonspecifically labeled particles (lipoprotein or protein sample contaminants) or by the presence of non-EV particles within the labeling mixture can be avoided, as demonstrated previously35,36.
In this paper, two cycles of purification were performed by size exclusion chromatography using columns. The first one may be substituted by sucrose gradient centrifugation. However, all EV populations were collected in the same eluted aliquot with this column and not subjected to high g forces encountered in high-speed centrifugation. However, avoiding centrifugation may lead to a slower process, especially if column washes are needed between separation cycles. Another limitation of this protocol is the need for sample concentration before starting the labeling process due to the limited volume of the column. This handicap may be overcome by using columns with higher loading capacity.
Other EV-labeling protocols describe the use of different dyes; however, their use of ultracentrifugation steps may damage EV integrity37. The protocol described here has allowed the monitoring of EV migration and uptake in cartilage explants easily with a confocal microscope without any particular function. Furthermore, this may be extrapolated to other tissues and lipidic samples or conditions, such as an in vivo assay.

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><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</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&amp;nbsp;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 &amp;micro;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&#039;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 &amp;micro;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 &amp;micro;g/&amp;micro;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 &amp;deg;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&amp;alpha;</td><td>R&amp;amp;D systems</td><td>210-TA-005</td><td>Prepare a stock solution to a final concentration of 0.01 &amp;micro;g/&amp;micro;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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;lambda;= 532 nm and camera sCMOS</td></tr><tr><td>Shandon Finesse 325 Manual Microtome</td><td>Thermo Scientific&amp;trade;</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 are used to treat osteoarthritic cartilage explants.
Once the cartilage explants are treated with EVs or the control group, they are fixed for different periods: 0, 1, 2, 3, 4, and 5 h. Each group is then paraffinized, sliced, and prepared for confocal microscopy with a mounting medium containing DAPI. Representative images for each group at each time point are presented in Figure 2, showing how EVs enter the tissue until they reach the chondrocytes and enter them over time.
As seen in Figure 2, labeled EVs are already localized around chondrocytes (shown in blue with DAPI staining) after 1 h of incubation (shown in red with PKH26 dye). Some background due to remnant dye can be observed for the control group, which does not have EVs but is processed following the same protocol as the EV sample. These results confirm the success of the protocol to label EVs, which can be used to monitor their migration through tissue in in vitro assays, as shown here, and in in vivo experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62780/62780fig01.jpg" /> 
Figure 1: Schematic overview for EV labeling and uptake monitoring protocol. Abbreviations: PBS = phosphate-buffered saline; EV = extracellular vesicle; RT = room temperature; BSA = bovine serum albumin; NTA = nanoparticle tracking analysis; OA = osteoarthritis; TNF&#945; = tumor necrosis factor-alpha; PL = platelet lysate; PFA = paraformaldehyde; DAPI = 4&#8242;,6-diamidino-2-phenylindole. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62780/62780fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62780/62780fig02.jpg" /> 
Figure 2: Representative images of EV uptake at different times. Confocal representative images taken after 0, 1, 2, 3, 4, and 5 h of osteoarthritic cartilage explants incubated with PKH-labeled EVs or with a control group. Images were taken at 400x. Scale bars = 50 &#181;m. Abbreviations: OA = osteoarthritis; PL = platelet lysate; EV = extracellular vesicle. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62780/62780fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Concentration (particles/mL)</td>
			<td>Particle size (nm)</td>
		</tr>
		<tr>
			<td>PL-EVs (initial)</td>
			<td>3.03 &#215; 1011</td>
			<td>134.0</td>
		</tr>
		<tr>
			<td>NTA-PL-EVs without PKH26 (after the protocol)</td>
			<td>8.30 &#215; 1010</td>
			<td>132.0</td>
		</tr>
	</tbody>
</table>
Table 1: Characterization by nanoparticle tracking analysis. Abbreviations: OA = osteoarthritis; PL = platelet lysate; EV = extracellular vesicle; NTA = nanoparticle tracking analysis.

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 ...

labeling-extracellular-vesicles-for-monitoring-migration-uptake

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

2.5K Views.  University of the Balearic Islands. 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. 

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

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biology

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Developmental Biology

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, proteins, and lipids from donor to acceptor cells, making them key signaling mediators between cells. In the central nervous system (CNS), sEVs can mediate intercellular signaling, including neuroimmune interactions. sEV functions can be studied by tracking the uptake of labeled sEVs in recipient cells both in vitro and in vivo. This paper describes the labeling of sEVs from the conditioned media of RAW 264.7 macrophage cells using a PKH membrane dye. It shows the uptake of different concentrations of labeled sEVs at multiple time points by Neuro-2a cells and primary astrocytes in vitro. Also shown is the uptake of sEVs delivered intrathecally in mouse spinal cord neurons, astrocytes, and microglia visualized by confocal microscopy. The representative results demonstrate time-dependent variation in the uptake of sEVs by different cells, which can help confirm successful sEVs delivery into the spinal cord.

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

We describe a protocol to label macrophage-derived small extracellular vesicles with PKH dyes and observe their uptake in vitro and in the spinal cord after intrathecal delivery.

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, ...

Neuroscience

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Medicine

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

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

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

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

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

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

Setting a Successful Sorting for Extracellular Vesicle Isolation

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

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

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

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

Summary

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Chapters in this video

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

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

Setting a Successful Sorting for Extracellular Vesicle Isolation