Name: Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants (Video) | JoVE Journal
Uploaded: 2021-10-04T14:00:00.000+00:00
Duration: 7 min 58 s
Description: Learn to label extracellular vesicles from platelet lysate and monitor their migration and uptake in cartilage explants using confocal microscopy. This protocol enables researchers to study EV distribution in osteoarthritis models.

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5 minutes

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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<li>Dehghani, M., Gulvin, S. M., Flax, J., Gaborski, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+evaluation+of+PKH+labelling+on+extracellular+vesicle+size+by+nanoparticle+tracking+analysis%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Labeling+extracellular+vesicles+for+nanoscale+flow+cytometry%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Confounding+factors+in+vesicle+uptake+studies+using+fluorescent+lipophilic+membrane+dyes%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">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/pubmed?term=((In+vitro+study+of+extracellular+vesicles+migration+in+cartilage-derived+osteoarthritis+samples+using+real-time+quantitative+multimodal+nonlinear+optics+imaging%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">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...

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

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...

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

Research

JoVE Journal

Bioengineering

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.

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

In this video, we demonstrate an assay to visualize and quantify the cellular uptake of extracellular vesicles or EVs. This assay utilizes 3D fluorescent imaging using a confocal microscope and allows for distinguishing internalized EVs from ones adhered to the cell surface.

Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique

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

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

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

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

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.

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

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.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

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.

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

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.

Setting a Successful Sorting for Extracellular Vesicle Isolation

Learn to label extracellular vesicles from platelet lysate and monitor their migration and uptake in cartilage explants using confocal microscopy. This protocol enables researchers to study EV distribution in osteoarthritis models.

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

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