Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants
October 4th, 2021
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.
This simple protocol allows one to label EVs and monitor them via confocal microscopy. For instance, EV imaging might help in evaluating EV distribution when use it in therapeutics. This technique allows one to monitor EV migration and uptake in cartilage explants easily, with confocal microscopy without any special function.
The same protocol can be used for EVs for in vitro or in vivo experiments. Make sure that the columns are prepared beforehand, and that the initial volume of EVs is enough to reach the desired final particle concentration, considering that around 10%of the particles will be lost during the purification step. Begin by concentrating the extracellular vesicle, or EV, sample and the control.
Place the samples in a 15 milliliter or 500 microliters concentrating tube, depending on the starting volume of the EV sample. Centrifuge the tubes according to the manufacturer's instructions until an almost dry sample is obtained. Re-suspend the concentrated EV samples with 200 microliters, and the control group with 100 microliters of diluent C, and transfer them to new 1.5-milliliter centrifuge tubes.
Separate the EV sample into two aliquots of 100 microliters each. Mark one of the aliquots with dye and use it as treatment. Leave the other unmarked, but process it like the EV sample, and use it to quantify the EV concentration by nanoparticle tracking analysis.
Prepare 2x dye solution such that the resulting concentration of PKH26 solution in diluent C is eight micromolar. Mix one microliter of one-millimolar PKH26 linker per 125 microliters of diluent C in the sample. Prepare the volume required to add to the samples in a 1:1 ratio.
Add 2x dye solution to PKH platelet lysate-derived EVs and control samples in a 1:1 ratio, to achieve 1x dye concentration, and four micromolar PKH26 concentration. Add the same volume of PBS to the NTA platelet lysate-derived EV sample. Incubate for five minutes at room temperature.
Add 5%bovine serum albumin-PBS solution to the samples in a 1:1 ratio, and ensure that the final volume is approximately 400 microliters. Proceed to separate the labeled EVs from the unbound dye and non-specific dye interactions with the column. Remove the cap from the column, add 400 microliters of PKH platelet lysate-derived EVs, NTA platelet lysate-derived EVs, or control and discard all eluted liquid.
Wait for the sample to enter the column completely before proceeding to the next step. Add 600 microliters of PBS and discard all eluted liquid. Wait for the PBS to enter the column completely before proceeding to the next step.
Add 600 microliters of PBS and collect a fraction of 600 microliters in a 1.5-milliliter centrifuge tube. For a new column, repeat the steps involving the concentration of the samples mentioned at the beginning of the protocol. Add 600 microliters of previously eluted EVs to the column and discard the eluted volume.
Then add 400 microliters of PBS and discard all eluted volumes. Add 600 microliters of PBS to the column and collect a fraction of 600 microliters in a 1.5-milliliter centrifuge tube. Wash the cartilage two times with PBS and excise it using a three-millimeter diameter biopsy punch under sterile conditions.
Place the explants in 96-well culture plates with DMEM-F12 medium, supplemented with 1%penicillin streptomycin at 37 degrees Celsius, 5%CO2, and 80%humidity. To establish an inflammation-driven OA model, supplement the cell culture medium with 10 nanograms per milliliter of oncostatin M and two nanograms per milliliter of tumor necrosis factor-alpha. Treat the explants with 1 times 10 to the 9th particles per well of PKH platelet lysate-derived EVs, or control, in cell culture medium supplemented with oncostatin M and tumor necrosis factor-alpha.
Remove the medium from the 96-well cell culture plates containing cartilage explants. Add 200 microliters of the cell culture medium to each well as described in the manuscript. Stop the in vitro assay every hour from zero to five hours.
Wash the cell culture wells containing the cartilage explants two times with 200 microliters of PBS. Add 100 microliters of 4%paraformaldehyde to the tissue to fix it for three hours at four degrees Celsius. Remove the PFA, add 100 microliters of PBS, store the fixed tissue at four degrees Celsius, and process the samples within 48 hours.
Images taken at different time points show how EVs enter the tissue until they reach the chondrocytes, and enter the chondrocytes over time. The labeled EVs were already localized around chondrocytes after one hour of incubation. Ensure that the initial volume of EVs is enough to reach the desired final particle concentration.
Another important thing to remember is to use the labeled EVs within the next 24 hours. If not use it within 24 hours, a decrease in fluorescence may occur. This technique will help researchers to improve EV imaging, and to study EVs in biology and therapeutics.
View the full transcript and gain access to thousands of scientific videos
Overview
This protocol describes a method for labeling extracellular vesicles (EVs) derived from platelet lysate to observe their migration and uptake in cartilage explants, a model for osteoarthritis. The technique utilizes confocal microscopy for monitoring EV distribution and behavior in therapeutic contexts.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Osteoarthritis Research
Background
- Extracellular vesicles play a crucial role in intercellular communication.
- Understanding EV behavior can provide insights into therapeutic applications.
- Monitoring EVs in cartilage can help elucidate mechanisms in osteoarthritis.
- Confocal microscopy is a powerful tool for visualizing EVs in biological samples.
Purpose of Study
- To develop a straightforward protocol for labeling EVs.
- To facilitate the monitoring of EV migration and uptake in cartilage explants.
- To enhance the understanding of EV distribution in therapeutic settings.
Methods Used
- Labeling of platelet lysate-derived EVs.
- Use of confocal microscopy for imaging.
- Preparation of columns and concentration of EV samples.
- Consideration of particle loss during purification.
Main Results
- The protocol allows for effective labeling and monitoring of EVs.
- Confocal microscopy provides clear imaging of EV distribution.
- EV migration and uptake can be easily assessed in cartilage explants.
- The method is applicable for both in vitro and in vivo experiments.
Conclusions
- This protocol is a valuable tool for researchers studying EVs in osteoarthritis.
- It simplifies the process of monitoring EV behavior in biological models.
- Future studies can leverage this technique for therapeutic evaluations.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables biopharma researchers to track extracellular vesicle (EV) migration and cellular uptake in cartilage explants, supporting mechanistic de-risking of EV-based therapeutics for osteoarthritis. By providing quantitative, time-resolved imaging of EV biodistribution in a disease-relevant system, the method enhances target validation and predictive confidence in preclinical development. It addresses a critical gap in assessing EV delivery efficiency before advancing candidates into later-stage studies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables direct visualization of EV internalization into chondrocytes, supporting mechanistic interrogation of therapeutic hypotheses.
- Operational Value: Uses standardized PKH26 labeling and column purification to reduce variability in EV preparation for consistent target engagement assessment.
Screening & Assay Development
- Scientific Value: Generates quantitative uptake data over time, enabling assay standardization for comparing EV formulations or dosing conditions.
- Operational Value: Integrates nanoparticle tracking analysis (NTA) for particle quantification, ensuring reproducible input concentrations despite ~10% loss during purification.
Translational & Preclinical Research
- Scientific Value: Models inflammation-driven osteoarthritis using oncostatin M and TNF-alpha, providing a disease-relevant system to evaluate EV behavior under pathophysiological conditions.
- Operational Value: Supports hourly fixation and confocal imaging, allowing kinetic analysis of EV migration and uptake to inform translational biomarker alignment.
Pipeline & Workflow Integration
The method fits within the discovery-to-preclinical continuum, enabling early assessment of EV biodistribution to guide lead identification and preclinical go/no-go decisions in osteoarthritis therapeutic development.
- Discovery Biology: Supports hypothesis testing by visualizing EV uptake kinetics, clarifying mechanisms of cellular internalization in target tissues.
- Screening: Delivers standardized, quantitative fluorescence readouts via confocal microscopy, enabling reliable comparison of EV preparations across experimental groups.
- Analytics: Combines NTA-based particle counting with imaging-derived uptake metrics, providing dual quantitative outputs for cross-condition comparison.
- Translational Research: Uses primary cartilage explants exposed to inflammatory cytokines, creating a bridge from discovery to preclinical validation in a clinically relevant osteoarthritis model.
- Enterprise Reuse: Requires only standard laboratory equipment (centrifuge, confocal microscope, NTA), facilitating adaptation across projects and teams as a reusable capability.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in EV therapeutics by providing direct evidence of target cell engagement in cartilage.
- Operational Value: Employs accessible, low-cost labeling (PKH26) and purification (column wash) steps, promoting reproducibility and scalability across EV batches.
- Strategic Value: Improves go/no-go decision-making by delivering early, visual proof of concept for EV delivery, reducing late-stage biological risk in osteoarthritis programs.
- Portfolio Impact: Enables risk-adjusted prioritization of EV candidates based on demonstrated uptake in disease-relevant explants, supporting capital-efficient advancement.
Implementation Considerations
- Requires expertise in EV isolation, fluorescent labeling, and confocal microscopy imaging.
- Depends on access to nanoparticle tracking analysis (NTA) for accurate particle quantification and column-based purification systems.
- Necessitates standardization of EV input concentrations across labs to account for ~10% particle loss during washing steps.
- Must be adapted for different EV sources or tissue models while maintaining labeling efficiency and minimizing dye aggregation artifacts.
- Limited by the 24-hour stability of PKH26-labeled EVs, requiring same-day use to avoid fluorescence degradation and ensure signal integrity.
