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

Podsumowanie

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.

Streszczenie

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 OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 µm 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.

Wprowadzenie

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 treatments^2,3,4, 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 authors^5,6. EVs are membranous bodies secreted by the majority of cell types, are involved in intercellular signaling, and have been shown to exert stem cells' therapeutic effects^7,8,9, 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 cells^11,12.

Platelet concentrates, such as platelet lysates (PLs), are used to enhance wound healing in different injuries, such as corneal ulcers^13,14,15 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/β-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.

Protokół

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

1. Equilibrate columns following the manufacturer's instructions or as follows:
   1. Remove the column cap and equilibrate the column. Remove the storage buffer by elution.
   2. 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.
      ​NOTE: Do not let the column dry.
   3. Cover the column with the cap after the last wash and until sample preparation.

2. EV labeling

NOTE: This EV labeling protocol uses a PL-EV sample previously isolated by size exclusion chromatography (SEC) with previously described conditions^21,22. However, any EV sample from any source may be used with this protocol.

1. Concentrate the EV sample and the control (PBS) using a concentrating tube.
   1. Place the samples in a 15 mL or 500 µL concentrating tube, depending on the starting volume of the EV sample starting. Centrifuge the tubes according to the manufacturer'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. 
2. Resuspend the concentrated samples with diluent C. Resuspend EV samples with 200 µL and the control group with 100 µL and transfer them to new 1.5 mL centrifuge tubes.
3. Separate the EV sample into two aliquots of 100 µL. 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.
4. Prepare 2x dye solution, resulting in 8 µM PKH26 solution in diluent C.
5. Mix 1 µL of 1 mM PKH26 linker per 125 µL of diluent C in the sample. Prepare a volume required to add to the samples in a 1:1 ratio.
6. Add 2x dye solution to PKH-PL-EV and control samples in a 1:1 ratio to achieve 1x dye concentration and 4 µM PKH26 concentration. Add the same volume of PBS to the NTA-PL-EV sample. Incubate for 5 min at room temperature.
7. Add 5% bovine serum albumin-PBS solution to the samples in a 1:1 ratio and ensure that the final volume is ~400 µL.
   NOTE: This step allows the removal of nonspecific dye interactions or unbound dye.
8. Proceed to separate the labeled EVs from the unbound dye and nonspecific interactions of the dye with the column.

3. Labeled-EV isolation

1. Remove the cap from the column, add 400 µL of the sample (PKH-PL-EV, NTA-PL-EV, or control), and discard all eluted liquid.
2. Wait for the sample to enter the column completely before proceeding to the next step. Add 600 µL of PBS and discard all eluted liquid.
3. Wait for the PBS to enter the column completely before proceeding to the next step. Add 600 µL of PBS and collect a fraction of 600 µL 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).
4. 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.
5. Add 600 µL 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.
6. Add 400 µL of PBS and discard all eluted volume. Wait for the liquid to enter the column completely before proceeding to the next step.
7. Add 600 µL of PBS and collect a fraction of 600 µL in a 1.5 mL centrifuge tube. Use the EVs and control samples for further analyses or store them overnight at 4 ᵒC.
8. Store the used columns for future use.
   1. 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.
   2. 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 ᵒC until subsequent use.

4. EV quantification

1. Prepare 1:1,000 dilutions of the NTA-PL-EV sample and the initial PL-EV sample as described by the following two steps.
   1. 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 µm filter.
   2. Prepare 1 mL of a 1:100 dilution of the previous diluted samples with PBS filtered through a 0.2 µm filter.
2. 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's instructions and recommendations for particle concentration and size distribution determination.
   ​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.

5. EVs used as a treatment for inflammation-driven OA

1. 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.
2. Place the explants in 96-well culture plates with DMEM-F12 medium supplemented with 1% penicillin-streptomycin at 37 ᵒC, 5% CO₂, and 80% humidity.
   1. 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 (TNFα).
3. Treat the explants with 1 × 10⁹ particles/well of labeled EVs (PKH-PL-EV) or control in cell culture medium supplemented with oncostatin M and TNFα.
   NOTE: Measure the volume of the sample containing 1 × 10⁹ particles/well and use the same volume for the control.
4. Remove the medium from the 96-well cell culture plates containing cartilage explants. Add 200 µL 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 µL of PBS to remove any EVs from the FBS.
5. Stop the in vitro assay at different times: 0, 1, 2, 3, 4, and 5 h.
6. Wash the cell culture wells containing the cartilage explants twice with 200 µL of PBS.
7. Add 100 µL of 4% paraformaldehyde (PFA) to the tissue to fix it for 3 h at 4 ᵒC.
   ​NOTE: Steps involving PFA should be performed in a fume hood following the Safety Data Sheet recommendations.
8. Remove the PFA, add 100 µL of PBS, store the fixed tissue at 4 °C, and process the samples within 48 h.

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.

1. Embed the fixed tissues in paraffin blocks. Cut the tissue into 6 µm-thick sections.
2. Deparaffinize the tissue sections.
   NOTE: All steps using xylene should be performed in a fume hood.
   1. 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.
3. Permeabilize the tissue sections.
   1. Prepare a 0.1% Triton-0.1% sodium citrate solution to permeabilize the tissue. Add a 20 µL drop to each tissue section and incubate for 10 min at room temperature. Wash each section twice with 20 µL of PBS.
4. On a microscopic slide, add a drop of mounting medium containing 4′,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.
5. Incubate the slides overnight at room temperature, protected from light.
6. Store at 4 °C, protected from light until confocal microscopy.

Wyniki

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...

Dyskusje

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 conditions^23,24...

Materiały

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