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

Summary

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.

Abstract

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.

Introduction

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 m....

Protocol

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 af....

Results

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.......

Discussion

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.......

Materials

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