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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Abstract

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Introduction

EVs are membrane-bound nanoparticles produced by nearly all cell types. For mammalian cells, EVs can be subdivided into two main groups with distinct production pathways1,2. Membrane vesicles, with a size range from roughly 100-1,000 nm, are produced by direct budding from the cell membrane. Exosomes, sized 30-200 nm, are derived from multivesicular bodies formed by inward budding into endosomes that subsequently fuse with the cell membrane to release multiple exosomes at once. The main function of these vesicles is the transport of information between cells3. For this purpose, cargos s....

Protocol

1. Cell culture and the production of cell-conditioned medium

  1. Generally, cultivate cells under the individual conditions required for the respective cell line.
  2. Cultivate the cells for 24-72 h in serum-free conditions or in medium containing EV-depleted fetal bovine serum (FBS).
    NOTE: If EV-depleted FBS is used, employ a method proven to efficiently deplete the serum, to prevent contamination with bovine serum-derived EVs26.
  3. Collect the medium from the flasks........

Representative Results

Figure 1 displays the storage characteristics of EVs isolated from HUVECs. EVs were isolated by UC, glucuronidase was encapsulated, and after SEC, the purified EVs were evaluated for their physicochemical properties by NTA. A sample of the vesicles was subsequently subjected to AF4 purification and the glucuronidase activity was measured.

The vesicles were then stored for 7 d at 4 °C or -80 °C and at 4 °C in lyophilized form, in the latter case with the addition.......

Discussion

In this manuscript, we present a comprehensive protocol to study the stability of EVs under different storage conditions. With the combination of encapsulated glucuronidase as a functional readout and the evaluation of the physicochemical parameters of the EVs, the protocol allows for a straightforward storage stability evaluation of EVs and the comparison of EVs from different cell lines. SEM and TEM as complementary methods allow an insight into changes of the EVs on the single-particle level. The results presented her.......

Acknowledgements

The NanoMatFutur Junior Research program from the Federal Ministry of Education and Research, Germany (grant number 13XP5029A) supported this work. Maximilian Richter was supported by Studienstiftung des Deutschen Volkes (German Academic Scholarship Foundation) through a Ph.D. fellowship.

....

Materials

NameCompanyCatalog NumberComments
1,2 dimyristoyl-sn glycero-3-phospho-choline (DMPC)Sigma-AldrichP2663-25MG
1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC)Sigma-AldrichP4329-25MG
225 cm² cell culture flasksCorning431082Used with 25 ml of medium
30 kDa regenerated cellulose membraneWyatt Technology Europe1854
350 µm spacerWyatt Technology Europe
Automated fraction collectorThermo Fisher Scientific
Beta-glucuronidaseSigma-AldrichG7646-100KU
ChloroformFisher scientificC/4966/17
Column ovenHitachi High-Technologies Europe
D-(+)-Trehalose dihydrateSigma-AldrichT9531-10G
DAWN HELEOS II, Multi-angle light scattering detector Wyatt Technology Europe
Durapore Membrane filter, PVDF,  0,1 µm, 47 mmMerckVVLP04700Used for the preparation of buffers for AF4
EBM-2Lonza Verviers, S.p.r.CC-3156Endothelial Cell Growth basal medium, used for the serum free culture of HUVEC cells
Eclipse dualtecWyatt Technology Europe
EGM-2Lonza Verviers, S.p.r.CC-3162Endothelial Cell Growth medium, used for the normal culture of HUVEC cells
ELISA Plate SealersR&D SystemsDY992used for sealing of 96-well plates for the glucuronidase assay
EthanolFisher scientificE/0665DF/17
Extruder Set With Holder/Heating BlockAvanti Polar Lipids610000-1EA
Filter supportAvanti Polar Lipids610014-1EAused for liposome preparation
Fluorescein di-β-D-glucoronideThermo Fisher ScientificF2915
Gibco PBS-tablets+CA10:F36Thermo Fisher Scientific18912014
Hettich Universal 320 RAndreas Hettich GmbH & Co.KGUsed for pelleting cells at 300 g
Hettich Rotina 420 RAndreas Hettich GmbH & Co.KGUsed for pelleting larger debris at 3000 g
HUVEC cellsLonza Verviers, S.p.r.C2517A
Kimble  FlexColumn 1X30CMKimble420401-1030
Lyophilizer ALPHA 2-4 LSCChrist
Microcentrifuge Tubes, PolypropyleneVWR international525-0255the tubes used for all EV-handling, found to be more favorable than comparable products from other suppliers regarding particle recovery
Nanosight LM14 equipped with a green laserMalvern Pananalytical
Nanosight-software version 3.1Malvern Pananalytical
Nucleopore 200 nm track-etch polycarbonate membranesWhatman/GE Healthcare110406used for liposome preparation
PEEK Inline filter holderWyatt Technology Europe
Phosphotungstic acid hydrateSigma-Aldrich79690-25G
Polycarbonate bottles for ultracentrifugationBeckman Coulter355622
QuantiPro BCA Assay KitSigma-AldrichQPBCA-1KT
SaponinSigma-Aldrich47036
Scanning electron microscopy Zeiss EVO HD 15Carl Zeiss AG
Sepharose Cl-2bGE Healthcare17014001
SEM copper grids with carbon filmPlanoS160-4
Small AF4 channelWyatt Technology Europe
Sputter-coater Q150R ESQuorum Technologies
Transmission electron microscopy JEOL JEM 2011Oxford Instruments
Type 45 Ti ultracentrifugation rotorBeckman Coulter339160
Ultimate 3000 Dionex autosamplerThermo Fisher Scientific
Ultimate 3000 Dionex isocratic pumpThermo Fisher Scientific
Ultimate 3000 Dionex online vacuum degasserThermo Fisher Scientific
Ultracentrifuge OptimaTM L-90 KBeckman Coulter
UV detectorThermo Fisher Scientific
Whatman 0.2 µm pore size mixed cellulose filterWhatman/GE Healthcare10401712Used for the filtration of all buffers used with the EVs and in SEC

References

  1. Stremersch, S., De Smedt, S. C., Raemdonck, K. Therapeutic and diagnostic applications of extracellular vesicles. Journal of Controlled Release. 244, 167-183 (2016).
  2. Fuhrmann, G., Herrmann, I. K., Stevens, M. M.

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Extracellular VesiclesStorage StabilityGlucuronidaseAsymmetric Flow Field Flow Fractionation AF4Cell CultureUltracentrifugationBeta glucuronidaseSaponin

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