Evaluation of the Storage Stability of Extracellular Vesicles

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 pathways^1,2. 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. Centrifuge at 300 x g for 10 min to pellet the cells. Carefully collect the cell-conditioned medium (CCM), without disturbing the pelleted c....

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.......

Materials

Name	Company	Catalog Number	Comments
1,2 dimyristoyl-sn glycero-3-phospho-choline (DMPC)	Sigma-Aldrich	P2663-25MG
1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC)	Sigma-Aldrich	P4329-25MG
225 cm² cell culture flasks	Corning	431082	Used with 25 ml of medium
30 kDa regenerated cellulose membrane	Wyatt Technology Europe	1854
350 µm spacer	Wyatt Technology Europe
Automated fraction collector	Thermo Fisher Scientific
Beta-glucuronidase	Sigma-Aldrich	G7646-100KU
Chloroform	Fisher scientific	C/4966/17
Column oven	Hitachi High-Technologies Europe
D-(+)-Trehalose dihydrate	Sigma-Aldrich	T9531-10G
DAWN HELEOS II, Multi-angle light scattering detector	Wyatt Technology Europe
Durapore Membrane filter, PVDF,  0,1 µm, 47 mm	Merck	VVLP04700	Used for the preparation of buffers for AF4
EBM-2	Lonza Verviers, S.p.r.	CC-3156	Endothelial Cell Growth basal medium, used for the serum free culture of HUVEC cells
Eclipse dualtec	Wyatt Technology Europe
EGM-2	Lonza Verviers, S.p.r.	CC-3162	Endothelial Cell Growth medium, used for the normal culture of HUVEC cells
ELISA Plate Sealers	R&D Systems	DY992	used for sealing of 96-well plates for the glucuronidase assay
Ethanol	Fisher scientific	E/0665DF/17
Extruder Set With Holder/Heating Block	Avanti Polar Lipids	610000-1EA
Filter support	Avanti Polar Lipids	610014-1EA	used for liposome preparation
Fluorescein di-β-D-glucoronide	Thermo Fisher Scientific	F2915
Gibco PBS-tablets+CA10:F36	Thermo Fisher Scientific	18912014
Hettich Universal 320 R	Andreas Hettich GmbH & Co.KG		Used for pelleting cells at 300 g
Hettich Rotina 420 R	Andreas Hettich GmbH & Co.KG		Used for pelleting larger debris at 3000 g
HUVEC cells	Lonza Verviers, S.p.r.	C2517A
Kimble  FlexColumn 1X30CM	Kimble	420401-1030
Lyophilizer ALPHA 2-4 LSC	Christ
Microcentrifuge Tubes, Polypropylene	VWR international	525-0255	the 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 laser	Malvern Pananalytical
Nanosight-software version 3.1	Malvern Pananalytical
Nucleopore 200 nm track-etch polycarbonate membranes	Whatman/GE Healthcare	110406	used for liposome preparation
PEEK Inline filter holder	Wyatt Technology Europe
Phosphotungstic acid hydrate	Sigma-Aldrich	79690-25G
Polycarbonate bottles for ultracentrifugation	Beckman Coulter	355622
QuantiPro BCA Assay Kit	Sigma-Aldrich	QPBCA-1KT
Saponin	Sigma-Aldrich	47036
Scanning electron microscopy Zeiss EVO HD 15	Carl Zeiss AG
Sepharose Cl-2b	GE Healthcare	17014001
SEM copper grids with carbon film	Plano	S160-4
Small AF4 channel	Wyatt Technology Europe
Sputter-coater Q150R ES	Quorum Technologies
Transmission electron microscopy JEOL JEM 2011	Oxford Instruments
Type 45 Ti ultracentrifugation rotor	Beckman Coulter	339160
Ultimate 3000 Dionex autosampler	Thermo Fisher Scientific
Ultimate 3000 Dionex isocratic pump	Thermo Fisher Scientific
Ultimate 3000 Dionex online vacuum degasser	Thermo Fisher Scientific
Ultracentrifuge OptimaTM L-90 K	Beckman Coulter
UV detector	Thermo Fisher Scientific
Whatman 0.2 µm pore size mixed cellulose filter	Whatman/GE Healthcare	10401712	Used for the filtration of all buffers used with the EVs and in SEC