Setting a Successful Sorting for Extracellular Vesicle Isolation

Summary

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Abstract

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications.

First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol.

In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique.

Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

Introduction

Extracellular Vesicles (EVs) are a heterogeneous group of membrane-structured vesicles released by almost all cells, delimited by a lipid bilayer, unable to replicate on their own¹. They can be found in several biofluids such as blood plasma, serum, saliva, breast milk, urine, bronchial lavage fluid, amniotic fluid, cerebrospinal fluid, and malignant ascites². One of the main functions of EVs is to transport various molecules, including nucleic acids, proteins, lipids, and carbohydrates, between a donor and a recipient cell. This can occur through various mechanisms, such as direct membrane fusion, receptor-ligand intera....

Protocol

The protocol here consists of four parts: (1) Sample preparation, (2) ASC-EVs characterization, (3) ASC-EVs sorting, and (4) Post sorting analysis. A schematic representing the workflow is shown in Figure 1.

Figure 1: Protocol Flow chart. The flow chart shows the steps involved in t.......

Discussion

Analyzing and sorting EVs is challenging due to their small size and the fact that they are near the detection limit of most flow cytometers. Our objective was to develop a protocol for isolating EVs derived from AMSCs labeled with CFSE. CFSE was selected as the staining method due to its reported high EVs labeling efficiency (≥90%), without the formation of unwanted particles such as protein aggregates given by antibodies. Nevertheless, it is possible that few EVs without esterase could be missed and future studie.......

Materials

Name	Company	Catalog Number	Comments
5(6)-Carboxyfluorescein diacetate N-succinimidyl ester	Merck	150347-59-4
Adipose Mesenchymal Stromal Cells	Wepredic, Parc d'affaires, 35760 Saint-Grégoire, France		Cells used in this study
Alexa 488 anti-Caveolin	R&D Systems	IC5736G	Flow cytometry antibody
APC anti-human CD44	BioLegend	338805	Flow cytometry antibody
APC anti-human CD63	BioLegend	353007	Flow cytometry antibody
APC anti-human CD81 (TAPA-1)	BioLegend	349509	Flow cytometry antibody
APC anti-human CD9	BioLegend	312107	Flow cytometry antibody
BC CytoFLEX S	Beckman Coulter		BC CytoFLEX S equipped with 3 lasers, Blue, Red and Violet
Flow-Check Pro Fluorospheres	Beckman Coulter	A63493	Fluorescent control beads for MoFLO Astrios EQ
FlowJo software (version 10.8.1)	BD	version 10.8.1	Analysis software
IntraSure kit	BD Biosciences	641776	Fixation and permeabilization for intracellular staining
Megamix-Plus FSC	BioCytex	7802	FSC polystyrene beads
MoFLO Astrios EQ	Beckman Coulter		MoFLO Astrios EQ equipped with 4 lasers, Blue, Yellow - Green, Violet and Red
Mouse anti-FLOT1 antibody	BD Transduction Laboratories	610820	Western Blot antibody
NanoSight NS300	Malvern	NS300
Rabbit anti-Calnexin antibody	Origene	TA336279	Western Blot antibody
Rabbit anti-CD9 and CD81 antibody (ExoAb antibody kit)	System Biosciences	EXOAB-KIT-1	Western Blot antibodies
Rabbit anti-TSG101 antibody	Merck	HPA006161	Western Blot antibody
Triton X-100	Merck	9036-19-5
Ultra Rainbow Fluorescent Particles	Spherotech	URFP-30-2
Ultracel 100 kDa MWCO	Merck	UFC910024
VER01 - Verity Shells	Exometry		Organo silica beads for scatter calibration