Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies

Abstract

This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.

Keywords: Bioluminescent labelling; CRISPR-Cas; Cre-loxP; EV tracking; Exosomes; Extracellular vesicles; Fluorescent labelling; In vivo imaging; Magnetic resonance; Radiolabelling.

Fig. 1

Strategies for labelling of exogenous extracellular vesicles

Fig. 2

An overview of frequently used fluorescent and bioluminescent labels comparing their maximum emission wavelengths

Fig. 3

Strategies for the labelling of endogenously produced EVs for the study of their functional transfer. A Cre-LoxP method. B CRISPR-Cas9 method. C Principle of Cre-LoxP system: Cre-mediated recombination activates a fluorescence switch from DsRed to eGFP in reporter⁺ cells after receiving Cre⁺-EVs produced by Cre⁺ cells. D Mechanism of CRISPR-Cas9: Reporter⁺ cells expressing Cas9 switch from mCherry to eGFP fluorescence after functional uptake of EVs. EVs produced by genetically manipulated donor cells carry a specific targeting single guide RNA (sgRNA) that navigates Cas9 with nuclease activity to generate cleavage of the target sequence