Orchestrating Extracellular Vesicle With Dual Reporters for Imaging and Capturing in Mammalian Cell Culture

Abstract

Background: Recent technological advancements have enabled live-cell imaging of intracellular organelles to monitor their biogenesis in mammalian cells. However, applying this method to gain insight into extracellular organelles, such as extracellular vesicles (EVs), presents unique challenges that require special considerations in design and engineering.Results: We have developed a dual-reporter system that combines genetic fusion, fluorescence microcopy and magnetic beads capture of EVs to study the biogenesis of EVs in mammalian cell cultures. First, we genetically produced a series of reporters by fusing a green fluorescent protein (GFP) and an affinity peptide (6xHis), with either the endogenous transmembrane protein, CD63, or EVs targeting vesicular stomatitis viral glycoprotein (VSVG). Transfection of these reporters into human 293T cells resulted in expression and integration of these reporters into pre-exosome compartments, which were subsequently released into the culture medium. Confocal imaging and nano-particle tracking analysis demonstrated that EVs were appropriately labeled and exhibited a single dominant peak in the 80–110 nm size range, indicating that isolated EVs were comprised of micro-vesicles and/or exosome subpopulations. Incubation of isolated EVs with nickel-coated magnetic beads resulted in successful capture of GFP-positive EVs. Finally, addition of EVs into culture medium was able to reveal the cellular uptake of GFP-labeled EVs by recipient cells. Taken together, our dual-reporter system provides a powerful method for both monitoring and capturing of EVs in mammalian cell culture systems.Conclusion: A dual-reporter system provides a robust tool to study the life cycle of EVs in mammalian cells from biogenesis and excretion to cellular uptake.

Unconventional Protein Secretion Dependent on Two Extracellular Vesicles: Exosomes and Ectosomes

“…In addition, recent evidence has demonstrated the ability of EVs to establish surface protein-protein interactions. It appears, therefore, that at least a fraction of EVs tends to show functionally integrated complexes ( Leidal et al, 2020 ; Levy et al, 2021 ; Nikoloff et al, 2021 ; Razzauti and Laurent, 2021 ). At present, populations of single EV type can be isolated by various techniques based on distinct approaches, including monoclonal antibodies ( Levy et al, 2021 ; Lim et al, 2021 ).…”

“…The state of knowledge and techniques are already advanced. Additional developments are expected for the future including their markers and signatures, useful for the identification of subtype-specific EVs and the unconventional secretion of their proteins ( Garcia-Martin et al, 2021 ; Levy et al, 2021 ; Nikoloff et al, 2021 ; Razzauti and Laurent, 2021 ).…”

“…The specific binding of a vesicle agonist to its receptor at the cell surface can be transient followed by generation of intracellular signals. In many cases, however, binding is followed by insertion of the vesicle membrane in the plasma membrane, with enlargement of the cell surface area and cargo discharged into the cell cytoplasm ( Levy et al, 2021 ; An et al, 2021 ). Upon the introduction of new techniques to reveal the progressive changes of the EV protein distribution, the membrane and cargo events have been intensively investigated ( Lim et al, 2021 ; Bost et al, 2021 ).…”

“…However, the disruption of discharged cargo molecules is not always the case. Rather, many proteins remain intact and functional, as it happens with receptors still active upon their discharge ( Hung and Leonard, 2016 ; Somiya, 2020 ; Levy et al, 2021 ). During and after such fusions also target cells release vesicles of their origin, containing at least part of the components received by their EV fusion.…”

Mesenchymal Stromal Cell-Derived Extracellular Vesicles as Biological Carriers for Drug Delivery in Cancer Therapy

“…Similarly, the SMART exos system is based on expressing distinct types of monoclonal antibodies on sEVs surfaces that bind specific surface proteins in target cells ( Shi et al, 2020 ). The genetic engineering approach can also be used to generate fusion proteins between the green fluorescent protein and sEVs-specific proteins –such as tetraspanins– to label sEVs for biodistribution analysis ( Levy et al, 2021 ). A limitation of this method is that only a small population of sEVs become fluorescent, inducing variability in the signal intensity ( Choi and Lee, 2016 ).…”