Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

Abstract

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

A functional corona around extracellular vesicles enhances angiogenesis during skin regeneration and signals in immune cells

“…Using multiplex bead-based flow cytometry [23] for EV surface marker profiling of three independent purified PLX-EV preparations confirmed high expression of tetraspanins CD81/CD63 and reduced CD9 expression. We found medium classified expression of the fibronectin receptor CD49e/CD29 and high/medium expression of the extracellular matrix interaction molecules CD44 and NG2 as well as the cytokine receptor CD105 (endoglin) and CD142 (coagulation factor III, tissue factor).…”

“…For this study, we used PLX cells derived from three individual donors, P150216R01, P250416R05 and P270114R27 (termed P15, P25 and P27, respectively). For EV production aliquots of 2.5 23 October 17, 2019 million PLX cells were propagated for one to two passages to avoid excess proliferation at moderate seeding density of 1,000 cells/cm² in 2,528 cm² cell factories (CF4, Thermo Fisher Scientific, USA) as established previously until approximately 70% confluence [19], [43]. For EV harvest PLX cells were washed twice with 37°C pre-warmed Dulbecco's PBS (Sigma Aldrich, USA) and cultured for an additional 48 h in particle depleted α-MEM*/TFF.…”

Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

“…Based on the robust MSC‐EV proteome signature that we were able to obtain from a concise and well‐controlled analysis of published proteomics datasets, a standardized approach for the characterization and verification, a quality control, could be envisioned. As the protein signature comprises several membrane‐ and extracellular proteins, a multiplex bead capture flow cytometry approach, for example, based on COL6A2 or COL6A3 and THY1 (CD90) could be envisioned as a standard, easy to use platform for MSC‐EV quality control, either in a research or clinical setting. Alternatively, such proteins could be tested in sandwich immunoassays or antibody arrays adapted specifically for this purpose.…”

Extracellular‐Vesicle Isolation from Different Biological Fluids by Size‐Exclusion Chromatography

“…Nevertheless, these methods have different strengths and pitfalls (Table 2), leading to a lack of agreement on a gold-standard technique to be used in EV research. The main problems with ultracentrifugation are its suboptimal yield and the low purity of the EVs obtained due to coprecipitation of contaminants and alteration of EV intactness (Linares, Tan, Gounou, Arraud, & Brisson, 2015;Wiklander et al, 2018); its time-consuming, divergent protocols, with deficient standardization of efficiency (Momen-Heravi et al, 2012); and the costly equipment required (Théry, Clayton, Amigorena, & Raposo, 2006). Precipitation-based protocols allegedly yield greater EV recovery but are actually linked to undesirable contaminant co-precipitation with EVs.…”

“…Immunoaffinitybased isolation is also a promising method, especially in combination with microfluidic methodologies, despite its high cost in terms of consumables. In this regard, efforts are being made to find pan-EV, specific markers expressed on EVs that circumvent heterogeneity, which has proven to be arduous work (Balaj et al, 2015;Ghosh et al, 2014;Kanwar, Dunlay, Simeone, & Nagrath, 2014;Morton, 2013;Nakai et al, 2016;Wiklander et al, 2018).…”