The generation and use of recombinant extracellular vesicles as biological reference material

Abstract

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.

Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration

“…Several efforts are being undertaken to develop reference materials as EV mimetics for use as validation of assays and quality controls [34][35][36][37]. Some come in the form of synthetic materials meant to mimic biological materials, such as hollow organosilica beads and liposomes, while others are derived from biological sources [34,35,37,38]. Synthetic materials such as hollow beads have the benefit of utilizing characteristics of EVs with fine control, such as low refractive indices and a coreshell structure, whilst being in the format of a tightly defined population which will likely be unambiguous in its detectability using optical methods.…”

“…Dispersion properties of polystyrene, silica and water were calculated using the Sellmeier equations for published materials [53][54][55]. Median refractive index (geometric mean in case of Gardiner et al) measurements for different EV sources were acquired from the literature [22,23,34,40,41].…”

Urinary Biomarkers in Bladder Cancer: Where Do We Stand and Potential Role of Extracellular Vesicles

“…This is mostly due to inter-laboratory variability, lack of widely accepted EV-marker normalization methodologies, backed up by the limited biological sample availability to test all these conditions in a single experiment. Recently, the use of “spike-in” fluorescent EVs standards were proposed to monitor the efficacy of pre-analytical methods and EV separation procedures [ 172 ]. Their use could enable data normalization across laboratories and a deeper comprehension on the causes of variability in the urinary EV cargo.…”

Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

“…Their success as immunogens and nanocarriers has been demonstrated in numerous preclinical and clinical trials over the last years (Charlton Hume et al, 2019). Among the different VLP candidates, enveloped human immunodeficiency virus‐1 (HIV‐1) Gag VLPs are in the spotlight as a scaffold for antigen presentation against different diseases (Cervera et al, 2019; Charlton Hume et al, 2019) and as a reference material for the production of extracellular vesicles (EVs; Geeurickx et al, 2019). The HIV‐1 structural polyprotein (Gag) has the capacity to form noninfective viral particles on their own (Göttlinger, 2001).…”