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.
Highlights
Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy
Results recombinant EV (rEV) bear EV-like physical and biochemical traits. rEV were produced in a well-characterized HEK293T cell culture model[16] by transient transfection with retroviral gag polyprotein C-terminally fused to EGFP17. rEV were separated from the conditioned medium (CM) 72 h after transfection of approximately 3 × 109 HEK293T cells by OptiPrep density gradient (ODG) centrifugation and consecutive pelleting resulting in ~5 × 1011 rEV (Fig. 1a–c)
We evaluated at least three biological replicates of rEV for their physical and biochemical characteristics that are principal to EV analysis and compared them to sample EV derived from various sources
Summary
Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. 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. EV research and its biomedical applications are hampered by the myriad of separation methods and measurement instruments and the lack of appropriate reference materials for accurate calibration, normalization and method development[4,5,6]. A reference material suitable for all those purposes should (1) have EV-like physical and biochemical characteristics, should be from biological origin; (2) be trackable and be distinguishable from sample EV and (3) behave as sample EV under various experimental conditions[7]. We define the physical and biochemical characteristics, trackability, stability and commutability of rEV, identify and test suitable read-out methods, provide tools to segregate rEV from sample EV for further downstream approaches and demonstrate the usability of rEV in various applications
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