Recombinant extracellular vesicles: biological reference material to standardize extracellular vesicle research

• Published in: Journal of extracellular vesicles 2018-01, Vol.7, p.268-268
• Main Authors: Geeurickx, Edward, Heyrman, Elisa, Van Deun, Jan, Lippens, Lien, Tulkens, Joeri, Dhondt, Bert, Vergauwen, Glenn, Gevaert, Kris, Impens, Francis, Miinalainen, Ikka, Van Bockstal, Pieter-Jan, De Beer, Thomas, Wauben, Marca H M, Nolte-'t-Hoen, Esther N M, Swinnen, Johan, Eyckerman, Sven, Mestdagh, Pieter, Vandesompele, Jo, Braems, Geert, Denys, Hannelore, De Wever, Olivier, Hendrix, An
• Format: Article
• Language: English
• Subjects: Biopsy; Breast cancer; Cell culture; Electron microscopy; Enzyme-linked immunosorbent assay; Extracellular vesicles; Flow cytometry; Freeze-thawing; Morphology; mRNA; Nanoparticles; Plasma; Proteomics; Reference materials; Size distribution; Ultracentrifugation; Zeta potential
• ISSN: 2001-3078

Background: Extracellular vesicles (EV) derived from liquid biopsies are emerging as potent biomarkers in health and disease. However, the complexity of liquid biopsies and the plethora of isolation and detection methods introduce variability that impedes interlaboratory concordance and clinical application. To evaluate and mitigate this variability, we developed recombinant EV (rEV) as a biological reference material with unique traceability, and physical and biochemical similarity to endogenous EV (eEV). Methods: rEV are purified by density gradient (DG) from cell culture supernatant of HEK293T cells expressing an eGFP-tagged self-assembling protein that directs its own release. We studied the similarity of rEV and eEV using electron microscopy, zeta potential analysis, nanoparticle tracking analysis (NTA), lipidomics and proteomics. We assessed the traceability, stability and commutability of rEV using fluorescent NTA (fNTA), flow cytometry (FC), fluorescent microplate reader, quantitative real time PCR (qRT-PCR) and ELISA. rEV was spiked in plasma to calculate the recovery efficiency of EV isolation methods and to normalize eEV numbers in plasma using fNTA and ELISA. Results: rEV shows biophysical and biochemical similarity to eEV such as morphology, zeta potential, size distribution, density and protein/lipid content. rEV can be accurately quantified by fNTA and FC in eEVcomprising samples. In addition, rEV behaves linearly with fluorescent intensity levels (R2 = 0.969) and ELISA concentrations (R2 = 0.978), and semi-logarithmic with qRT-PCR for eGFP mRNA (R2 = 0.938). rEV is stable during multiple freeze-thaw cycles at -80°C and can be lyophilized without changes in morphology, concentration and aggregation. EV recoveries from plasma for size-exclusion chromatography, differential ultracentrifugation, DG and ExoQuick were respectively 100%, 10%, 30% and 100%. For the first time, we could calculate the normalized EV concentration for breast cancer patients, which was significantly higher than healthy individuals (1.77E11 vs 6.51E10 particles/mL plasma). Summary/Conclusion: We developed rEV, a biological reference material for EV research which can be used as positive control, spike-in material or calibrator to ensure standardized EV measurements in various applications.