Biological reference materials for extracellular vesicle studies.
Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.
EV Group, Biochemistry and Biotechnology, Faculty of Biological and Environmental Sciences, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Finland; Finnish Red Cross Blood Service, Helsinki, Finland. Electronic address: sami.valkonen@helsinki.fi.
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Valkonen
Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, The Netherlands; Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, The Netherlands.
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van der Pol
Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
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Böing
Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
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Yuana
Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Finland; Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy.
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Yliperttula
Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
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Nieuwland
Finnish Red Cross Blood Service, Helsinki, Finland.
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Laitinen
EV Group, Biochemistry and Biotechnology, Faculty of Biological and Environmental Sciences, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Finland.
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Siljander
D004912:Erythrocytes / Q000166:cytology*; D000067128:Extracellular Vesicles / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D046529:Microscopy, Electron, Transmission; D049329:Nanostructures / Q000737:chemistry / Q000592:standards* / Q000648:ultrastructure; D011510:Proteolipids / Q000737:chemistry / Q000592:standards*; D012015:Reference Standards
D011510:Proteolipids; C020485:proteoliposomes
Characterization; Extracellular vesicles; Nanoerythrosome; Quantification; Reference material; Standardization