Chapter 7 176 ENGLISH SUMMARY Extracellular Vesicles (EVs) are submicron-sized particles released by all cells. EVs carry proteins on their surface and a variety of macromolecules as cargo – which are thought to reflect the status of their cell of origin. As ‘snapshots’ of the releasing cells, EVs gain more and more interest as potential biomarkers for various diseases. In the context of clinical kidney transplantation, allograft status may be deduced through the analysis of EVs in perfusion fluids, blood plasma, or urine. The current gold standard for the analysis of EVs in such (complex) biological samples involves the separation of EVs from “contaminating” components. In practice, this is complicated as the nature of these components differ according to the sample of interest, but often overlap with EVs in terms of their biophysical properties (e.g. size, density). In recent years it has become clear that isolation methods such as ultracentrifugation (the most used EV-isolation method todate) may alter EV properties – thus influencing down-stream analysis and data interpretation. Additionally, as numerous methodologies to isolate and analyze EVs are employed, the EV-field struggles with the reproducibility of EV research – which is a prerequisite for understanding the biological significance of EVs. Introductory chapter 1 outlines these challenges, as well as the limitations of current EV detection techniques, and identifies the unmet need for an EV analysis platform which allows accurate, reproducible quantitation and characterization of single EVs in complex biological samples. The chapter then continues to describe a set of criteria for a reproducible EV analysis platform, and postulates Imaging Flow Cytometry (IFCM) as a potential technique for the analysis of single EVs in suspension. Before exploring IFCM as a potential EV detection platform, in chapter 2 we first describe the quantitation of nanoparticles released into the perfusion fluids by Expanded-Criteria Donor (ECD) kidneys during Normothermic Machine Perfusion (NMP). The term ‘nanoparticles’ is used to indicate all uncharacterized sub-micron particles (including, but not limited to EVs) in a given sample. NMP is an experimental organ-preservation technique in which a 37 oC, oxygenated perfusion fluid is pumped through the donor kidney. Consequently, cellular metabolism is activated, and we demonstrate that ECD kidneys release nanoparticles during NMP. However, the analysis platform used in these experiments (nanoparticle tracking analysis – NTA) has limited phenotyping capabilities, and consequently, is
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