PhD defence W.W. (Wouter) Woud

Summary:

Extracellular Vesicles (EVs) are a heterogeneous group of sub-micron sized lipid bilayered membrane structures. EVs are ubiquitously present in biofluids (e.g. blood plasma and urine), and their biochemical composition is thought to reflect the status of their cell of origin. Though the current gold standard for EV analysis involves the isolation of EVs, emerging evidence suggests that EV isolation techniques may alter EV properties. To allow the direct monitoring of an individuals’ status (as reflected in EV concentration, size, protein makeup and content), new methods for EV analysis – which do not require EV isolation – are needed.

Part I of this thesis presents Imaging Flow Cytometry (IFCM) as a suitable technique that can detect single EVs, discriminate distinct EV subpopulations, and distinguish EVs from non-EV particles in complex biofluids without prior EV isolation.  Part II of this thesis demonstrates the characterization of EV subpopulations in complex biofluids in the context of clinical kidney transplantation using the developed IFCM-based methodology. This thesis demonstrates that marginal donor kidneys release different EV subsets “on the pump” (before transplantation), and that some of these subsets are correlated with well-established parameters of kidney quality. Additionally, this thesis describes the identification and monitoring of donor-derived EVs in blood samples obtained from kidney transplant recipients (after transplantation), demonstrating the ability of the developed methodology to identify clinically relevant EV subsets. 

In summary, the research as described in this thesis represents a technological advancement for the EV-field by demonstrating 1) the first-ever full calibration of IFCM for sub-micron particle detection, and 2) the detection of individual, single EVs in complex, clinically relevant biofluids.