Chapter 5 122 ABSTRACT Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. The characterization of EVs released by kidney grafts during Normothermic Machine Perfusion (NMP) may present a promising avenue to assess graft status prior to transplantation. Here, we phenotyped and determined the concentrations of EVs in the perfusate of 8 discarded expanded-criteria donor (ECD) human kidneys during 6 hours of NMP. Perfusate samples were taken at 0/60/180/360 minutes and examined with Nanoparticle Tracking Analysis (NTA) and Imaging Flow Cytometry (IFCM). Using IFCM, EVs were identified by their expression of common EV markers CD9, CD63 and CD81 (tetraspanins) in combination with either platelet endothelial cell adhesion molecule (CD31), pan-leukocyte protein (CD45) or CFSE fluorescence. NTA measurements revealed the release of nanoparticles <400 nm into the perfusate during NMP. With IFCM, tetraspanin protein signatures of the released nanoparticles were characterized, and the majority (~75%) of CFSE+ EV were found to be CD81+ while ~16% were CD9+ and ~8% CD63+. Correlation analysis of concentrations of identified EV subset with crude donor characteristics and NMP viability characteristics revealed significant correlations with cold ischemia time, donor age, and renal flow. Our findings demonstrate that discarded ECD kidney grafts release distinct subsets of EV during NMP. As these subsets correlate with well-established indicators of transplant outcome, EV might represent new potential candidates for assessment of kidney graft quality.
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