Ex Vivo Lung Perfusion Model to Study Pulmonary Tissue Extracellular Microvesicle Profiles

Extracellular microvesicles (EVs) are being increasingly studied for their diagnostic potential. We investigated the feasibility of studying the kinetics and tissue-specific profiles of pulmonary EVs in the context of ex vivo lung perfusion (EVLP) used for salvaging marginal lungs for transplantation.

Perfusate from six marginal donor lungs placed on EVLP was collected at the following time points: 0, 10, and 60 minutes after and after perfusate exchange with Steen Solution; 120 and 180 minutes. Three lungs were successfully recovered for transplantation (transplant group), and three were not recoverable (nontransplant group). Perfusate EVs were isolated using methods of size exclusion chromatography, ultrafiltration, and ultracentrifugation. EVs were analyzed on NanoSight nanoparticle detector for quantity, size distribution, and surface expression of pulmonary tissue-specific markers. EV cargoes were profiled using mass spectrometry, reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis.

Time course analysis showed EV presence by 10-minute time point. EV median size was smaller in the transplant group (165 nm versus 212 nm, p = 0.04). EV cargo analysis on Western blot analysis, RT-PCR, and NanoSight showed contribution from monocytes (CD14), endothelium (platelet endothelial cell adhesion molecule 1), and pulmonary parenchyma (epithelial cell adhesion molecule) into the perfusate total EV pool. Mass spectrometry showed differences in the EV protein cargoes of the transplant group versus the nontransplant group.

EVLP system provides a platform to understand the kinetics of pulmonary EVs in an isolated fashion. Donor lung recovery may be associated with changes in EV size distribution and proteomic profiles. Pulmonary tissue-specific EV profiling using the EVLP system may provide insights into EV contribution to pulmonary pathologic processes.