1. Ex Vivo Lung Perfusion Model to Study Pulmonary Tissue Extracellular Microvesicle Profiles 
Prashanth Vallabhajosyula, MD, MS, Laxminarayana Korutla, PhD, Andreas Habertheuer, MD, PhD, Sanjana Reddy, BS, Christian Schaufler, BS, Jared Lasky, BS, Joshua Diamond, MD, Edward Cantu, MD, PhD 
The Annals of Thoracic Surgery 
Volume 103, Issue 6, Pages (June 2017)
DOI: /j.athoracsur
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

2. Fig 1
Steen Solution, used to perfuse donor lungs on ex vivo lung perfusion (EVLP) circuit, is devoid of extracellular microvesicles (EVs). To validate that EVs isolated from EVLP perfusate are strictly from the donor lungs, Steen Solution was analyzed for EV presence. (A) Steen Solution placed on NanoSight light scatter did not show any exosomes/EVs at baseline. We then performed column chromatography of Steen Solution and checked the eluent fractions where exosomes/EVs are typically present, and we also checked the later fractions. In both sets of fractions, Steen Solution did not show EVs on NanoSight. (B) Western blot analysis of Steen Solution and its eluent fractions showed a single intensity protein band on a Ponceau S blue stain, unlike EVLP at the 60 min pre time point and lung tissue control. (C) Western blot analysis of Steen Solution showed the high presence of albumin at baseline but the absence of exosome markers CD63 and flotillin-1. EVLP 60 min pre time point and lung tissue served as positive controls. (SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

3. Fig 2
Extracellular microvesicles (EVs) isolated from ex vivo lung perfusion (EVLP) circuit express exosome markers. (A) Western blot analysis of EV cargo confirmed expression of canonical exosome markers CD63 and flotillin-1, along with the absence of apoptotic body marker cytochrome c. (B) NanoSight light scatter analysis showed no difference in quantity of EVs between the transplant and nontransplant groups. In each NanoSight panel, EV particle concentration (108/mL) and number of nanoparticles tracked (Tracks) are shown for 1 μg of EV protein. Representative images from the 60-min post time point from the transplant and nontransplant groups are shown.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

4. Fig 3
Extracellular microvesicles (EVs) are detectable in the ex vivo lung perfusion (EVLP) circuit. (A) Representative EV profiles on the NanoSight nanoparticle detector for various time points after starting donor EVLP are shown (minutes). In each NanoSight panel, time point, EV particle concentration (108/mL), and number of nanoparticles tracked (Tracks) for 1 μg of EV protein equivalent are shown. (B) Time course analysis revealed EV presence after 10 minutes on the EVLP circuit with increasing concentrations until 60 minutes and statistically plateauing, nondifferent concentrations thereafter. Mean EV particle concentrations seen per 1 μg of protein for each time point are shown. (C) Box-whisker plot of EV size is shown for the transplant and nontransplant groups. Overall, EV median size was smaller in the transplant group (165 nm versus 212 nm, p = 0.04).
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

5. Fig 4
Lungs on ex vivo lung perfusion EVLP release tissue-specific extracellular microvesicles (EVs) into the perfusate. EVs were tested for expression of tissue-specific marker proteins representing contribution from donor lung epithelial cells (human epithelial cell adhesion molecule [hEpCAM]), monocytes (CD14), endothelial cells (platelet endothelial cell adhesions molecule 1 [PECAM1]), and major histocompatibility complex (MHC) class I and class II expressing cells on the NanoSight. For surface marker expression, EVs were labeled with primary antibody, followed by secondary antibody-conjugated quantum dot (605 nm emission). Tissue-specific EV subsets were detected on the NanoSight fluorescence mode. For each sample, light scatter mode represents the total EV particle concentration, and the fluorescence mode represents the subset of particles that fluoresced for the particular primary antibody. Immunoglobulin G (IgG) isotype served as negative control.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

6. Fig 5
Extracellular microvesicle (EV) protein cargo analysis. EVs express lung epithelial cell protein markers (human epithelial cell adhesion molecule [hEpCAM]) and endothelial cell protein markers (vascular endothelial [VE]-cadherin and platelet endothelial cell adhesion molecule 1 [PECAM-1]). Subpopulation of EVs also express leukocyte monocyte marker, CD14. Canonical exosome markers (CD63 and flotillin-1) and β-actin are shown as controls.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

7. Fig 6
Extracellular microvesicle (EV) mRNA cargo analysis. (A) Expression of lung endothelial and epithelial markers vascular endothelial (VE)-cadherin and human epithelial cell adhesion molecule (hEpCAM) was seen in EVs. mRNA expression of CD14 monocyte marker was also noted. (B) All four surfactant mRNAs (SP-A, SP-B, SP-C, and SP-D) were seen in EVs, suggesting that lung parenchymal tissue releases EVs into the perfusate.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

8. Fig 7
Heatmap. Mass spectrometry analysis of extracellular microvesicle (EV) protein cargo for the transplant group versus the nontransplant group.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions