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1 advances.sciencemag.org/cgi/content/full/3/8/e /dc1 Supplementary Materials for Functional vascularized lung grafts for lung bioengineering N. Valerio Dorrello, Brandon A. Guenthart, John D. O Neill, Jinho Kim, Katherine Cunningham, Ya-Wen Chen, Mauer Biscotti, Theresa Swayne, Holly M. Wobma, Sarah X. L. Huang, Hans-Willem Snoeck, Matthew Bacchetta, Gordana Vunjak-Novakovic The PDF file includes: Published 30 August 2017, Sci. Adv. 3, e (2017) DOI: /sciadv table S1. Primary antibodies. table S2. Secondary antibodies. fig. S1. Lung de-epithelialization. fig. S2. Optimization of de-epithelialization solution in rodent lungs on EVLP. fig. S3. Efficiency of de-epithelialization. fig. S4. Preservation of lung structure and ECM. fig. S5. Dynamic lung compliance. fig. S6. Vascular viability and function after lung de-epithelialization. fig. S7. Cell attachment and viability in de-epithelialized lung. fig. S8. Schematic of ex vivo lung regeneration. fig. S9. Outline of stereologic analysis. Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/3/8/e /dc1) movie S1 (.mp4 format). HFOV before de-epithelialization on EVLP. movie S2 (.mp4 format). HFOV during de-epithelialization on EVLP. movie S3 (.mov format). Native lung perfusion with microspheres. movie S4 (.mov format). De-epithelialized lung perfusion with microspheres.
2 table S1. Primary antibodies.
3 table S2. Secondary antibodies.
4 fig. S1. Lung de-epithelialization. (A) Double lung cannulation demonstrating Pulmonary Artery (PA) cannula (blue) and Left Ventricle (LV) cannula (red). Characteristics of PA and LV cannulas (boxed area). (B) Experimental setup of EVLP circuit. (C) Haemogas analysis of rodent blood (n = 6) in EVLP circuit. Photos of blood reservoir demonstrate color change in blood prior to EVLP (de-oxygenated) and during EVLP (oxygenated). Mean and ± s.d. of experimental values. (D) Single lung isolation and de-epithelialization (left lung, blue). Blue dye was added to de-epithelialization solution to confirm isolation of left lung and distribution of deepithelialization solution.
5 fig. S2. Optimization of de-epithelialization solution in rodent lungs on EVLP. (A) Summary of solutions used for de-epithelialization. (B) Lung map used for sampling. Lung was divided into 3 regions for each side. (C) Histological analysis of different treatments: native; 4mM CHAPS 0.3M NaCl; 4mM CHAPS 0.5M NaCl (de-epithelialization solution chosen); 8mM CHAPS 0.5M NaCl; 4mM CHAPS 1M NaCl.
6 fig. S3. Efficiency of de-epithelialization. (A to D) Immunofluorescence staining confirming preservation of fibroblasts in de-epithelialized lung (B and D) indicated by vimentin. Vessel (star); airway (arrow). (E and F) Immunofluorescence staining showing loss of EpCAM in the respiratory zone of de-epithelialized lung. (G and I) Western blot of lung epithelial cell-specific markers (Aquaporin 5, SPC, CC-10, Acetyl-tubulin) from native and de-epithelialized lung (G). Ponceau red staining showing similar protein loading of native and de-epithelialized lung samples. (H) Quantification data indicate de-epithelialized lungs contained significantly reduced amounts of all epithelial cell markers (n = 3, values normalized to levels in native lung). Error bars, s.d. ± mean of experimental values. (I) Western blot of CD31 and EpCAM from native and de-epithelialized lungs (asterisk indicates non-specific band). Small airway epithelial cells (SAEC) and human umbilical vein endothelial cells (HUVEC) as EpCAM and CD31 positive controls, respectively. Human CD31 runs higher in electrophoresis due to higher molecular weight compared to rat CD31. (J to M) Transmission electron microscopy of native and deepithelialized lung. In J, an intact alveolus surrounded by capillaries is shown; in L endothelial cells (asterisk), ATII (arrowhead) and ATI (cross) are shown. In K, alveoli are de-epithelialized, while capillaries with endothelial cells are preserved; in M, only endothelial cells (asterisk) within a capillary are visualized between alveoli. A
7 fig. S4. Preservation of lung structure and ECM. (A) Immunostaining and special staining of the respiratory zone of native and de-epithelialized lung: Collagen I, Alcian blue, Trichrome, van Gieson. (B to G) Ultrastructural morphology of native and de-epithelialized lungs. Scanning electron microscopy of native lung (B, D, F) and de-epithelialized lung (C, E, G) showing preservation of airway and vascular architecture. Airway (arrowhead); vessel (asterisk).
8 fig. S5. Dynamic lung compliance. (A) Schematic of experimental lungs showing native right lung and de-epithelialized left lung. Pressure-volume loops of (B) native lung (control) (n = 3, ml/cm H 2O ± ). Compliance of the native lung did not change while the contralateral lung was de-epithelialized. (C) de-epithelialized lung pre and post-treatment (n = 3, ml/cm H 2O ± , after treatment ml/cm H 2O ± Compliance decreased by ~61.82 % ± 3.31). (D to G) Methacholine challenge experiment. Schematic of experimental lungs during methacholine infusion (D). Pressure-volume loops of native (E) and deepithelialized lung (F and G), pre and post-methacholine. For de-epithelialized lung, higher magnification of dynamic lung compliance after methacholine treatment is shown in the insert (G). Dynamic compliance data explained in the main text. Values expressed as mean ± s.d..
9 fig. S6. Vascular viability and function after lung de-epithelialization. (A) Immunofluorescence staining demonstrating the preservation of endothelial cells (vwf, CD31), vascular smooth muscle (SMA), pericytes (NG2), tight and gap junction proteins (ZO-1, Connexin 43) of the vascular bed following de-epithelialization. Vessel (asterisk). (B and C) Viability of endothelial cells by capture of acetylated LDL (aldl) in macro-vasculature of native and de-epithelialized lungs. Vessel (asterisk); airway (arrowhead). (D and E) Apoptotic endothelial cells shown by TUNEL and vwf co-staining in native and de-epithelialized lungs. Vessel (asterisk); apoptotic cells (arrowhead). (F and G) Pressure traces following infusion of endothelin-1 (E) and treprostinil (F) in de-epithelialized lung.
10 fig. S7. Cell attachment and viability in de-epithelialized lung. (A to D) SAECs attachment and vascular preservation following recellularization of de-epithelialized lung. Preservation of macro- and micro-vasculature (A and B). Vessel (asterisk). Recellularization of conductive and respiratory zones with reconstitution of bronchial and alveolar lining (C and D). Bronchi (full arrowhead), Alveoli (empty arrowhead). (E and F) CSFE-labeled SAECs attachment in alveoli, and Ki67 staining. Ki67 positive SAECs (full arrowhead); Ki67 positive endogenous cells (empty arrowhead) (E). DAPI, Ki67, CSFE staining and merge of SAECs cells in recellularized lung (F). (G and H) Day human ipsc-derived lung-specified epithelial cells attached in deepithelialized lung. (I and J) Day human ipsc-derived lung-specified epithelial cells attached in de-epithelialized lung expressing human alveolar type II cell marker HT J shows in detail the punctate pattern of HT2-280 staining. (K and L) Similar staining pattern of HT2-280 can also be observed in adult human lung tissue. J and L show higher magnification of boxed areas.
11 fig. S8. Schematic of ex vivo lung regeneration. Envisioned clinical application of lung deepithelialization to recover and regenerate injured lungs prior to transplantation.
12 fig. S9. Outline of stereologic analysis. (A) Lung section divided into grid of square fields. (B) Random fields selected for analysis. (C) Random field with line grid for surface measurements. (D) Magnified view of boxed area in C, showing marked intercepts with septa. (E) Random field with point grid for volume fraction measurements. (F) Magnified view of boxed area in E showing magenta point probes marked as alveolar airspace (dark blue), alveolar duct airspace (yellow), and septa (light blue). Scale bars: A and B, 1000 µm; C and E, 50 µm; E and F, 20 µm.
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