Dhruva K. Mishra, PhD, Michael J. Thrall, MD, Brandi N

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Human Lung Cancer Cells Grown on Acellular Rat Lung Matrix Create Perfusable Tumor Nodules Dhruva K. Mishra, PhD, Michael J. Thrall, MD, Brandi N. Baird, MS, Harald C. Ott, MD, Shanda H. Blackmon, MD, MPH, Jonathan M. Kurie, MD, Min P. Kim, MD The Annals of Thoracic Surgery Volume 93, Issue 4, Pages 1075-1081 (April 2012) DOI: 10.1016/j.athoracsur.2012.01.011 Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Decellularization unit and bioreactor. (A) Three customized decellularization chambers connected to a pump.(B) Four customized bioreactors inside an incubator connected to a pump and an oxygenator. The Annals of Thoracic Surgery 2012 93, 1075-1081DOI: (10.1016/j.athoracsur.2012.01.011) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Decellularized rat lung.(A) Freshly harvested intact native rat lung with heart block. (B) Hematoxylin and eosin staining of native rat lung showing cellularized alveoli with pneumocytes and endothelial cells (×20 magnification). (C) Translucent acellular lung after sodium dodecyl sulfate and Triton-X perfusion through pulmonary artery. (D) Hematoxylin and eosin staining of acellular lung lobe showing absence of any cells (×20 magnification). The Annals of Thoracic Surgery 2012 93, 1075-1081DOI: (10.1016/j.athoracsur.2012.01.011) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 DNA concentration of native and acellular lung. Equal amounts of tissue samples were taken for DNA extraction using Qiagen kit. DNA concentration was significantly reduced to less than 5% of native lung tissue. The Annals of Thoracic Surgery 2012 93, 1075-1081DOI: (10.1016/j.athoracsur.2012.01.011) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 A549 human lung cancer cell line grown on acellular rat lung matrix. (A) Lung matrix with A549 cells on day 11 showing tumor nodules. (B) Hematoxylin and eosin staining of left upper lobe on day 3 showing cells attached to matrix in airways, terminal bronchioles, alveolar ducts, and alveoli with no cells in vasculature (×20 magnification). (C, D) Immunohistochemistry staining of lung seeded with A549 cells using Ki-67 (C) shows high proliferative index and using vimentin (×20 magnification) (D) shows presence of intermediate filaments (×20 magnification). The Annals of Thoracic Surgery 2012 93, 1075-1081DOI: (10.1016/j.athoracsur.2012.01.011) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Lung matrix seeded with H460 cells. (A) Numerous tumor nodules on day 7 in lung scaffold. (B, C) Hematoxylin and eosin staining of lung section in low-power field (B) and high-power field (×10 magnification) (C) showing poorly differentiated non–small cell lung cancer with sheetlike growth along airways and alveoli (×20 magnification). The Annals of Thoracic Surgery 2012 93, 1075-1081DOI: (10.1016/j.athoracsur.2012.01.011) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Lung matrix seeded with H1299 cells. (A) Numerous tumor nodules on day 7 in lung scaffold. (B, C) Hematoxylin and eosin staining of lung section in low-power field (B) and high-power field (×4 magnification) (C) showing very poorly differentiated non–small cell lung cancer cells in disorganized fashion (×20 magnification). The Annals of Thoracic Surgery 2012 93, 1075-1081DOI: (10.1016/j.athoracsur.2012.01.011) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions