Production and assessment of decellularized pig and human lung scaffolds.

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.

[1]  Angela Panoskaltsis-Mortari,et al.  Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. , 2010, Tissue engineering. Part A.

[2]  L. Bonassar,et al.  Autologous tissue-engineered trachea with sheep nasal chondrocytes. , 2002, The Journal of thoracic and cardiovascular surgery.

[3]  S. Erzurum,et al.  Human primary lung endothelial cells in culture. , 2012, American journal of respiratory cell and molecular biology.

[4]  Massoud Motamedi,et al.  In vivo multimodal nonlinear optical imaging of mucosal tissue. , 2004, Optics express.

[5]  Korkut Uygun,et al.  Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. , 2011, Annual review of biomedical engineering.

[6]  Jean A. Niles,et al.  Production and utilization of acellular lung scaffolds in tissue engineering , 2012, Journal of cellular biochemistry.

[7]  K. McCurry,et al.  Lung Transplantation in the United States, 1998–2007 , 2009, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[8]  F. de Robertis,et al.  Early outcomes of bilateral sequential single lung transplantation after ex-vivo lung evaluation and reconditioning. , 2011, The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation.

[9]  W. Webb,et al.  Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  A. Pena,et al.  Second harmonic imaging and scoring of collagen in fibrotic tissues. , 2007, Optics express.

[11]  P. Jaksch,et al.  The role of chemokine receptors in acute lung allograft rejection , 2009, European Respiratory Journal.

[12]  K. Wood,et al.  Role of T cells in graft rejection and transplantation tolerance , 2010, Expert review of clinical immunology.

[13]  N. Roberts,et al.  Human macrophage responses to vaccine strains of influenza virus: synthesis of viral proteins, interleukin-1β, interleukin-6, tumour necrosis factor-α and interleukin-1 inhibitor , 1993 .

[14]  Mengyan Li,et al.  Engineering three-dimensional pulmonary tissue constructs. , 2006, Tissue engineering.

[15]  Sergey Plotnikov,et al.  Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure , 2012, Nature Protocols.

[16]  Joan E Nichols,et al.  Engineering of a complex organ: progress toward development of a tissue-engineered lung. , 2008, Proceedings of the American Thoracic Society.

[17]  Sara Mantero,et al.  Clinical transplantation of a tissue-engineered airway , 2008, The Lancet.

[18]  H. Ott,et al.  Enhanced in vivo function of bioartificial lungs in rats. , 2011, The Annals of thoracic surgery.

[19]  J. Belperio,et al.  Chemokines and Transplant Vasculopathy , 2008, Circulation research.

[20]  Jean A. Niles,et al.  Human Lymphocyte Apoptosis after Exposure to Influenza A Virus , 2001, Journal of Virology.

[21]  Qiuming Liao,et al.  First human transplantation of a nonacceptable donor lung after reconditioning ex vivo. , 2007, The Annals of thoracic surgery.

[22]  F. Jatene,et al.  Histologic and functional evaluation of lungs reconditioned by ex vivo lung perfusion. , 2011, The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation.

[23]  Joan E. Nichols,et al.  Engineering Complex Synthetic Organs , 2011 .

[24]  K. König,et al.  Multiphoton autofluorescence imaging of intratissue elastic fibers. , 2005, Biomaterials.

[25]  Jean A. Niles,et al.  Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation. , 2010, Tissue engineering. Part A.

[26]  Zhen W. Zhuang,et al.  Tissue-Engineered Lungs for in Vivo Implantation , 2010, Science.

[27]  Charles A Vacanti,et al.  Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth. , 2006, Tissue engineering.

[28]  B. Tromberg,et al.  Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Zlotnik,et al.  Recent advances in chemokines and chemokine receptors. , 1999, Critical reviews in immunology.

[30]  Leslie M Loew,et al.  Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms , 2003, Nature Biotechnology.

[31]  D. Ingbar,et al.  Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro. A novel system for type II pneumocyte culture. , 1986, Experimental cell research.

[32]  S. Steen,et al.  Transplantation of lungs from a non-heart-beating donor , 2001, The Lancet.

[33]  Christian Schuetz,et al.  Regeneration and orthotopic transplantation of a bioartificial lung , 2010, Nature Medicine.

[34]  Marie-Claire Schanne-Klein,et al.  Three‐dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy , 2007, Microscopy research and technique.

[35]  J. Orens,et al.  Lung transplant outcomes: a review of survival, graft function, physiology, health-related quality of life and cost-effectiveness , 2004, European Respiratory Journal.

[36]  A. Luster,et al.  Chemokines--chemotactic cytokines that mediate inflammation. , 1998, The New England journal of medicine.