Perfusion decellularization of whole organs

The native extracellular matrix (ECM) outlines the architecture of organs and tissues. It provides a unique niche of composition and form, which serves as a foundational scaffold that supports organ-specific cell types and enables normal organ function. Here we describe a standard process for pressure-controlled perfusion decellularization of whole organs for generating acellular 3D scaffolds with preserved ECM protein content, architecture and perfusable vascular conduits. By applying antegrade perfusion of detergents and subsequent washes to arterial vasculature at low physiological pressures, successful decellularization of complex organs (i.e., hearts, lungs and kidneys) can be performed. By using appropriate modifications, pressure-controlled perfusion decellularization can be achieved in small-animal experimental models (rat organs, 4–5 d) and scaled to clinically relevant models (porcine and human organs, 12–14 d). Combining the unique structural and biochemical properties of native acellular scaffolds with subsequent recellularization techniques offers a novel platform for organ engineering and regeneration, for experimentation ex vivo and potential clinical application in vivo.

[1]  V. Barocas,et al.  Effects of Freezing and Cryopreservation on the Mechanical Properties of Arteries , 2006, Annals of Biomedical Engineering.

[2]  S. Tanase,et al.  Activation of Human Matrix Metalloproteinases by Various Bacterial Proteinases* , 1997, The Journal of Biological Chemistry.

[3]  Yun Chen,et al.  Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. , 2010, Tissue engineering. Part C, Methods.

[4]  Laura E Niklason,et al.  Decellularized tissue-engineered blood vessel as an arterial conduit , 2011, Proceedings of the National Academy of Sciences.

[5]  Hiroshi Yagi,et al.  Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix , 2010, Nature Medicine.

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

[7]  Aline M. Betancourt,et al.  A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells. , 2012, Tissue engineering. Part A.

[8]  B. Sicari,et al.  Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering. , 2013, Biomaterials.

[9]  A. Lichtenberg,et al.  Detergent decellularization of heart valves for tissue engineering: toxicological effects of residual detergents on human endothelial cells. , 2010, Artificial organs.

[10]  Guoping Chen,et al.  Decellularized matrices for tissue engineering , 2010, Expert opinion on biological therapy.

[11]  C. Howe,et al.  Removal of unbound sodium dodecyl sulfate (SDS) from proteins in solution by electrophoresis through triton x-100-agarose. , 1978, Journal of immunological methods.

[12]  W. Boyd,et al.  Pericardial reconstruction using an extracellular matrix implant correlates with reduced risk of postoperative atrial fibrillation in coronary artery bypass surgery patients. , 2010, The heart surgery forum.

[13]  Donald O Freytes,et al.  Reprint of: Extracellular matrix as a biological scaffold material: Structure and function. , 2015, Acta biomaterialia.

[14]  T. Ashikaga,et al.  The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs. , 2013, Biomaterials.

[15]  Anthony Atala,et al.  Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system. , 2012, Biomaterials.

[16]  C. Werner,et al.  Dewaxed ECM: A simple method for analyzing cell behaviour on decellularized extracellular matrices , 2015, Journal of tissue engineering and regenerative medicine.

[17]  Xi Ren,et al.  Perfusion decellularization of human and porcine lungs: bringing the matrix to clinical scale. , 2014, The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation.

[18]  A. Lichtenberg,et al.  The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities. , 2011, Tissue engineering. Part C, Methods.

[19]  Yitao Ding,et al.  Evaluation of two decellularization methods in the development of a whole‐organ decellularized rat liver scaffold , 2013, Liver international : official journal of the International Association for the Study of the Liver.

[20]  G. Vunjak‐Novakovic,et al.  Decellularization of human and porcine lung tissues for pulmonary tissue engineering. , 2013, The Annals of thoracic surgery.

[21]  Lei Yang,et al.  Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells , 2013, Nature Communications.

[22]  Michael J. Thrall,et al.  Human lung cancer cells grown on acellular rat lung matrix create perfusable tumor nodules. , 2012, The Annals of thoracic surgery.

[23]  P. Bornstein,et al.  Matricellular proteins: extracellular modulators of cell function. , 2002, Current opinion in cell biology.

[24]  Donald O Freytes,et al.  Preparation of cardiac extracellular matrix from an intact porcine heart. , 2010, Tissue engineering. Part C, Methods.

[25]  T. Jensen,et al.  A rapid lung de-cellularization protocol supports embryonic stem cell differentiation in vitro and following implantation. , 2012, Tissue engineering. Part C, Methods.

[26]  Joseph P. Vacanti,et al.  Bio-engineered endocrine pancreas based on decellularized pancreatic matrix and mesenchymal stem cell/islet cell coculture , 2010 .

[27]  M. Dalsing,et al.  Effectiveness of an extracellular matrix graft (OASIS Wound Matrix) in the treatment of chronic leg ulcers: a randomized clinical trial. , 2005, Journal of vascular surgery.

[28]  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.

[29]  S. Badylak,et al.  Extracellular matrix as a biological scaffold material: Structure and function. , 2009, Acta biomaterialia.

[30]  Stephen F Badylak,et al.  An overview of tissue and whole organ decellularization processes. , 2011, Biomaterials.

[31]  J. Guyette,et al.  Regeneration and Experimental Orthotopic Transplantation of a Bioengineered Kidney , 2013, Nature Medicine.

[32]  BurkJanina,et al.  Freeze-thaw cycles enhance decellularization of large tendons. , 2014 .

[33]  P. Iaizzo,et al.  Freeze–Thaw Induced Biomechanical Changes in Arteries: Role of Collagen Matrix and Smooth Muscle Cells , 2010, Annals of Biomedical Engineering.

[34]  Ernst Wolner,et al.  Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells. , 2004, The Journal of thoracic and cardiovascular surgery.

[35]  Jean A. Niles,et al.  Production and assessment of decellularized pig and human lung scaffolds. , 2013, Tissue engineering. Part A.

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

[37]  Harald C Ott,et al.  Perspectives on whole-organ assembly: moving toward transplantation on demand. , 2012, The Journal of clinical investigation.

[38]  M. Conconi,et al.  Structural and morphologic evaluation of a novel detergent-enzymatic tissue-engineered tracheal tubular matrix. , 2009, The Journal of thoracic and cardiovascular surgery.

[39]  D. Navajas,et al.  Effects of freezing/thawing on the mechanical properties of decellularized lungs. , 2013, Journal of biomedical materials research. Part A.

[40]  M. Bissell,et al.  Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. , 2006, Annual review of cell and developmental biology.

[41]  Anthony Callanan,et al.  Comparison of methods for whole-organ decellularization in tissue engineering of bioartificial organs. , 2013, Tissue engineering. Part B, Reviews.

[42]  K. Yamamoto,et al.  Quantitative determination by derivative spectrophotometry of Triton X-100 in solubilized preparations of membrane proteins. , 1985, Analytical biochemistry.

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

[44]  A. Landsman,et al.  Extracellular matrix biomaterials for soft tissue repair. , 2009, Clinics in podiatric medicine and surgery.

[45]  Min Yang,et al.  Favorable effects of the detergent and enzyme extraction method for preparing decellularized bovine pericardium scaffold for tissue engineered heart valves. , 2009, Journal of biomedical materials research. Part B, Applied biomaterials.

[46]  Stephen F Badylak,et al.  Decellularization of tissues and organs. , 2006, Biomaterials.

[47]  J. M. Wallis,et al.  Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization. , 2012, Tissue engineering. Part C, Methods.

[48]  F. Rusconi,et al.  Quantification of sodium dodecyl sulfate in microliter-volume biochemical samples by visible light spectroscopy. , 2001, Analytical biochemistry.

[49]  Doris A Taylor,et al.  Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart , 2008, Nature Medicine.

[50]  Hélène A. Simon,et al.  FLUID MECHANICS OF ARTIFICIAL HEART VALVES , 2009, Clinical and experimental pharmacology & physiology.

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

[52]  Timo K. Korhonen,et al.  Bacterial plasminogen receptors: in vitro evidence for a role in degradation of the mammalian extracellular matrix , 1995, Infection and immunity.

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

[54]  Paolo De Coppi,et al.  Production and Implantation of Renal Extracellular Matrix Scaffolds From Porcine Kidneys as a Platform for Renal Bioengineering Investigations , 2012, Annals of surgery.