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Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts. Impact Statement For patients undergoing laryngectomy, no reconstruction methods are available to restore the complex functions of the human larynx. The first promising preclinical results have been achieved with the use of biological scaffolds fabricated from decellularized tissue. However, the complexity of laryngeal tissue composition remains a hurdle to create functional viable grafts, since previously each cell type requires tailored culture conditions. In this study, we report the de novo formation of three humanized laryngeal tissue compartments under one uniform culture condition, a possible keystone in creating vital composite tissue grafts for laryngeal regeneration.

Islet transplantation is superior to extrinsic insulin supplementation in the treating severe Type 1 diabetes. However, its efficiency and longevity are limited by substantial islet loss post-transplantation due to lack of engraftment and vascular supply. To overcome these limitations, we developed a novel approach to bio-fabricate functional, vascularized islet organs (VIOs) ex vivo. We endothelialized acellular lung matrixes to provide a biocompatible multicompartment scaffold with an intact hierarchical vascular tree as a backbone for islet engraftment. Over seven days of culture, islets anatomically and functionally integrated into the surrounding bio-engineered vasculature, generating a functional perfusable endocrine organ. When exposed to supra-physiologic arterial glucose levels in vivo and ex vivo, mature VIOs responded with a physiologic insulin release from the vein and provided more efficient reduction of hyperglycemia compared to intraportally transplanted fresh islets. In long-term transplants in diabetic mice, subcutaneously implanted VIOs achieved normoglycemia significantly faster and more efficiently compared to islets that were transplanted in deviceless fashion. We conclude that ex vivo bio-fabrication of VIOs enables islet engraftment and vascularization before transplantation, and thereby helps to overcome limited islet survival and function observed in conventional islet transplantation. Given recent progress in stem cells, this technology may enable assembly of functional personalized endocrine organs., (Copyright © 2019. Published by Elsevier Ltd.)

Full-thickness skin loss is a challenging problem due to limited reconstructive options, demanding 75 million surgical procedures annually in the United States. Autologous skin grafting is the gold standard treatment, but results in donor-site morbidity and poor aesthetics. Numerous skin substitutes are available on the market to date, however, none truly functions as full-thickness skin due to lack of a vascular network. The creation of an autologous full-thickness skin analogue with a vascular pedicle would result in a paradigm shift in the management of wounds and in reconstruction of full-thickness skin defects. To create a clinically relevant foundation, we generated an acellular skin flap scaffold (SFS) with a perfusable vascular pedicle of clinically relevant size by perfusion decellularization of porcine fasciocutaneous flaps. We then analyzed the yielded SFS for mechanical properties, biocompatibility, and regenerative potential in vitro and in vivo. Furthermore, we assessed the immunological response using an in vivo model. Finally, we recellularized the vascular compartment of an SFS and reconnected it to a recipient's blood supply to test for perfusability. Perfusion decellularization removed all cellular components with preservation of native extracellular matrix composition and architecture. Biaxial testing revealed preserved mechanical properties. Immunologic response and biocompatibility assessed via implantation and compared with native xenogenic skin and commercially available dermal substitutes revealed rapid neovascularization and complete tissue integration. Composition of infiltrating immune cells showed no evidence of allorejection and resembled the inflammatory phase of wound healing. Implantation into full-thickness skin defects demonstrated good tissue integration and skin regeneration without cicatrization. We have developed a protocol for the generation of an SFS of clinically relevant size, containing a vascular pedicle, which can be utilized for perfusion decellularization and, ultimately, anastomosis to the recipient vascular system after precellularization. The observed favorable immunological response and good tissue integration indicate the substantial regenerative potential of this platform.

Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5(+)TP63(+) basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Whole lung extracellular matrix scaffolds can be created by perfusion of cadaveric organs with decellularizing detergents, providing a platform for organ regeneration. Lung epithelial engineering must address both the proximal airway cells that function to metabolize toxins and aid mucociliary clearance and the distal pneumocytes that facilitate gas exchange. Engineered pulmonary vasculature must support in vivo blood perfusion with low resistance and intact barrier function and be antithrombotic. Repopulating the native lung matrix with sufficient cell numbers in appropriate anatomic locations is required to enable organ function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rationale: More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure., Objective: The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell-derived cardiomyocytes., Methods and Results: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell-derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function., Conclusions: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell-derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities., (© 2015 American Heart Association, Inc.)

The primary treatment for end-stage lung disease is lung transplantation. However, donor organ shortage remains a major barrier for many patients. In recent years, techniques for maintaining lungs ex vivo for evaluation and short-term (<12 h) resuscitation have come into more widespread use in an attempt to expand the donor pool. In parallel, progress in whole organ engineering has provided the potential perspective of patient derived grafts grown on demand. As both of these strategies advance to more complex interventions for lung repair and regeneration, the need for a long-term organ culture system becomes apparent. Herein we describe a novel clinical scale bioreactor capable of maintaining functional porcine and human lungs for at least 72 h in isolated lung culture (ILC). The fully automated, computer controlled, sterile, closed circuit system enables physiologic pulsatile perfusion and negative pressure ventilation, while gas exchange function, and metabolism can be evaluated. Creation of this stable, biomimetic long-term culture environment will enable advanced interventions in both donor lungs and engineered grafts of human scale., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

Both substrate topography and substrate mechanical properties are known to influence cell behavior, but little is known about how they act in concert. Here, a method is presented to introduce topographical features into PA hydrogel substrates that span a wide range of physiological E values. Gel swelling plays a significant role in the fidelity of protruding micromolded features, with the most efficient pattern transfer occurring at a crosslinking concentration equal to or greater than ≈5%. In contrast, swelling does not influence the spacing fidelity of microcontact printed islands of collagen on 2D PA substrates. BAECs cultured on micromolded PA substrates exhibit contact guidance along ridges patterned for all E tested., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)