Orthotopic transplantation of the bioengineered lung using a mouse-scale perfusion-based bioreactor and human primary endothelial cells.

Whole lung engineering and the transplantation of its products is an ambitious goal and ultimately a viable solution for alleviating the donor-shortage crisis for lung transplants. There are several limitations currently impeding progress in the field with a major obstacle being efficient revascularization of decellularized scaffolds, which requires an extremely large number of cells when using larger pre-clinical animal models. Here, we developed a simple but effective experimental pulmonary bioengineering platform by utilizing the lung as a scaffold. Revascularization of pulmonary vasculature using human umbilical cord vein endothelial cells was feasible using a novel in-house developed perfusion-based bioreactor. The endothelial lumens formed in the peripheral alveolar area were confirmed using a transmission electron microscope. The quality of engineered lung vasculature was evaluated using box-counting analysis of histological images. The engineered mouse lungs were successfully transplanted into the orthotopic thoracic cavity. The engineered vasculature in the lung scaffold showed blood perfusion after transplantation without significant hemorrhage. The mouse-based lung bioengineering system can be utilized as an efficient ex-vivo screening platform for lung tissue engineering.

Emerging Paradigms in Bioengineering the Lungs.

In end-stage lung diseases, the shortage of donor lungs for transplantation and long waiting lists are the main culprits in the significantly increasing number of patient deaths. New strategies to curb this issue are being developed with the help of recent advancements in bioengineering technology, with the generation of lung scaffolds as a steppingstone. There are various types of lung scaffolds, namely, acellular scaffolds that are developed via decellularization and recellularization techniques, artificial scaffolds that are synthesized using synthetic, biodegradable, and low immunogenic materials, and hybrid scaffolds which combine the advantageous properties of materials in the development of a desirable lung scaffold. There have also been advances in the design of bioreactors in terms of providing an optimal regenerative environment for the maturation of functional lung tissue over time. In this review, the emerging paradigms in the field of lung tissue bioengineering will be discussed.

Advanced single-cell technologies to guide the development of bioengineered lungs.

Lung transplantation remains the only viable option for individuals suffering from end-stage lung failure. However, a number of current limitations exist including a continuing shortage of suitable donor lungs and immune rejection following transplantation. To address these concerns, engineering a decellularized biocompatible lung scaffold from cadavers reseeded with autologous lung cells to promote tissue regeneration is being explored. Proof-of-concept transplantation of these bioengineered lungs into animal models has been accomplished. However, these lungs were incompletely recellularized with resulting epithelial and endothelial leakage and insufficient basement membrane integrity. Failure to repopulate lung scaffolds with all of the distinct cell populations necessary for proper function remains a significant hurdle for the progression of current engineering approaches and precludes clinical translation. Advancements in 3D bioprinting, lung organoid models, and microfluidic device and bioreactor development have enhanced our knowledge of pulmonary lung development, as well as important cell-cell and cell-matrix interactions, all of which will help in the path to a bioengineered transplantable lung. However, a significant gap in knowledge of the spatiotemporal interactions between cell populations as well as relative quantities and localization within each compartment of the lung necessary for its proper growth and function remains. This review will provide an update on cells currently used for reseeding decellularized scaffolds with outcomes of recent lung engineering attempts. Focus will then be on how data obtained from advanced single-cell analyses, coupled with multiomics approaches and high-resolution 3D imaging, can guide current lung bioengineering efforts for the development of fully functional, transplantable lungs.

Multiday maintenance of extracorporeal lungs using cross-circulation with conscious swine.

OBJECTIVES: Lung remains the least-utilized solid organ for transplantation. Efforts to recover donor lungs with reversible injuries using ex vivo perfusion systems are limited to <24 hours of support. Here, we demonstrate the feasibility of extending normothermic extracorporeal lung support to 4 days using cross-circulation with conscious swine. METHODS: A swine behavioral training program and custom enclosure were developed to enable multiday cross-circulation between extracorporeal lungs and recipient swine. Lungs were ventilated and perfused in a normothermic chamber for 4 days. Longitudinal analyses of extracorporeal lungs (ie, functional assessments, multiscale imaging, cytokine quantification, and cellular assays) and recipient swine (eg, vital signs and blood and tissue analyses) were performed. RESULTS: Throughout 4 days of normothermic support, extracorporeal lung function was maintained (arterial oxygen tension/inspired oxygen fraction >400 mm Hg; compliance >20 mL/cm H2O), and recipient swine were hemodynamically stable (lactate <3 mmol/L; pH, 7.42 ± 0.05). Radiography revealed well-aerated lower lobes and consolidation in upper lobes of extracorporeal lungs, and bronchoscopy showed healthy airways without edema or secretions. In bronchoalveolar lavage fluid, granulocyte-macrophage colony-stimulating factor, interleukin (IL) 4, IL-6, and IL-10 levels increased less than 6-fold, whereas interferon gamma, IL-1α, IL-1ß, IL-1ra, IL-2, IL-8, IL-12, IL-18, and tumor necrosis factor alpha levels decreased from baseline to day 4. Histologic evaluations confirmed an intact blood-gas barrier and outstanding preservation of airway and alveolar architecture. Cellular viability and metabolism in extracorporeal lungs were confirmed after 4 days. CONCLUSIONS: We demonstrate feasibility of normothermic maintenance of extracorporeal lungs for 4 days by cross-circulation with conscious swine. Cross-circulation approaches could support the recovery of damaged lungs and enable organ bioengineering to improve transplant outcomes.

Molecular, cellular, and bioengineering approaches to stimulate lung regeneration after injury.

The lung is susceptible to damage from a variety of sources throughout development and in adulthood. As a result, the lung has great capacities for repair and regeneration, directed by precisely controlled sequences of molecular and signaling pathways. Impairments or alterations in these signaling events can have deleterious effects on lung structure and function, ultimately leading to chronic lung disorders. When lung injury is too severe for the normal pathways to repair, or if those pathways do not function properly, lung regenerative medicine is needed to restore adequate structure and function. Great progress has been made in recent years in the number of regenerative techniques and their efficacy. This review will address recent progress in lung regenerative medicine focusing on pharmacotherapy including the expanding role of nanotechnology, stem cell-based therapies, and bioengineering techniques. The use of these techniques individually and collectively has the potential to significantly improve morbidity and mortality associated with congenital and acquired lung disorders.

Utilization of Natural Detergent Potassium Laurate for Decellularization in Lung Bioengineering.

IMPACT STATEMENT: Recent advances in tissue engineering using decellularized organ scaffolds have expanded the possibilities for organ replacement therapy. However, detergent-based decellularization itself damages the extracellular matrix (ECM), which results in failure associated with the transplanted bioengineered organ. This study determined that potassium laurate (PL), a natural detergent, significantly reduces lung ECM damage during the decellularization process compared with protocols using sodium dodecyl sulfate. PL-decellularized lungs showed better microarchitecture preservation and low biological reactions after subcutaneous implantation. PL-decellularized scaffolds supported rat lung endothelial cell attachment/proliferation and the bioengineered lungs significantly reduced lung congestion after transplantation.

Lung tissue bioengineering for chronic obstructive pulmonary disease: overcoming the need for lung transplantation from human donors.

Introduction: Chronic obstructive pulmonary disease (COPD) affects more than 380 million people, causing more than 3 million deaths annually worldwide. Despite this enormous burden, currently available therapies are largely limited to symptom control. Lung transplant is considered for end-stage disease but is severely limited by the availability of human organs. Furthermore, the pre-transplant course is a complex orchestration of locating and harvesting suitable lungs, and the post-transplant course is complicated by rejection and infection. Lung tissue bioengineering has the potential to relieve the organ shortage and improve the post-transplant course by generating patient-specific lungs for transplant. Additionally, emerging progenitor cell therapies may facilitate in vivo regeneration of pulmonary tissue, obviating the need for transplant. Areas Covered: We review several lung tissue bioengineering approaches including the recellularization of decellularized scaffolds, 3D bioprinting, genetically-engineered xenotransplantation, blastocyst complementation, and direct therapy with progenitor cells. Articles were identified by searching relevant terms (see Key Words) in the PubMed database and selected for inclusion based on novelty and uniqueness of their approach. Expert Opinion: Lung tissue bioengineering research is in the early stages. Of the methods reviewed, only direct cell therapy has been investigated in humans. We anticipate a minimum of 5-10 years before human therapy will be feasible.

Cell replacement in human lung bioengineering.

BACKGROUND: As the number of patients with end-stage lung disease continues to rise, there is a growing need to increase the limited number of lungs available for transplantation. Unfortunately, attempts at engineering functional lung de novo have been unsuccessful, and artificial mechanical devices have limited utility as a bridge to transplant. This difficulty is largely due to the size and inherent complexity of the lung; however, recent advances in cell-based therapeutics offer a unique opportunity to enhance traditional tissue-engineering approaches with targeted site- and cell-specific strategies. METHODS: Human lungs considered unsuitable for transplantation were procured and supported using novel cannulation techniques and modified ex-vivo lung perfusion. Targeted lung regions were treated using intratracheal delivery of decellularization solution. Labeled mesenchymal stem cells or airway epithelial cells were then delivered into the lung and incubated for up to 6 hours. RESULTS: Tissue samples were collected at regular time intervals and detailed histologic and immunohistochemical analyses were performed to evaluate the effectiveness of native cell removal and exogenous cell replacement. Regional decellularization resulted in the removal of airway epithelium with preservation of vascular endothelium and extracellular matrix proteins. After incubation, delivered cells were retained in the lung and showed homogeneous topographic distribution and flattened cellular morphology. CONCLUSIONS: Our findings suggest that targeted cell replacement in extracorporeal organs is feasible and may ultimately lead to chimeric organs suitable for transplantation or the development of in-situ interventions to treat or reverse disease, ultimately negating the need for transplantation.

Pressure- and flow-controlled media perfusion differently modify vascular mechanics in lung decellularization.

Organ biofabrication is a potential future alternative for obtaining viable organs for transplantation. Achieving intact scaffolds to be recellularized is a key step in lung bioengineering. Perfusion of decellularizing media through the pulmonary artery has shown to be effective. How vascular perfusion pressure and flow vary throughout lung decellularization, which is not well known, is important for optimizing the process (minimizing time) while ensuring scaffold integrity (no barotrauma). This work was aimed at characterizing the pressure/flow relationship at the pulmonary vasculature and at how effective vascular resistance depends on pressure- and flow-controlled variables when applying different methods of media perfusion for lung decellularization. Lungs from 43 healthy mice (C57BL/6; 7-8 weeks old) were investigated. After excision and tracheal cannulation, lungs were inflated at 10 cmH2O airway pressure and subjected to conventional decellularization with a solution of 1% sodium dodecyl sulfate (SDS). Pressure (PPA) and flow (V'PA) at the pulmonary artery were continuously measured. Decellularization media was perfused through the pulmonary artery: (a) at constant PPA=20 cmH2O or (b) at constant V'PA=0.5 and 0.2 ml/min. Effective vascular resistance was computed as Rv=PPA/V'PA. Rv (in cmH2O/(ml/min)); mean±SE) considerably varied throughout lung decellularization, particularly for pressure-controlled perfusion (from 29.1±3.0 in baseline to a maximum of 664.1±164.3 (p<0.05), as compared with flow-controlled perfusion (from 49.9±3.3 and 79.5±5.1 in baseline to a maximum of 114.4±13.9 and 211.7±70.5 (p<0.05, both), for V'PA of 0.5 and 0.2 ml/min respectively. Most of the media infused to the pulmonary artery throughout decellularization circulated to the airways compartment across the alveolar-capillary membrane. This study shows that monitoring perfusion mechanics throughout decellularization provides information relevant for optimizing the process time while ensuring that vascular pressure is kept within a safety range to preserve the organ scaffold integrity.

Using nature's platform to engineer bio-artificial lungs.

Native lung extracellular matrix can be isolated from cadaveric organs via perfusion decellularization and provides a novel scaffold material for lung engineering. Based on this platform, several proof-of-principle studies have demonstrated the feasibility of whole organ recellularization and culture in rodent models and have helped us better understand the numerous challenges in up-scaling to clinically relevant tissues. Standardized protocols to generate whole lung scaffolds of porcine and human scale have been reported, but our understanding of the remaining extracellular matrix components and their properties is incomplete. Effective recellularization will require the isolation and in vitro expansion of clinically relevant cell sources, either from primary or stem cell-derived populations, and techniques to effectively deliver these populations throughout the lung scaffold. Ultimately, only tightly controlled recapitulation of tissue development and repair in vitro will enable us to mature lung grafts to function before implantation. Although substantial progress has been made, we are only beginning to grasp the complexity of this exciting new technology.

Low oxygen tension enhances the generation of lung progenitor cells from mouse embryonic and induced pluripotent stem cells.

Whole-organ decellularization technology has emerged as a new alternative for the fabrication of bioartificial lungs. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are potentially useful for recellularization since they can be directed to express phenotypic marker genes of lung epithelial cells. Normal pulmonary development takes place in a low oxygen environment ranging from 1 to 5%. By contrast, in vitro ESC and iPSC differentiation protocols are usually carried out at room-air oxygen tension. Here, we sought to determine the role played by oxygen tension on the derivation of Nkx2.1+ lung/thyroid progenitor cells from mouse ESC and iPSC. A step-wise differentiation protocol was used to generate Nkx2.1+ lung/thyroid progenitors under 20% and 5% oxygen tension. On day 12, gene expression analysis revealed that Nkx2.1 and Foxa2 (endodermal and early lung epithelial cell marker) were significantly upregulated at 5% oxygen tension in ESC and iPSC differentiated cultures compared to 20% oxygen conditions. In addition, quantification of Foxa2+Nkx2.1+Pax8- cells corresponding to the lung field, with exclusion of the potential thyroid fate identified by Pax8 expression, confirmed that the low physiologic oxygen tension exerted a significant positive effect on early pulmonary differentiation of ESC and iPSC. In conclusion, we found that 5% oxygen tension enhanced the derivation of lung progenitors from mouse ESC and iPSC compared to 20% room-air oxygen tension.

Mechanical properties of acellular mouse lungs after sterilization by gamma irradiation.

Lung bioengineering using decellularized organ scaffolds is a potential alternative for lung transplantation. Clinical application will require donor scaffold sterilization. As gamma-irradiation is a conventional method for sterilizing tissue preparations for clinical application, the aim of this study was to evaluate the effects of lung scaffold sterilization by gamma irradiation on the mechanical properties of the acellular lung when subjected to the artificial ventilation maneuvers typical within bioreactors. Twenty-six mouse lungs were decellularized by a sodium dodecyl sulfate detergent protocol. Eight lungs were used as controls and 18 of them were submitted to a 31kGy gamma irradiation sterilization process (9 kept frozen in dry ice and 9 at room temperature). Mechanical properties of acellular lungs were measured before and after irradiation. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. After irradiation lungs presented higher values of resistance and elastance than before irradiation: RL increased by 41.1% (room temperature irradiation) and 32.8% (frozen irradiation) and EL increased by 41.8% (room temperature irradiation) and 31.8% (frozen irradiation). Similar increases were induced by irradiation in Est and Edyn. Scanning electron microscopy showed slight structural changes after irradiation, particularly those kept frozen. Sterilization by gamma irradiation at a conventional dose to ensure sterilization modifies acellular lung mechanics, with potential implications for lung bioengineering.

Mechanical properties of mouse lungs along organ decellularization by sodium dodecyl sulfate.

Lung decellularization is based on the use of physical, chemical, or enzymatic methods to break down the integrity of the cells followed by a treatment to extract the cellular material from the lung scaffold. The aim of this study was to characterize the mechanical changes throughout the different steps of lung decellularization process. Four lungs from mice (C57BL/6) were decellularized by using a conventional protocol based on sodium dodecyl sulfate. Lungs resistance (R(L)) and elastance (E(L)) were measured along decellularization steps and were computed by linear regression fitting of tracheal pressure, flow, and volume during mechanical ventilation. Transients differences found were more distinct in an intermediate step after the lungs were rinsed with deionized water and treated with 1% SDS, whereupon the percentage of variation reached approximately 80% for resistance values and 30% for elastance values. In conclusion, although a variation in extracellular matrix stiffness was observed during the decellularization process, this variation can be considered negligible overall because the resistance and elastance returned to basal values at the final decellularization step.

Effects of freezing/thawing on the mechanical properties of decellularized lungs.

Lung bioengineering based on decellularized organ scaffolds is a potential alternative for transplantation. Freezing/thawing, a usual procedure in organ decellularization and storage could modify the mechanical properties of the lung scaffold and reduce the performance of the bioengineered lung when subjected to the physiological inflation-deflation breathing cycles. The aim of this study was to determine the effects of repeated freezing/thawing on the mechanical properties of decellularized lungs in the physiological pressure-volume regime associated with normal ventilation. Fifteen mice lungs (C57BL/6) were decellularized using a conventional protocol not involving organ freezing and based on sodium dodecyl sulfate detergent. Subsequently, the mechanical properties of the acellular lungs were measured before and after subjecting them to three consecutive cycles of freezing/thawing. The resistance (RL ) and elastance (EL ) of the decellularized lungs were computed by linear regression fitting of the recorded signals (tracheal pressure, flow, and volume) during mechanical ventilation. RL was not significantly modified by freezing-thawing: from 0.88 ± 0.37 to 0.90 ± 0.38 cmH2 O·s·mL(-1) (mean ± SE). EL slightly increased from 64.4 ± 11.1 to 73.0 ± 16.3 cmH2 O·mL(-1) after the three freeze-thaw cycles (p = 0.0013). In conclusion, the freezing/thawing process that is commonly used for both organ decellularization and storage induces only minor changes in the ventilation mechanical properties of the organ scaffold.