Lung bioengineering: advances and challenges in lung decellularization and recellularization.

PURPOSE OF REVIEW: Bioengineering the lung based on its natural extracellular matrix (ECM) offers novel opportunities to overcome the shortage of donors, to reduce chronic allograft rejections, and to improve the median survival rate of transplanted patients. During the last decade, lung tissue engineering has advanced rapidly to combine scaffolds, cells, and biologically active molecules into functional tissues to restore or improve the lung's main function, gas exchange. This review will inspect the current progress in lung bioengineering using decellularized and recellularized lung scaffolds and highlight future challenges in the field. RECENT FINDINGS: Lung decellularization and recellularization protocols have provided researchers with tools to progress toward functional lung tissue engineering. However, there is continuous evolution and refinement particularly for optimization of lung recellularization. These further the possibility of developing a transplantable bioartificial lung. SUMMARY: Bioengineering the lung using recellularized scaffolds could offer a curative option for patients with end-stage organ failure but its accomplishment remains unclear in the short-term. However, the state-of-the-art of techniques described in this review will increase our knowledge of the lung ECM and of chemical and mechanical cues which drive cell repopulation to improve the advances in lung regeneration and lung tissue engineering.

Probing Micromechanical Properties of the Extracellular Matrix of Soft Tissues by Atomic Force Microscopy.

The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell-matrix mechanical crosstalk and disease-induced tissue stiffness alterations. J. Cell. Physiol. 232: 19-26, 2017. © 2016 Wiley Periodicals, Inc.

Lung bioengineering: physical stimuli and stem/progenitor cell biology interplay towards biofabricating a functional organ.

A current approach to obtain bioengineered lungs as a future alternative for transplantation is based on seeding stem cells on decellularized lung scaffolds. A fundamental question to be solved in this approach is how to drive stem cell differentiation onto the different lung cell phenotypes. Whereas the use of soluble factors as agents to modulate the fate of stem cells was established from an early stage of the research with this type of cells, it took longer to recognize that the physical microenvironment locally sensed by stem cells (e.g. substrate stiffness, 3D architecture, cyclic stretch, shear stress, air-liquid interface, oxygenation gradient) also contributes to their differentiation. The potential role played by physical stimuli would be particularly relevant in lung bioengineering since cells within the organ are physiologically subjected to two main stimuli required to facilitate efficient gas exchange: air ventilation and blood perfusion across the organ. The present review focuses on describing how the cell mechanical microenvironment can modulate stem cell differentiation and how these stimuli could be incorporated into lung bioreactors for optimizing organ bioengineering.

Regional and disease specific human lung extracellular matrix composition.

Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), are characterized by regional extracellular matrix (ECM) remodeling which contributes to disease progression. Previous proteomic studies on whole decellularized lungs have provided detailed characterization on the impact of COPD and IPF on total lung ECM composition. However, such studies are unable to determine the differences in ECM composition between individual anatomical regions of the lung. Here, we employ a post-decellularization dissection method to compare the ECM composition of whole decellularized lungs (wECM) and specific anatomical lung regions, including alveolar-enriched ECM (aECM), airway ECM (airECM), and vasculature ECM (vECM), between non-diseased (ND), COPD, and IPF human lungs. We demonstrate, using mass spectrometry, that individual regions possess a unique ECM signature characterized primarily by differences in collagen composition and basement-membrane associated proteins, including ECM glycoproteins. We further demonstrate that both COPD and IPF lead to alterations in lung ECM composition in a region-specific manner, including enrichment of type-III collagen and fibulin in IPF aECM. Taken together, this study provides methodology for future studies, including isolation of region-specific lung biomaterials, as well as a dataset that may be applied for the identification of novel ECM targets for therapeutics.

Human alveolar hydrogels promote morphological and transcriptional differentiation in iPSC-derived alveolar type 2 epithelial cells.

Alveolar type 2 epithelial cells (AT2s) derived from human induced pluripotent stem cells (iAT2s) have rapidly contributed to our understanding of AT2 function and disease. However, while iAT2s are primarily cultured in three-dimensional (3D) Matrigel, a matrix derived from cancerous mouse tissue, it is unclear how a physiologically relevant matrix will impact iAT2s phenotype. As extracellular matrix (ECM) is recognized as a vital component in directing cellular function and differentiation, we sought to derive hydrogels from decellularized human lung alveolar-enriched ECM (aECM) to provide an ex vivo model to characterize the role of physiologically relevant ECM on iAT2 phenotype. We demonstrate aECM hydrogels retain critical in situ ECM components, including structural and basement membrane proteins. While aECM hydrogels facilitate iAT2 proliferation and alveolosphere formation, a subset of iAT2s rapidly change morphology to thin and elongated ring-like cells. This morphological change correlates with upregulation of recently described iAT2-derived transitional cell state genetic markers. As such, we demonstrate a potentially underappreciated role of physiologically relevant aECM in iAT2 differentiation.

Decellularization and Recellularization Methods for Avian Lungs: An Alternative Approach for Use in Pulmonary Therapeutics.

The shortage of compatible allogeneic organs and an increase in the number of patients requiring long-term lung assist devices while waiting for lung transplantation have motivated scientists to explore alternatives to bioengineer new lungs, including through decellularization and recellularization processes. A novel approach for bioengineering an extracorporeal membrane oxygenator is based on the parenchymal structure of avian lungs which utilizes a cross-current unidirectional flow of air and blood rather than bidirectional airflow, and thus eliminates dead-space ventilation. This provides more efficient gas exchange than mammalian lungs. The novel approach utilized is to decellularize avian lungs and then to recellularize with patient-derived human lung epithelial and vascular endothelial cells with the goal of creating a fully functional structure that can be used as a gas-exchange device. Here, we present avian lung decellularization and recellularization methods for chicken and emu lungs, in order to study both small- and large-scale avian lung models. For decellularization, a detergent-based protocol is utilized, and different techniques are used to validate the de- and recellularization of those lungs, including microscopy, mass spectrometry, and immunohistochemical analyses. For recellularization, techniques for seeding different human lung cell types into the decellularized scaffolds are presented.

Bioprintable Lung Extracellular Matrix Hydrogel Scaffolds for 3D Culture of Mesenchymal Stromal Cells.

Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell-matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.

Development of alginate and gelatin-based pleural and tracheal sealants.

Pleural and tracheal injuries remain significant problems, and an easy to use, effective pleural or tracheal sealant would be a significant advance. The major challenges are requirements for adherence, high strength and elasticity, dynamic durability, appropriate biodegradability, and lack of cell or systemic toxicity. We designed and evaluated two sealant materials comprised respectively of alginate methacrylate and of gelatin methacryloyl, each functionalized by conjugation with dopamine HCl. Both compounds are cross-linked into easily applied as pre-formed hydrogel patches or as in situ hydrogels formed at the wound site utilizing FDA-approved photo-initiators and oxidants. Material testing demonstrates appropriate adhesiveness, tensile strength, burst pressure, and elasticity with no significant cell toxicity in vitro assessments. Air-leak was absent after sealant application to experimentally-induced injuries in ex-vivo rat lung and tracheal models and in ex vivo pig lungs. Sustained repair of experimentally-induced pleural injury was observed for up to one month in vivo rat models and for up to 2 weeks in vivo rat tracheal injury models without obvious air leak or obvious toxicities. The alginate-based sealant worked best in a pre-formed hydrogel patch whereas the gelatin-based sealant worked best in an in situ formed hydrogel at the wound site thus providing two potential approaches. These studies provide a platform for further pre-clinical and potential clinical investigations. STATEMENT OF SIGNIFICANCE: Pneumothorax and pleural effusions resulting from trauma and a range of lung diseases and critical illnesses can result in lung collapse that can be immediately life-threatening or result in chronic leaking (bronchopleural fistula) that is currently difficult to manage. This leads to significantly increased morbidity, mortality, hospital stays, health care costs, and other complications. We have developed sealants originating from alginate and gelatin biomaterials, each functionalized by methacryloylation and by dopamine conjugation to have desired mechanical characteristics for use in pleural and tracheal injuries. The sealants are easily applied, non-cytotoxic, and perform well in vitro and in vivo model systems of lung and tracheal injuries. These initial proof of concept investigations provide a platform for further studies.

Functional role of glycosaminoglycans in decellularized lung extracellular matrix.

Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGFß1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2, HGF, and TGFß1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. STATEMENT OF SIGNIFICANCE: Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGs) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives.

Avian lungs: A novel scaffold for lung bioengineering.

Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics. To initially assess this in both small and large avian lung models, chicken (Gallus gallus domesticus) and emu (Dromaius novaehollandiae) lungs were decellularized using modifications of a detergent-based protocol, previously utilized with mammalian lungs. Light and electron microscopy, vascular and airway resistance, quantitation and gel analyses of residual DNA, and immunohistochemical and mass spectrometric analyses of remaining extracellular matrix (ECM) proteins demonstrated maintenance of lung structure, minimal residual DNA, and retention of major ECM proteins in the decellularized scaffolds. Seeding with human bronchial epithelial cells, human pulmonary vascular endothelial cells, human mesenchymal stromal cells, and human lung fibroblasts demonstrated initial cell attachment on decellularized avian lungs and growth over a 7-day period. These initial studies demonstrate that decellularized avian lungs may be a feasible approach for generating functional lung tissue for clinical therapeutics.

Effects of two different decellularization routes on the mechanical properties of decellularized lungs.

Considering the limited number of available lung donors, lung bioengineering using whole lung scaffolds has been proposed as an alternative approach to obtain lungs suitable for transplantation. However, some decellularization protocols can cause alterations on the structure, composition, or mechanical properties of the lung extracellular matrix. Therefore, the aim of this study was to compare the acellular lung mechanical properties when using two different routes through the trachea and pulmonary artery for the decellularization process. This study was performed by using the lungs excised from 30 healthy male C57BL/6 mice, which were divided into 3 groups: tracheal decellularization (TDG), perfusion decellularization (PDG), and control groups (CG). Both decellularized groups were subjected to decellularization protocol with a solution of 1% sodium dodecyl sulfate. The behaviour of mechanical properties of the acellular lungs was measured after decellularization process. Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. TDG and PDG showed reduced Est and Edyn elastances after lung decellularization. Scanning electron microscopy showed no structural changes after lung decellularization of the TDG and PDG. In conclusion, was demonstrated that there is no significant difference in the behaviour of mechanical properties and extracellular matrix of the decellularized lungs by using two different routes through the trachea and pulmonary artery.

Early Impairment of Lung Mechanics in a Murine Model of Marfan Syndrome.

Early morbidity and mortality in patients with Marfan syndrome (MFS) -a connective tissue disease caused by mutations in fibrillin-1 gene- are mainly caused by aorta aneurysm and rupture. However, the increase in the life expectancy of MFS patients recently achieved by reparatory surgery promotes clinical manifestations in other organs. Although some studies have reported respiratory alterations in MFS, our knowledge of how this connective tissue disease modifies lung mechanics is scarce. Hence, we assessed whether the stiffness of the whole lung and of its extracellular matrix (ECM) is affected in a well-characterized MFS mouse model (FBN1C1039G/+). The stiffness of the whole lung and of its ECM were measured by conventional mechanical ventilation and atomic force microscopy, respectively. We studied 5-week and 9-month old mice, whose ages are representative of early and late stages of the disease. At both ages, the lungs of MFS mice were significantly more compliant than in wild type (WT) mice. By contrast, no significant differences were found in local lung ECM stiffness. Moreover, histopathological lung evaluation showed a clear emphysematous-like pattern in MFS mice since alveolar space enlargement was significantly increased compared with WT mice. These data suggest that the mechanism explaining the increased lung compliance in MFS is not a direct consequence of reduced ECM stiffness, but an emphysema-like alteration in the 3D structural organization of the lung. Since lung alterations in MFS are almost fully manifested at an early age, it is suggested that respiratory monitoring could provide early biomarkers for diagnosis and/or follow-up of patients with the Marfan syndrome.

Residual Detergent Detection Method for Nondestructive Cytocompatibility Evaluation of Decellularized Whole Lung Scaffolds.

The development of reliable tissue engineering methods using decellularized cadaveric or donor lungs could potentially provide a new source of lung tissue. The vast majority of current lung decellularization protocols are detergent based and incompletely removed residual detergents may have a deleterious impact on subsequent scaffold recellularization. Detergent removal and quality control measures that rigorously and reliably confirm removal, ideally utilizing nondestructive methods, are thus critical for generating optimal acellular scaffolds suitable for potential clinical translation. Using a modified and optimized version of a methylene blue-based detergent assay, we developed a straightforward, noninvasive method for easily and reliably detecting two of the most commonly utilized anionic detergents, sodium deoxycholate (SDC) and sodium dodecyl sulfate (SDS), in lung decellularization effluents. In parallel studies, we sought to determine the threshold of detergent concentration that was cytotoxic using four different representative human cell types utilized in the study of lung recellularization: human bronchial epithelial cells, human pulmonary vascular endothelial cells (CBF12), human lung fibroblasts, and human mesenchymal stem cells. Notably, different cells have varying thresholds for either SDC or SDS-based detergent-induced cytotoxicity. These studies demonstrate the importance of reliably removing residual detergents and argue that multiple cell lines should be tested in cytocompatibility-based assessments of acellular scaffolds. The detergent detection assay presented here is a useful nondestructive tool for assessing detergent removal in potential decellularization schemes or for use as a potential endpoint in future clinical schemes, generating acellular lungs using anionic detergent-based decellularization protocols.

Behavior of vascular resistance undergoing various pressure insufflation and perfusion on decellularized lungs.

Bioengineering of functional lung tissue by using whole lung scaffolds has been proposed as a potential alternative for patients awaiting lung transplant. Previous studies have demonstrated that vascular resistance (Rv) could be altered to optimize the process of obtaining suitable lung scaffolds. Therefore, this work was aimed at determining how lung inflation (tracheal pressure) and perfusion (pulmonary arterial pressure) affect vascular resistance. This study was carried out using the lungs excised from 5 healthy male Sprague-Dawley rats. The trachea was cannulated and connected to a continuous positive airway pressure (CPAP) device to provide a tracheal pressure ranging from 0 to 15cmH2O. The pulmonary artery was cannulated and connected to a controlled perfusion system with continuous pressure (gravimetric level) ranging from 5 to 30cmH2O. Effective Rv was calculated by ratio of pulmonary artery pressure (PPA) by pulmonary artery flow (V'PA). Rv in the decellularized lungs scaffolds decreased at increasing V'PA, stabilizing at a pulmonary arterial pressure greater than 20cmH2O. On the other hand, CPAP had no influence on vascular resistance in the lung scaffolds after being subjected to pulmonary artery pressure of 5cmH2O. In conclusion, compared to positive airway pressure, arterial lung pressure markedly influences the mechanics of vascular resistance in decellularized lungs.

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.

In vitro comparative study of two decellularization protocols in search of an optimal myocardial scaffold for recellularization.

INTRODUCTION: Selection of a biomaterial-based scaffold that mimics native myocardial extracellular matrix (ECM) architecture can facilitate functional cell attachment and differentiation. Although decellularized myocardial ECM accomplishes these premises, decellularization processes may variably distort or degrade ECM structure. MATERIALS AND METHODS: Two decellularization protocols (DP) were tested on porcine heart samples (epicardium, mid myocardium and endocardium). One protocol, DP1, was detergent-based (SDS and Triton X-100), followed by DNase I treatment. The other protocol, DP2, was focused in trypsin and acid with Triton X-100 treatments. Decellularized myocardial scaffolds were reseeded by embedding them in RAD16-I peptidic hydrogel with adipose tissue-derived progenitor cells (ATDPCs). RESULTS: Both protocols yielded acellular myocardial scaffolds (~82% and ~94% DNA reduction for DP1 and DP2, respectively). Ultramicroscopic assessment of scaffolds was similar for both protocols and showed filamentous ECM with preserved fiber disposition and structure. DP1 resulted in more biodegradable scaffolds (P = 0.04). Atomic force microscopy revealed no substantial ECM stiffness changes post-decellularization compared to native tissue. The Young's modulus did not differ between heart layers (P = 0.69) or decellularization protocols (P = 0.15). After one week, recellularized DP1 scaffolds contained higher cell density (236 ± 106 and 98 ± 56 cells/mm(2) for recellularized DP1 and DP2 scaffolds, respectively; P = 0.04). ATDPCs in both DP1 and DP2 scaffolds expressed the endothelial marker isolectin B4, but only in the DP1 scaffold ATDPCs expressed the cardiac markers GATA4, connexin43 and cardiac troponin T. CONCLUSIONS: In our hands, DP1 produced myocardial scaffolds with higher cell repopulation and promotes ATDPCs expression of endothelial and cardiomyogenic markers.

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.

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.

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.