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Evaluation of scaffold produced from smokers' lungs and alveolar epithelial cells (AECs) isolated from smokers' lungs. (a–c) Adult lungs from heavy smoker: (b) AC single-lung scaffold produced from the lungs of a heavy smoker; (c) carbon deposits in the AC smoker's lung scaffold. (d) Close up of adult non-smoker AC scaffold produced, showing minimal carbon deposition. (e, f) Paediatric whole double lung, (e) prior to and (f) following decellularization. (g) Results for AECs from five non-smoking donors, cultured on 2.5 cm3 pieces of either AC human non-smoker's lung (NS) or smoker's lung (SM) scaffold: total and viable cells were evaluated; significantly fewer viable cells were isolated from smoker's lung scaffolds compared to those from non-smoker's lung (*p < 0.0002)
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We report, for the first time, the development of an organ culture system and protocols to support recellularization of whole acellular (AC) human paediatric lung scaffolds. The protocol for paediatric lung recellularization was developed using human transformed or immortalized cell lines and single human AC lung scaffolds. Using these surrogate ce...
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... prototype bioreactor was built using star board highdensity polyethylene plastic (HDPE) as the stand to support the three stepper motors that run the peristaltic pump heads (see supporting information, Figure S1a-c). Nema 23 435 oz 4.2A stepper motors (Longs Motor, Changzhou City, China) were purchased as a kit to build a small computer numerical control (CNC) machining mill 9 (see supporting information, Figure S1a, b). ...
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... prototype bioreactor was built using star board highdensity polyethylene plastic (HDPE) as the stand to support the three stepper motors that run the peristaltic pump heads (see supporting information, Figure S1a-c). Nema 23 435 oz 4.2A stepper motors (Longs Motor, Changzhou City, China) were purchased as a kit to build a small computer numerical control (CNC) machining mill 9 (see supporting information, Figure S1a, b). These stepper motors were controlled by CNC machining software and a break-out board purchased from Probotox (Peoria, IL, USA). ...
Context 3
... lid to the chamber was made from the star board, and plumbing hardware was purchased at Lowes Home Improvement Store (Houston, TX, USA). Photographs and a diagram of the bioreactor are shown in Figure S1 (see supporting information). ...
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... data exist regarding the use of cells or scaffold from smokers' lungs for bioengineering lungs, and, because of this, we felt the need to investigate the use of cells or scaffolds from smokers in this procedure. Carbon depositslungs (Figure 1e) or scaffolds produced from paediatric lungs (Figure 1f). Following thawing, the average viability of cells collected from smokers' and non-smokers' lungs was similar (88 ± 2.6% vs 89 ± 1.2%, respectively). ...
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... data exist regarding the use of cells or scaffold from smokers' lungs for bioengineering lungs, and, because of this, we felt the need to investigate the use of cells or scaffolds from smokers in this procedure. Carbon depositslungs (Figure 1e) or scaffolds produced from paediatric lungs (Figure 1f). Following thawing, the average viability of cells collected from smokers' and non-smokers' lungs was similar (88 ± 2.6% vs 89 ± 1.2%, respectively). ...
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... following 7 days of plate culture, the average viability of primary lung cells isolated from non-smokers' lungs (862.2%) was always significantly greater than that from smokers' lungs (60 ± 6.6%, p < 0.005). The viability of non-smokers' primary lung cells cultured on smokers' scaffolds was significantly less (p < 0.0002) than that for primary lung cells cultured on non-smokers' lung scaffolds (Figure 1g). For these reasons, we did not use primary lung cells or lung scaffolds from smokers' lungs to bioengineer lungs. ...
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... Authors declare no conflict of interest. of Texas John Sealy Memorial Fund Research Pilot Grant. We thank John D. Barajas for production of the lung diagrams in Figure 1. The authors wish to thank Dame Julia Polak (26 June 1939-11 August 2014), who received a heart and lung transplant in 1995 and who was the inspiration for the work presented in this manuscript. ...
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... prototype bioreactor was built using star board highdensity polyethylene plastic (HDPE) as the stand to support the three stepper motors that run the peristaltic pump heads (see supporting information, Figure S1a-c). Nema 23 435 oz 4.2A stepper motors (Longs Motor, Changzhou City, China) were purchased as a kit to build a small computer numerical control (CNC) machining mill 9 (see supporting information, Figure S1a, b). ...
Context 9
... prototype bioreactor was built using star board highdensity polyethylene plastic (HDPE) as the stand to support the three stepper motors that run the peristaltic pump heads (see supporting information, Figure S1a-c). Nema 23 435 oz 4.2A stepper motors (Longs Motor, Changzhou City, China) were purchased as a kit to build a small computer numerical control (CNC) machining mill 9 (see supporting information, Figure S1a, b). These stepper motors were controlled by CNC machining software and a break-out board purchased from Probotox (Peoria, IL, USA). ...
Context 10
... lid to the chamber was made from the star board, and plumbing hardware was purchased at Lowes Home Improvement Store (Houston, TX, USA). Photographs and a diagram of the bioreactor are shown in Figure S1 (see supporting information). ...
Context 11
... data exist regarding the use of cells or scaffold from smokers' lungs for bioengineering lungs, and, because of this, we felt the need to investigate the use of cells or scaffolds from smokers in this procedure. Carbon depositslungs (Figure 1e) or scaffolds produced from paediatric lungs (Figure 1f). Following thawing, the average viability of cells collected from smokers' and non-smokers' lungs was similar (88 ± 2.6% vs 89 ± 1.2%, respectively). ...
Context 12
... data exist regarding the use of cells or scaffold from smokers' lungs for bioengineering lungs, and, because of this, we felt the need to investigate the use of cells or scaffolds from smokers in this procedure. Carbon depositslungs (Figure 1e) or scaffolds produced from paediatric lungs (Figure 1f). Following thawing, the average viability of cells collected from smokers' and non-smokers' lungs was similar (88 ± 2.6% vs 89 ± 1.2%, respectively). ...
Context 13
... following 7 days of plate culture, the average viability of primary lung cells isolated from non-smokers' lungs (862.2%) was always significantly greater than that from smokers' lungs (60 ± 6.6%, p < 0.005). The viability of non-smokers' primary lung cells cultured on smokers' scaffolds was significantly less (p < 0.0002) than that for primary lung cells cultured on non-smokers' lung scaffolds (Figure 1g). For these reasons, we did not use primary lung cells or lung scaffolds from smokers' lungs to bioengineer lungs. ...
Context 14
... Authors declare no conflict of interest. of Texas John Sealy Memorial Fund Research Pilot Grant. We thank John D. Barajas for production of the lung diagrams in Figure 1. The authors wish to thank Dame Julia Polak (26 June 1939-11 August 2014), who received a heart and lung transplant in 1995 and who was the inspiration for the work presented in this manuscript. ...
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Background:
It has been difficult to perform tracheal allotransplantation without immunosuppression. To determine whether decellularized trachea can be used in tracheal replacement, we evaluated the viability of decellularized tracheal allografts in a rabbit model of immunosuppressant-free transplantation.
Method:
Half allograft (Group 1, n?=?7)...
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. T...
Citations
... However, several groups have reported the use of humanderived dECM obtained from donated, damaged, diseased, or cadaveric tissues. These include skin, 129,130 teeth, 131 lungs, 132 liver, 133,134 kidney, 135 heart, 136 cartilage, 137 ovarian, 138 among others. 62,139 Xenografts have been a suitable solution to the shortage of human tissue for research or clinical applications. ...
Tissue development, wound healing, pathogenesis, regeneration, and homeostasis rely upon coordinated and dynamic spatial and temporal remodeling of extracellular matrix (ECM) molecules. ECM reorganization and normal physiological tissue function, require the establishment and maintenance of biological, chemical, and mechanical feedback mechanisms directed by cell-matrix interactions. To replicate the physical and biological environment provided by the ECM in vivo, methods have been developed to decellularize and solubilize tissues which yield organ and tissue-specific bioactive hydrogels. While these biomaterials retain several important traits of the native ECM, the decellularizing process, and subsequent sterilization, and solubilization result in fragmented, cleaved, or partially denatured macromolecules. The final product has decreased viscosity, moduli, and yield strength, when compared to the source tissue, limiting the compatibility of isolated decellularized ECM (dECM) hydrogels with fabrication methods such as extrusion bioprinting. This review describes the physical and bioactive characteristics of dECM hydrogels and their role as biomaterials for biofabrication. In this work, critical variables when selecting the appropriate tissue source and extraction methods are identified. Common manual and automated fabrication techniques compatible with dECM hydrogels are described and compared. Fabrication and post-manufacturing challenges presented by the dECM hydrogels decreased mechanical and structural stability are discussed as well as circumvention strategies. We further highlight and provide examples of the use of dECM hydrogels in tissue engineering and their role in fabricating complex in vitro 3D microenvironments.
... Several studies have developed large-scale bioreactors for human size lung tissue engineering, many of which led to the implantation of animal models [81,82]. An organ culture system and protocols to support recellularization of whole acellular human pediatric lung scaffold were developed whereby the bioreactor was used for both decellularization and recellularization. ...
... An organ culture system and protocols to support recellularization of whole acellular human pediatric lung scaffold were developed whereby the bioreactor was used for both decellularization and recellularization. After 30 days of bioreactor culture, type I and type II alveolar epithelial cells and alveolar-capillary junctions were present, and the static compliance of engineered and normal lungs were similar [82]. Furthermore, to culture rhesus macaque lung tissue with bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) and lung microvascular endothelial cells, a bioreactor providing mechanical stretch and strain by negative pressure ventilation and pulsatile perfusion through the vasculature was utilized. ...
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.
... Various tissues and organs from the human body have been used to create dECM. For example, the entire heart [77,78], cartilage [79e81], ovarian tissue [82], adipose tissue [83,84], pancreas [85], kidney [86], liver [87], skin [88], teeth [89] and lungs [90] have been successfully decellularized. ...
Decellularized extracellular matrix (dECM) is widely used in regenerative medicine as a scaffold material due to its unique biological activity and good biocompatibility. Hydrogel is a three-dimensional network structure polymer with high water content and high swelling that can simulate the water environment of human tissues, has good biocompatibility, and can exchange nutrients, oxygen, and waste with cells. At present, hydrogel is the ideal biological material for tissue engineering. In recent years, rapid development of the hydrogel theory and technology and progress in the use of dECM to form hydrogels have attracted considerable attention to dECM hydrogels as an innovative method for tissue engineering and regenerative medicine. This article introduces the classification of hydrogels, and focuses on the history and formation of dECM hydrogels, the source of dECM, the application of dECM hydrogels in tissue engineering and the commercial application of dECM materials.
... There are a variety types of decellularization methods, including enzymatic, physical, and chemical methods [18,19]. Also, several protocols are existed for obtaining scaffolds of acellular human lung [20][21][22][23][24][25][26][27][28][29]. The aim of this chapter is an overview of the techniques of decellularized lung tissue engineering purposes. ...
Lung transplantation may be considered as a final treatment option for diseases such as chronic lung disease, pulmonary hypertension, bronchopulmonary dysplasia, pulmonary fibrosis, and end-stage lung disease. The five-year survival rate of lung transplants is nearly 50%. Unfortunately, many patients will die before a suitable lung donor can be found. Importantly, the shortage of donor organs has been a significant problem in lung transplantation. The tissue engineering approach uses de- and recellularization of lung tissue to create functional lung substitutes to overcome donor lung limitations. Decellularization is hope for generating an intact ECM in the development of the engineered lung. The goal of decellularization is to prepare a suitable scaffold of lung tissue that contains an appropriate framework for the functionality of regenerated lung tissue. In this chapter, we aim to describe the decellularization protocols for lung tissue regenerative purposes.
... Within the same system, cellular seeding has also been done with either vascular or airway media flow, continuously moving cells and media. This method can increase cell dispersal; however, suspension cultures can disrupt cell attachment rates [Karuri et al., 2004;Nichols et al., 2017]. Alternatively, with RWV systems, cells are first perfused throughout the lung, and then the seeded tissue is statically cultured for 1-4 days before initiating rotational culture [Cortiella et al., 2010;Crabbé et al., 2015;Radtke and Herbst-Kralovetz, 2012]. ...
Bioreactors for the reseeding of decellularized lung scaffolds have evolved with various advancements, including biomimetic mechanical stimulation, constant nutrient flow, multi-output monitoring, and large mammal scaling. Although dynamic bioreactors are not new to the field of lung bioengineering, ideal conditions during cell seeding have not been extensively studied or controlled. To address the lack of cell dispersal in traditional seeding methods, we have designed a two-step bioreactor. The first step is a novel system that rotates a seeded lung every 20 min at different angles in a sequence designed to anchor 20% of cells to a particular location based on the known rate of attachment. The second step involves perfusion-ventilation culture to ensure nutrient dispersion and cellular growth. Compared to statically seeded lungs, rotationally seeded lungs had significantly increased dsDNA content and more uniform cellular distribution after perfusion and ventilation had been administered. The addition of this novel seeding system before traditional culture methods will aid in recellularizing the lung and other geometrically complex organs for tissue engineering.
... Porcine organs may be a promising source of xenografts because of their appropriate size and human-compatible biological characteristics. 29 In our previous study, we compared several detergents, including SDS, Triton X-100, peracetic acid (PAA), and SDC, for porcine liver and kidney perfusion and finally developed an optimal SDS/Triton X-100 perfusion method. 30 SDS is an ionic detergent that is, more effective than others in dense organs. ...
Whole‐organ engineering is emerging as an alternative source for xenotransplantation in end‐stage diseases. Utilization of decellularized whole lung scaffolds created by detergent perfusion is an effective strategy for organ replacement. In the current study, we attempted to decellularize porcine whole lungs to generate an optimal and reproducible decellularized matrix for future clinical use. Porcine whole lungs were decellularized via perfusion of various detergents (sodium dodecyl sulfate (SDS)/Triton X‐100, sodium lauryl ether sulfate (SLES)/Triton X‐100, dextrose/SDS/Triton X‐100 and dextrose/SLES/Triton X‐100) through the pulmonary artery and bronchus of the lung. The decellularized scaffolds were evaluated for decellularization efficiency, extracellular matrix (ECM) component preservation, xenoantigen removal and compatibility. The resulting lung scaffolds obtained from treatment with the dextrose/SLES/Triton X‐100 cocktail showed minimal residual cellular components and xenoantigens, including DNA and protein, and good preservation of ECM composition. Evaluation of the porcine lung ECM by specific staining and immunofluorescence confirmed that the three‐dimensional ultrastructure of the ECM was noticeably preserved in the SLES‐treated groups. In addition, the decellularized lung scaffolds originating from the dextrose/SLES/Triton X‐100 cocktail supported cell adhesion and growth. In summary, the novel detergent SLES alleviated the damage to retain a better‐preserved ECM than SDS. Sequential Triton X‐100 perfusion eliminated SLES. Moreover, performing dextrose perfusion in advance further protected scaffold components, especially collagen. We developed an optimal dextrose/SLES/Triton X‐100 cocktail method that can be used for the decellularization of porcine whole lung to obtain a clinical‐scale bioengineered scaffold.
... 52,116 Whole organs can be decellularized for the bioengineering of transplantable organs. 11,82,117 Perfusion of decellularization agents could be performed through the native vasculature of organs such as the kidney, 82 liver, 118 and lung 119 which results in a 3D ECM scaffold that can be repopulated with patient-derived cells to engineer transplantable human organs. ...
Due to the current lack of innovative and effective therapeutic approaches, tissue engineering (TE) has attracted much attention during the last decades providing new hopes for the treatment of several degenerative disorders. Tissue engineering is a complex procedure, which includes processes of decellularization and recellularization of biological tissues or functionalization of artificial scaffolds by active cells. In this review, we have first discussed those conventional steps, which have led to great advancements during the last several years. Moreover, we have paid special attention to the new methods of post-decellularization that can significantly ameliorate the efficiency of decellularized cartilage extracellular matrix (ECM) for the treatment of osteoarthritis (OA). We propose a series of post-decellularization procedures to overcome the current shortcomings such as low mechanical strength and poor bioactivity to improve decellularized ECM scaffold towards much more efficient and higher integration.
... Within the same system, cellular seeding has also been done in the presence of either vascular or airway flow, continuously moving cells and media. This method would increase the dispersal of cells; however, suspension cultures can disrupt cell attachment rates (20,21). The RWV system initially disperses cells throughout the lung and then statically cultures the seeded tissue for 1 to 4 days before initiating rotational culture (11,22,23). ...
... Even if the rotation began during the time of seeding, constant rotation at 20 RPM would not allow cells to remain stationary for long enough for cells to adhere to the matrix. These systems have been designed and commercialized for both rodent lung culture and more complex porcine and human-sized organs (2,8,20). Advances in both commercially available and custom lung bioreactor systems include artificial pleural membranes, gas exchange units, waste removal, real-time monitoring of oxygen and lung mechanics, and highthroughput automation (12). ...
Bioreactors for reseeding of decellularized lung scaffolds have evolved with a wide variety of advancements. These include biomimetic mechanical stimulation, constant nutrient flow, multi-output monitoring, and large mammal scaling. Although dynamic bioreactors are not new to the field of bioengineered lungs, ideal conditions during cell seeding have not been extensively studied or controlled. To address the lack of cell dispersal in traditional seeding methods, we have designed a two-step bioreactor. The first step rotates a seeded lung every 20 minutes at different angles to ensure 20 percent of cells are anchored to a particular location based on the known rate of attachment. The second step involves perfusion culture to ensure nutrient dispersion and cellular growth. Compared to statically seeded lungs followed by conventional perfusion, rotationally seeded lungs followed by perfusion had significantly increased dsDNA content and more uniform cellular distribution. This new bioreactor system will aid in recellularizing the lung and other geometrically complex organs for tissue engineering.
... An additional major advance in the field of whole lung engineering has been the translation and adaptation of rodent lung engineering strategies to human-sized lungs, with a vision toward future clinical application. Perfusion detergentbased decellularization protocols have been successfully scaled up to whole lungs of larger animals including pig (15,83,159,183,212,234), nonhuman primate (14,27,212), and human (14,83,158,159). Concomitantly, bioreactors capable of providing physiologically relevant perfusion and ventilation have been developed for these larger organs (43,187). ...
... Several proof-of-concept studies have been published of the recellularization, and in some cases, implantation of human-scale engineered lungs. Most of these studies use either primary porcine (157) or human (82,158,277) cells to repopulate either porcine or human single lung scaffolds, and in general have demonstrated the successful adhesion and proliferation of both epithelial and endothelial cells. It remains an unresolved question in the field as to which cell type(s) may ultimately prove effective for regenerating lungs for transplantation, both because of the logistical challenges of acquiring sufficient cell numbers [upwards of 15 billion cells will be necessary to repopulate a full human lung scaffold, even with 4 population doublings after seeding (215,216)], and because what constitutes a lung stem cell, and in which settings, is an ongoing and evolving field of inquiry (119,126). ...
... Human donor lungs that go unused, which outnumber those transplanted by fourfold (85), could potentially serve as engineered lung scaffolds. Unfortunately, conditions that currently preclude transplantation may also make decellularized scaffolds from these organs unusable: lung scaffolds derived from smokers and from those with chronic lung disease have been shown to negatively impact the behavior and viability of reseeded cells (28,158,193,208,233). The use of porcine lungs as xenograft scaffolds is an appealing alternative given their potentially unlimited availability. ...
The pulmonary blood-gas barrier represents a remarkable feat of engineering. It achieves the exquisite thinness needed for gas exchange by diffusion, the strength to withstand the stresses and strains of repetitive and changing ventilation, and the ability to actively maintain itself under varied demands. Understanding the design principles of this barrier is essential to understanding a variety of lung diseases, and to successfully regenerating or artificially recapitulating the barrier ex vivo. Many classical studies helped to elucidate the unique structure and morphology of the mammalian blood-gas barrier, and ongoing investigations have helped to refine these descriptions and to understand the biological aspects of blood-gas barrier function and regulation. This article reviews the key features of the blood-gas barrier that enable achievement of the necessary design criteria and describes the mechanical environment to which the barrier is exposed. It then focuses on the biological and mechanical components of the barrier that preserve integrity during homeostasis, but which may be compromised in certain pathophysiological states, leading to disease. Finally, this article summarizes recent key advances in efforts to engineer the blood-gas barrier ex vivo, using the platforms of lung-on-a-chip and tissue-engineered whole lungs. © 2020 American Physiological Society. Compr Physiol 10:415-452, 2020.
... For generating ECs from stem cells, Cortiella's group showed that mouse embryonic stem cells (ESCs) differentiated into CD31 positive cells on pulmonary ECM ( Cortiella et al., 2010;Nichols et al., 2017). More recently, induced pluripotent stem cells (iPSCs) have been broadly investigated as a promising cell source for ECs ( Ren et al., 2015a). ...
Biomaterials have been used for a long time in the field of medicine. Since the success of “tissue engineering” pioneered by Langer and Vacanti in 1993, tissue engineering studies have advanced from simple tissue generation to whole organ generation with three-dimensional reconstruction. Decellularized scaffolds have been widely used in the field of reconstructive surgery because the tissues used to generate decellularized scaffolds can be easily harvested from animals or humans. When a patient’s own cells can be seeded onto decellularized biomaterials, theoretically this will create immunocompatible organs generated from allo- or xeno-organs. The most important aspect of lung tissue engineering is that the delicate three-dimensional structure of the organ is maintained during the tissue engineering process. Therefore, organ decellularization has special advantages for lung tissue engineering where it is essential to maintain the extremely thin basement membrane in the alveoli. Since 2010, there have been many methodological developments in the decellularization and recellularization of lung scaffolds, which includes improvements in the decellularization protocols and the selection and preparation of seeding cells. However, early transplanted engineered lungs terminated in organ failure in a short period. Immature vasculature reconstruction is considered to be the main cause of engineered organ failure. Immature vasculature causes thrombus formation in the engineered lung. Successful reconstruction of a mature vasculature network would be a major breakthrough in achieving success in lung engineering. In order to regenerate the mature vasculature network, we need to remodel the vascular niche, especially the microvasculature, in the organ scaffold. This review highlights the reconstruction of the vascular niche in a decellularized lung scaffold. Because the vascular niche consists of endothelial cells (ECs), pericytes, extracellular matrix (ECM), and the epithelial–endothelial interface, all of which might affect the vascular tight junction (TJ), we discuss ECM composition and reconstruction, the contribution of ECs and perivascular cells, the air–blood barrier (ABB) function, and the effects of physiological factors during the lung microvasculature repair and engineering process. The goal of the present review is to confirm the possibility of success in lung microvascular engineering in whole organ engineering and explore the future direction of the current methodology.
