Acellular Lung Research Articles

Effect of three-dimensional acellular fibrotic lung matrices on fibroblast-to-myofibroblast differentiation

To explore the effect of three-dimensional (3D) and two-dimensional (2D) acellular fibrotic lung matrices on fibroblast-to-myofibroblast differentiation. Twenty adult SD rats were randomly divided into control group and idiopathic pulmonary fibrosis (IPF) group. The pulmonary fibrosis was induced by Bleomycin. Normal and fibrotic decellularized lungs were made, then sections with 500 µm (3D) and 10 µm (2D) thick were cut by a standard Vibratome. The lung fibroblasts were isolated from the lungs of SD rats at postnatal day 1, which were seeded dropwise into different slices (2D/3D normal and fibrotic scaffolds), then cultured for 72 hours in vitro. The protein expression of alpha smooth muscle actin (α-SMA) was measured by immunofluorescence staining and western blot. There was no spatial structure both in 2D normal scaffold and fibrotic scaffold. The spatial structure was different between 3D normal scaffold and fibrotic scaffold. The relative expression amount of α-SMA in 2D normal and fibrotic scaffold group and none scaffold group were (4.64 ± 0.36, 4.86 ± 0.37, 4.67 ± 0.35, respectively), there were no difference among them (P>0.05). In 3D fibrotic scaffold group, the relative expression amount of α-SMA was significantly increased compared with those in 3D normal scaffold group and 2D fibrotic scaffold group (6.29 ± 0.85 vs 4.85 ± 0.56, 4.86 ± 0.37, both P<0.05). 3D acellular fibrotic lung scaffold can promote fibroblast-to-myofibroblast differentiation.

Production of decellularized porcine lung scaffolds for use in tissue engineering

There is a growing body of work dedicated to producing acellular lung scaffolds for use in regenerative medicine by decellularizing donor lungs of various species. These scaffolds typically undergo substantial matrix damage due to the harsh conditions required to remove cellular material (e.g., high pH, strong detergents), lengthy processing times, or pre-existing tissue contamination from microbial colonization. In this work, a new decellularization technique is described that maintains the global tissue architecture, key matrix components, mechanical composition and cell-seeding potential of lung tissue while effectively removing resident cellular material. Acellular lung scaffolds were produced from native porcine lungs using a combination of Triton X-100 and sodium deoxycholate (SDC) at low concentrations in 24 hours. We assessed the effect of matrix decellularization by measuring residual DNA, biochemical composition, mechanical characteristics, tissue architecture, and recellularization capacity.

Gas Transfer in Cellularized Collagen-Membrane Gas Exchange Devices.

Chronic lower respiratory disease is highly prevalent in the United States, and there remains a need for alternatives to lung transplant for patients who progress to end-stage lung disease. Portable or implantable gas oxygenators based on microfluidic technologies can address this need, provided they operate both efficiently and biocompatibly. Incorporating biomimetic materials into such devices can help replicate native gas exchange function and additionally support cellular components. In this work, we have developed microfluidic devices that enable blood gas exchange across ultra-thin collagen membranes (as thin as 2 μm). Endothelial, stromal, and parenchymal cells readily adhere to these membranes, and long-term culture with cellular components results in remodeling, reflected by reduced membrane thickness. Functionally, acellular collagen-membrane lung devices can mediate effective gas exchange up to ∼288 mL/min/m(2) of oxygen and ∼685 mL/min/m(2) of carbon dioxide, approaching the gas exchange efficiency noted in the native lung. Testing several configurations of lung devices to explore various physical parameters of the device design, we concluded that thinner membranes and longer gas exchange distances result in improved hemoglobin saturation and increases in pO2. However, in the design space tested, these effects are relatively small compared to the improvement in overall oxygen and carbon dioxide transfer by increasing the blood flow rate. Finally, devices cultured with endothelial and parenchymal cells achieved similar gas exchange rates compared with acellular devices. Biomimetic blood oxygenator design opens the possibility of creating portable or implantable microfluidic devices that achieve efficient gas transfer while also maintaining physiologic conditions.

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

The use of extracellular matrix (ECM) scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs.

Extended Biomimetic Culture and Functional Assessment of Recellularized Human Lungs

RationaleNative lung scaffolds prepared by decellularization provide a novel platform for organ engineering. Recellularization with specific cell types requires ex vivo culture for evaluation and restoration of organ function prior to transplantation.MethodsHuman lung‐derived epithelial and endothelial cells were isolated and expanded in vitro. Immunostaining, flow cytometery, and qPCR confirmed cell identity. Epithelial and endothelial cells were delivered to lung scaffold airways and vasculature respectively (&gt;350x106).Constructs were cultured for 10 days in a custom automated, closed‐circuit bioreactor, providing physiologic perfusion and negative pressure ventilation. Perfusate was analyzed daily by iSTAT. Tissue was analyzed by histology and qPCR.ResultsDonor‐derived epithelium was a population of primarily basal‐cell phenotype (70% p63+), with 20% CC10+, and minor pneumocytes (&lt;10% SP‐B/C) by flow cytometry. Basal cell ciliogenesis was confirmed by ALI. Endothelium was sorted to CD31 purity.Biocompatibility to lung matrix confirmed maintenance of cell phenotype, viability, and proliferation (Ki67+) in slice co‐culture.Recellularized constructs were cultured for 6 days with constant perfusion, plus 4 days with additional fluid or air ventilation. Increasing rate of glucose metabolism and lactate production in the perfusate was quantified over time. Measured pO2, pCO2, and pH remained consistent.Cell distribution and viability was confirmed by Resazurin metabolism, and by histology/qPCR of serial tissue biopsies.ConclusionIntact acellular lung scaffolds can be repopulated with primary tissue‐derived cells and maintained in extended ex‐vivo biomimetic culture, supporting cell viability and function.image

Ex Vivo Four-Dimensional Lung Cancer Model Mimics Metastasis

We have developed a four-dimensional (4D) lung cancer model that forms perfusable tumor nodules. We determined if the model could be modified to mimic metastasis. We modified the 4D lung cancer model by seeding H1299, A549, or H460 cells through the trachea only to the left lobes of the acellular lung matrix. The model was modified so that the tumor cells can reach the right lobes of the acellular lung matrix only through the pulmonary artery as circulating tumor cells (CTC). We determined the gene expressions of the primary tumor, CTCs, and metastatic lesions using the Human OneArray chip. All cell lines formed a primary tumor in the left lobe of the exvivo 4D lung cancer model. The CTCs were identified in the media and increased over time. All cell lines formed metastatic lesions with H460 forming significantly more metastatic lesions than H1299 and A549 cells. The CTC gene signature predicted poor survival in lung cancer patients. Unique genes were significantly expressed in CTC compared with the primary tumor and metastatic lesion. The 4D lung cancer model can isolate tumor cells in 3 phases of tumor progression. This 4D lung cancer model may mimic the biology of lung cancer metastasis and may be used to determine its mechanism and potential therapy in the future.

Acellular Lung Scaffolds Direct Differentiation of Endoderm to Functional Airway Epithelial Cells: Requirement of Matrix-Bound HS Proteoglycans

SummaryEfficient differentiation of pluripotent cells to proximal and distal lung epithelial cell populations remains a challenging task. The 3D extracellular matrix (ECM) scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone. Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

REVASCULARIZATION OF ACELLULAR RAT LUNG SCAFFOLDS USING THE ENDOTHELIAL COLONY FORMING CELLS

s S157 revascularization of acellular lung scaffolds using ECFCs as a critical first step in engineering truly functional grafts. OBJECTIVES: 1) Compare decellularization Methods to achieve optimal decellularization with maintained vascular perfusion. 2) Regenerate vascular endothelium using ECFCs. METHODS: Acellular rat lung scaffolds were generated by static incubation and perfusions Methods of decellularization. For static incubation, lungs were submerged into Triton-X 100 (0.1%) and sodium deoxycholate (2%) based decellularization buffer. For perfusion decellularization, isolated rat lungs were perfused using CHAPS buffer (8mM CHAPS, 1M NaCl, 25mM EDTA in 1X PBS) or SDS-Triton-X buffer (0.1% SDS followed by 1% Triton-X). Decellularization of lungs was evaluated by tissue histology, total DNA measurement and immuno-blotting. For revascularization, ECFCs were generated from human peripheral blood or rat bone marrow derived mononuclear cells and characterized for ECFCmarkers. Further, we also studied initial engraftment of human ECFCs in the rat lung scaffolds. RESULTS: Static incubation method of decellularization generated acellular lung scaffolds with no evidence of intact cells; however, presence of intracellular protein (GAPDH) was evident. Vascular perfusion could not be established in these scaffolds. On the other side, perfusion decellularization using CHAPS buffer produced acellular lung scaffolds with maintained extracellular structure. Moreover, perfusion decellularization with CHAPS buffer consistently demonstrated complete absence of cells, below detectable DNA remnants and intracellular protein, with more optimal vascular perfusion. ECFCs were successfully derived from human peripheral blood and rat bone-marrow mononuclear cells. ECFCs were positive for endothelial specific markers such as Ac-LDL uptake, lectin binding and expressed CD31, von Willbrand Factor (vWF) and VEGFR2. Moreover, these cells did not express pan-leukocyte marker CD45. Initial engraftment study of human ECFCs in rat lung scaffolds showed engraftment of human ECFCs in vascular compartment of the lung scaffolds. CONCLUSION: We successfully prepared acellular lung scaffolds and identified ECFC, which can be utilized to generate truly functional grafts. HSF, CIHR 204 GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION M Nakhaei-Nejad, AG Murray, N Jahroudi

IDENTIFICATION OF A SUB-FRACTION OF INDUCED PLURIPOTENT STEM CELLS WITH HIGH PROPENSITY FOR DIFFERENTIATION INTO ENDOTHELIAL CELLS

Pulmonary arterial hypertension (PAH) is characterized by progressive remodeling and obliteration of distal lung arterioles, eventually requiring lung transplantation. Unfortunately, there is a shortage of donor organs and many patients who need lung transplantation never receive this life saving therapy. Transplantation with a bioartificial lung generated by seeding acellular lung scaffolds with patient-specific cell types represents a novel therapeutic option. However, inadequate re-endothelialization hampers current attempts to recellularize lung scaffolds, leading to thrombosis-induced organ failure. The use of patient-specific induced pluripotent stem cells (iPSCs) capable of multilineage differentiation provides a viable resolution to this challenge. Seeding decellularised lung scaffold with endothelial progenitor cells (EPCs) derived from iPSCs could potentially provide more complete re-endothelialization of biological scaffolds. We hypothesized that different sub-fractions within a heterogeneous population of iPSCs possess inherent predisposition to differentiate down certain lineages. Specifically, we are interested in identifying the sub-fraction which demonstrates the greatest propensity to differentiate into cells arising from the mesodermal lineages such as EPCs. Double FACS was performed with pluripotency markers CD9 and EpCAM, four sub-fractions arbitrarily termed R3 (CD9Hi-EpCAMHi, 24.3%), R4 (CD9Mid-EpCAMMid; 18.3%), R5 (CD9Low-EpCAMLow; 40%) and R6 (CD9Neg-EpCAMNeg; 12.6%) were generated. Subsequent Q-PCR with pluripotency genes OCT-4, Nanog and GDF-3 demonstrated a decrease in expression level moving across the R3 to R6 sub-fractions, confirming that the R3 sub-fraction contains the highest proportion of pluripotent cells whilst R6 contains predominantly differentiated cell types. Q-PCR performed on R3-R6 samples seven days post-FACS, MIXL1 and Brachyury were selectively increased in the R4 sub-fraction. Importantly, gene expression trends of mesodermal markers across the four sub-fractions strongly mirrored that of endothelial cell associated gene, eNOS. Relative to R3, the R4 sub-fraction showed 1.3-fold, 1.9-fold and 2.6-fold increases in expression of Brachyury, MIXL1 and eNOS respectively. In contrast, relative to R3, the endodermal marker, GATA4 (17-fold increase), and ectodermal marker, PAX6 (6.4-fold increase), are preferentially expressed in R6 sub-fraction. Gene expression trend for mesodermal and endothelial genes was maintained following differentiation into EPCs with R4 sub-fraction demonstrating increase of 11.4-fold, 1.3-fold and 2.5-fold in Brachyury, MIXL1 and eNOS respectively, relative to R3. Importantly, results from preliminary flow cytometric analysis was congruous with Q-PCR results, with the R4 sub-fraction demonstrating highest expression of surface markers typically used to characterize EPCs. Sub-fractions demonstrate distinct preferential lineage commitment with the R4 sub-fraction showing most promise for differentiation to the mesodermal lineage and consequently into EPCs.

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.

Enhanced Lung Epithelial Specification of Human Induced Pluripotent Stem Cells on Decellularized Lung Matrix

Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs). Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position. Activin-A treatment, followed by transforming growth factor-β inhibition, induced differentiation ofhuman iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after5 days. Cells expressing Nkx2.1 were identified at 40%to 60% efficiency. Within whole-lung scaffolds andunder perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways. Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further invivo graft maturation.

Influence of pH on extracellular matrix preservation during lung decellularization.

The creation of decellularized organs for use in regenerative medicine requires the preservation of the organ extracellular matrix (ECM) as a means to provide critical cues for differentiation and migration of cells that are seeded onto the organ scaffold. The purpose of this study was to assess the influence of varying pH levels on the preservation of key ECM components during the decellularization of rat lungs. Herein, we show that the pH of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-based decellularization solution influences ECM retention, cell removal, and also the potential for host response upon implantation of acellular lung tissue. The preservation of ECM components, including elastin, fibronectin, and laminin, were better retained in the lower pH conditions that were tested (pH ranges tested: 8, 10, 12); glycosaminoglycans were preserved to a higher extent in the lower pH groups as well. The DNA content following decellularization of the rat lung was inversely correlated with the pH of the decellularization solution. Despite detectible levels of cyotoskeletal proteins and significant residual DNA, tissues decellularized at pH 8 demonstrated the greatest tissue architecture maintenance and the least induction of host response of all acellular conditions. These results highlight the effect of pH on the results obtained by organ decellularization and suggest that altering the pH of the solutions used for decellularization may influence the ability of cells to properly differentiate and home to appropriate locations within the scaffold, based on the preservation of key ECM components and implantation results.

Inhomogeneity of local stiffness in the extracellular matrix scaffold of fibrotic mouse lungs

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

Epithelial Cell Differentiation of Human Mesenchymal Stromal Cells in Decellularized Lung Scaffolds

Identification of appropriate donor cell types is important for lung cell therapy and for lung regeneration. Previous studies have indicated that mesenchymal stromal cells derived from human bone marrow (hBM-MSCs) and from human adipose tissue (hAT-MSCs) may have the ability to trans-differentiate into lung epithelial cells. However, these data remain controversial. Herein, the ability of hBM-MSCs and hAT-MSCs to repopulate acellular rodent lung tissue was evaluated. hBM-MSCs and hAT-MSCs were isolated from bone marrow aspirate and lipoaspirate, respectively. Rat lungs were decellularized with CHAPS detergent, followed by seeding the matrix with hBM-MSCs and hAT-MSCs. Under appropriate culture conditions, both human MSC populations attached to and proliferated within the lung tissue scaffold. In addition, cells were capable of type 2 pneumocyte differentiation, as assessed by marker expression of surfactant protein C (pro-SPC) at the protein and the RNA level, and by the presence of lamellar bodies by transmission electron microscopy. Additionally, hAT-MSCs contributed to Clara-like cells that lined the airways in the lung scaffolds, whereas the hBM-MSCs did not. We also tested the differentiation potential of MSCs on different extracellular matrix components in vitro, and found that protein substrate influences MSC epithelial differentiation. Together our data show the capacity for human MSCs to differentiate toward lung epithelial phenotypes, and the possibility of using these cells for lung cell therapies and tissue engineering.

Design and Synthesis of an Artificial Pulmonary Pleura for High Throughput Studies in Acellular Human Lungs.

Whole organ decellularization of complex organs, such as lungs, presents a unique opportunity for use of acellular scaffolds for ex vivo tissue engineering or for studying cell-extracellular matrix interactions ex vivo. A growing body of literature investigating decellularizing and recellularizing rodent lungs has provided important proof of concept models and rodent lungs are readily available for high throughput studies. In contrast, comparable progress in large animal and human lungs has been impeded owing to more limited availability and difficulties in handling larger tissue. While the use of smaller segments of acellular large animal or human lungs would maximize usage from a single lung, excision of small acellular segments compromises the integrity of the pleural layer, leaving the terminal ends of blood vessels and airways exposed. We have developed a novel pleural coating using non-toxic ionically crosslinked alginate or photocrosslinked methacrylated alginate which can be applied to excised acellular lung segments, permits inflation of small segments, and significantly enhances retention of cells inoculated through cannulated airways or blood vessels. Further, photocrosslinking methacrylated alginate, using eosin Y and triethanolamine at 530nm wavelength, results in a mechanically stable pleural coating that permits effective cyclic 3-dimensional stretch, i.e., mechanical ventilation, of individual segments.

Comparative decellularization and recellularization of normal versus emphysematous human lungs

Acellular whole human lung scaffolds represent a unique opportunity for ex vivo tissue engineering. However, it remains unclear whether lungs from individuals with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) can be appropriately decellularized and recellularized. To assess this, cadaveric human lungs from normal (non-smoking) patients and from patients with COPD (smoking history) were decellularized and found by histochemical and immunohistochemical staining, electron microscopy, and mass spectrometry to retain characteristic histological architecture and extracellular matrix components (ECM) reflecting either normal or COPD, particularly emphysematous, origin. Inoculation of human bronchial epithelial cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, and lung fibroblasts via airway or vascular routes into small, excised segments of the decellularized lungs demonstrated that normal lung scaffolds robustly supported initial engraftment and growth of each cell type for up to one month. In contrast, despite initial binding, all cell types inoculated into decellularized emphysematous lungs did not survive beyond one week. However, cell attachment and proliferation on solubilized ECM homogenates of decellularized normal and emphysematous lungs coated onto tissue culture plates was comparable and not impaired, suggesting that the 3-dimensional decellularized emphysematous scaffolds may lack the necessary ECM architecture to support sustained cell growth.

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ's natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use. The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ's natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use. The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Effects of the Decellularization Method on the Local Stiffness of Acellular Lungs

Lung bioengineering, a novel approach to obtain organs potentially available for transplantation, is based on decellularizing donor lungs and seeding natural scaffolds with stem cells. Various physicochemical protocols have been used to decellularize lungs, and their performance has been evaluated in terms of efficient decellularization and matrix preservation. No data are available, however, on the effect of different decellularization procedures on the local stiffness of the acellular lung. This information is important since stem cells directly sense the rigidity of the local site they are engrafting to during recellularization, and it has been shown that substrate stiffness modulates cell fate into different phenotypes. The aim of this study was to assess the effects of the decellularization procedure on the inhomogeneous local stiffness of the acellular lung on five different sites: alveolar septa, alveolar junctions, pleura, and vessels' tunica intima and tunica adventitia. Local matrix stiffness was measured by computing Young's modulus with atomic force microscopy after decellularizing the lungs of 36 healthy rats (Sprague-Dawley, male, 250-300 g) with four different protocols with/without perfusion through the lung circulatory system and using two different detergents (sodium dodecyl sulfate [SDS] and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [CHAPS]). The local stiffness of the acellular lung matrix significantly depended on the site within the matrix (p<0.001), ranging from ∼ 15 kPa at the alveolar septum to ∼ 60 kPa at the tunica intima. Acellular lung stiffness (p=0.003) depended significantly, albeit modestly, on the decellularization process. Whereas perfusion did not induce any significant differences in stiffness, the use of CHAPS resulted in a ∼ 35% reduction compared with SDS, the influence of the detergent being more important in the tunica intima. In conclusion, lung matrix stiffness is considerably inhomogeneous, and conventional decellularization procedures do not result in substantially different local stiffness in the acellular lung.

Human iPS cell–derived alveolar epithelium repopulates lung extracellular matrix

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.