Vessel network extraction and analysis of mouse pulmonary vasculature via X-ray micro-computed tomographic imaging.

In this work, non-invasive high-spatial resolution three-dimensional (3D) X-ray micro-computed tomography (µCT) of healthy mouse lung vasculature is performed. Methodologies are presented for filtering, segmenting, and skeletonizing the collected 3D images. Novel methods for the removal of spurious branch artefacts from the skeletonized 3D image are introduced, and these novel methods involve a combination of distance transform gradients, diameter-length ratios, and the fast marching method (FMM). These new techniques of spurious branch removal result in the consistent removal of spurious branches without compromising the connectivity of the pulmonary circuit. Analysis of the filtered, skeletonized, and segmented 3D images is performed using a newly developed Vessel Network Extraction algorithm to fully characterize the morphology of the mouse pulmonary circuit. The removal of spurious branches from the skeletonized image results in an accurate representation of the pulmonary circuit with significantly less variability in vessel diameter and vessel length in each generation. The branching morphology of a full pulmonary circuit is characterized by the mean diameter per generation and number of vessels per generation. The methods presented in this paper lead to a significant improvement in the characterization of 3D vasculature imaging, allow for automatic separation of arteries and veins, and for the characterization of generations containing capillaries and intrapulmonary arteriovenous anastomoses (IPAVA).

MRI method for labeling and imaging decellularized extracellular matrix scaffolds for tissue engineering.

PURPOSE: To develop a facile method for labeling and imaging decellularized extracellular matrix (dECM) scaffolds intended for regenerating 3D tissues. METHODS: A small molecule manganese porphyrin, MnPNH2 , was synthesized and used to label dECM scaffolds made from porcine bladder and trachea and murine whole lungs. The labeling protocol was optimized on bladder dECM, and imaging on a 3T clinical scanner was performed to assess reductions in T1 and T2 relaxation times. In vivo MRI was performed on dECM injected in the rat dorsum to verify sensitivity of detection. Toxicity assays for cell viability, metabolism, and proliferation were performed on human umbilical vein endothelial cells. The incorporation of MnPNH2 and its long-term retention in dECM were assessed on transmission electron microscopy and ultraviolet absorbance of eluted MnPNH2 over time. RESULTS: All tissues, including thick whole 3D organs, were uniformly labeled and demonstrated high signal-to-noise on MRI. A nearly 10-fold reduction in T1 was consistently obtained at a labeling dose of 0.4 mM, and even 0.2 mM provided sufficient contrast in vivo and ex vivo. No toxicity was observed up to 0.4 mM, the maximum tested. Binding studies suggested nonspecific association, and retention studies in the labeled whole decellularized lungs revealed less than 20% MnPNH2 loss over 30 days, the majority occurring in the first 3 days after labeling. CONCLUSION: The proposed labeling method is the first report for visualizing dECM on MRI and has the potential for long-term monitoring and optimization of dECM-based organ tissue engineering.

Cell-Based Therapeutic Approaches for Cystic Fibrosis.

Cystic Fibrosis (CF) is a chronic autosomal recessive disease caused by defects in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Cystic Fibrosis affects multiple organs but progressive remodeling of the airways, mucus accumulation, and chronic inflammation in the lung, result in lung disease as the major cause of morbidity and mortality. While advances in management of CF symptoms have increased the life expectancy of this devastating disease, and there is tremendous excitement about the potential of new agents targeting the CFTR molecule itself, there is still no curative treatment. With the recent advances in the identification of endogenous airway progenitor cells and in directed differentiation of pluripotent cell sources, cell-based therapeutic approaches for CF have become a plausible treatment method with the potential to ultimately cure the disease. In this review, we highlight the current state of cell therapy in the CF field focusing on the relevant autologous and allogeneic cell populations under investigation and the challenges associated with their use. In addition, we present advances in induced pluripotent stem (iPS) cell approaches and emerging new genetic engineering methods, which have the capacity to overcome the current limitations hindering cell therapy approaches.

Partial Restoration of CFTR Function in cftr-Null Mice following Targeted Cell Replacement Therapy.

Cystic fibrosis (CF) is a fatal recessive genetic disorder caused by a mutation in the gene encoding CF transmembrane conductance regulator (CFTR) protein. Alteration in CFTR leads to thick airway mucus and bacterial infection. Cell therapy has been proposed for CFTR restoration, but efficacy has been limited by low engraftment levels. In our previous studies, we have shown that using a pre-conditioning regimen in combination with optimization of cell number and time of delivery, we could obtain greater bone marrow cell (BMC) retention in the lung. Here, we found that optimized delivery of wild-type (WT) BMC contributed to apical CFTR expression in airway epithelium and restoration of select ceramide species and fatty acids in CFTR-/- mice. Importantly, WT BMC delivery delayed Pseudomonas aeruginosa lung infection and increased survival of CFTR-/- recipients. Only WT BMCs had a beneficial effect beyond 6 months, suggesting a dual mechanism of BMC benefit: a non-specific effect early after cell delivery, possibly due to the recruitment of macrophages and neutrophils, and a late beneficial effect dependent on long-term CFTR expression. Taken together, our results suggest that BMC can improve overall lung function and may have potential therapeutic benefit for the treatment of CF.

Cisplatin-resistant cells in malignant pleural mesothelioma cell lines show ALDH(high)CD44(+) phenotype and sphere-forming capacity.

BACKGROUND: Conventional chemotherapy in malignant pleural mesothelioma (MPM) has minimal impact on patient survival due to the supposed chemoresistance of cancer stem cells (CSCs). We sought to identify a sub-population of chemoresistant cells by using putative CSC markers, aldehyde dehydrogenase (ALDH) and CD44 in three MPM cell lines; H28, H2052 and Meso4. METHODS: The Aldefluor assay was used to measure ALDH activity and sort ALDH(high) and ALDH(low) cells. Drug-resistance was evaluated by cell viability, anchorage-independent sphere formation, flow-cytometry and qRT-PCR analyses. RESULTS: The ALDH(high) - and ALDH(low) -sorted fractions were able to demonstrate phenotypic heterogeneity and generate spheres, the latter being less efficient, and both showed an association with CD44. Cis- diamminedichloroplatinum (II) (cisplatin) treatment failed to reduce ALDH activity and conferred only a short-term inhibition of sphere generation in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. Induction of drug sensitivity by an ALDH inhibitor, diethylaminobenzaldehyde (DEAB) resulted in significant reductions in cell viability but not a complete elimination of the sphere-forming cells, suggestive of the presence of a drug-resistant subpopulation. At the transcript level, the cisplatin + DEAB-resistant cells showed upregulated mRNA expression levels for ALDH1A2, ALDH1A3 isozymes and CD44 indicating the involvement of these markers in conferring chemoresistance in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. CONCLUSIONS: Our study shows that ALDH(high) CD44(+) cells are implicated in conveying tolerance to cisplatin in the three MPM cell lines. The combined use of CD44 and ALDH widens the window for identification and targeting of a drug-resistant population which may improve the current treatment modalities in mesothelioma.

Bioengineering of vascularized porcine flaps using perfusion-recellularization.

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Mesenchymal cells support the early retention of primary alveolar type 2 cells on acellular mouse lung scaffolds.

Objectives: Tissue engineering approaches via repopulation of acellular biological grafts provide an exciting opportunity to generate lung grafts for transplantation. Alveolar type 2 (AT2) cells are a promising cell source for re-epithelialization. There are however inherent limitations with respect to their survival and growth, thus impeding their usability for tissue engineering applications. This study investigates the use of mesenchymal stromal cells to support primary AT2 cells for recellularization of mouse lung scaffolds. Methods: AT2 cells and bone marrow-derived mesenchymal cells (BMC) were co-delivered to decellularized mouse lung scaffolds. Recellularized lungs were evaluated for cell surface coverage, viability, and differentiation at 1 and 4 days after cell seeding. Recellularization was evaluated via histological analysis and immunofluorescence. Results: Simultaneous delivery of AT2 and BMC into acellular lung scaffolds resulted in enhanced cell surface coverage and reduced AT2 cell apoptosis in the recellularized scaffolds at Day 1 but not Day 4. AT2 cell number decreased after 4 days in both of AT2 only and codelivery groups suggesting limited expansion potential in the scaffold. After retention in the scaffold, AT2 cells differentiated into Aqp5-expressing cells. Conclusions: Our results indicate that BMC support AT2 cell survival during the initial attachment and engraftment phase of recellularization. While our findings suggest only a short-term beneficial effect of BMC, our study demonstrates that AT2 cells can be delivered and retained in acellular lung scaffolds; thus with preconditioning and supporting cells, may be used for re-epithelialization. Selection and characterization of appropriate cell sources for use in recellularization, will be critical for ultimate clinical application.

In silico model development and optimization of in vitro lung cell population growth.

Tissue engineering predominantly relies on trial and error in vitro and ex vivo experiments to develop protocols and bioreactors to generate functional tissues. As an alternative, in silico methods have the potential to significantly reduce the timelines and costs of experimental programs for tissue engineering. In this paper, we propose a methodology to formulate, select, calibrate, and test mathematical models to predict cell population growth as a function of the biochemical environment and to design optimal experimental protocols for model inference of in silico model parameters. We systematically combine methods from the experimental design, mathematical statistics, and optimization literature to develop unique and explainable mathematical models for cell population dynamics. The proposed methodology is applied to the development of this first published model for a population of the airway-relevant bronchio-alveolar epithelial (BEAS-2B) cell line as a function of the concentration of metabolic-related biochemical substrates. The resulting model is a system of ordinary differential equations that predict the temporal dynamics of BEAS-2B cell populations as a function of the initial seeded cell population and the glucose, oxygen, and lactate concentrations in the growth media, using seven parameters rigorously inferred from optimally designed in vitro experiments.

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

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

Evaluation of a decellularized bronchial patch transplant in a porcine model.

Biological scaffolds for airway reconstruction are an important clinical need and have been extensively investigated experimentally and clinically, but without uniform success. In this study, we evaluated the use of a decellularized bronchus graft for airway reconstruction. Decellularized left bronchi were procured from decellularized porcine lungs and utilized as grafts for airway patch transplantation. A tracheal window was created and the decellularized bronchus was transplanted into the defect in a porcine model. Animals were euthanized at 7 days, 1 month, and 2 months post-operatively. Histological analysis, immunohistochemistry, scanning electron microscopy, and strength tests were conducted in order to evaluate epithelialization, inflammation, and physical strength of the graft. All pigs recovered from general anesthesia and survived without airway obstruction until the planned euthanasia timepoint. Histological and electron microscopy analyses revealed that the decellularized bronchus graft was well integrated with native tissue and covered by an epithelial layer at 1 month. Immunostaining of the decellularized bronchus graft was positive for CD31 and no difference was observed with immune markers (CD3, CD11b, myeloperoxidase) at two months. Although not significant, tensile strength was decreased after one month, but recovered by two months. Decellularized bronchial grafts show promising results for airway patch reconstruction in a porcine model. Revascularization and re-epithelialization were observed and the immunological reaction was comparable with the autografts. This approach is clinically relevant and could potentially be utilized for future applications for tracheal replacement.

Procurement and Decellularization of Rat Hindlimbs using an Ex Vivo Perfusion-based Bioreactor for Vascularized Composite Allotransplantation.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Bioreactor-Based De-epithelialization of Long-Segment Tracheal Grafts.

Tissue engineering techniques to generate a graft ex vivo is an exciting field of research. In particular, the use of biological scaffolds has shown to be promising in a clinical setting. In this approach, decellularized donor scaffolds are obtained following detergent-based enzymatic treatment to remove donor cells and subsequently repopulated with recipient specific cells. Herein, we describe our bioreactor-based partial decellularization approach to generate hybrid tracheal grafts. Using a short detergent-based treatment with sodium dodecyl sulfate (SDS), we remove the epithelium and maintain the structural integrity of the donor grafts by keeping the cartilage alive. The following will be a step-by-step description of the bioreactor system setup and partial decellularization protocol to obtain a de-epithelialized tracheal graft.

Perfusion decellularization for vascularized composite allotransplantation.

Vascularized composite allotransplantation is becoming the emerging standard for reconstructive surgery treatment for patients with limb trauma and facial injuries involving soft tissue loss. Due to the complex immunogenicity of composite grafts, patients who undergo vascularized composite allotransplantation are reliant on lifelong immunosuppressive therapy. Decellularization of donor grafts to create an extracellular matrix bio-scaffold provides an immunomodulatory graft that preserves the structural and bioactive function of the extracellular matrix. Retention of extracellular matrix proteins, growth factors, and signaling cascades allow for cell adhesion, migration, proliferation, and tissue regeneration. Perfusion decellularization of detergents through the graft vasculature allows for increased regent access to all tissue layers, and removal of cellular debris through the venous system. Grafts can subsequently be repopulated with appropriate cells through the vasculature to facilitate tissue regeneration. The present work reviews methods of decellularization, process parameters, evaluation of adequate cellular and nuclear removal, successful applications of perfusion decellularization for use in vascularized composite allotransplantation, and current limitations.

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient's quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells. This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor. Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

Planar organization of airway epithelial cell morphology using hydrogel grooves during ciliogenesis fails to induce ciliary alignment.

Topographical cues are known to influence cell organization both in native tissues and in vitro. In the trachea, the matrix beneath the epithelial lining is composed of collagen fibres that run along the long axis of the airway. Previous studies have shown that grooved topography can induce morphological and cytoskeletal alignment in epithelial cell lines. In the present work we assessed the impact of substrate topography on the organization of primary human tracheal epithelial cells (HTECs) and human induced pluripotent stem cell (hiPSC)-derived airway progenitors and the resulting alignment of cilia after maturation of the airway cells under Air-Liquid-Interface (ALI) culture. Grooves with optimized dimensions were imprinted into collagen vitrigel membranes (CVM) to produce gel inserts for ALI culture. Grooved CVM substrates induced cell alignment in HTECs and hiPSC airway progenitors in submerged culture. Further, both cell types were able to terminally differentiate into a multi-ciliated epithelium on both flat and groove CVM substrates. When exposed to ALI conditions, HTECs lost alignment after 14 days. Meanwhile, hiPSC-derived airway progenitors maintained their alignment throughout 31 days of ALI culture. Interestingly, neither initial alignment on the grooves, nor maintained alignment on the grooves induced alignment of cilia basal bodies, an indication of the direction of ciliary beating direction in the airway cells. Planar organization of airway cells during or prior to ciliogenesis therefore does not appear to be a feasible strategy to control cilia organization and subsequent airway epithelial function and additional cues are likely necessary to produce cilia alignment.

Cell Inertia: Predicting Cell Distributions in Lung Vasculature to Optimize Re-endothelialization.

We created a transient computational fluid dynamics model featuring a particle deposition probability function that incorporates inertia to quantify the transport and deposition of cells in mouse lung vasculature for the re-endothelialization of the acellular organ. Our novel inertial algorithm demonstrated a 73% reduction in cell seeding efficiency error compared to two established particle deposition algorithms when validated with experiments based on common clinical practices. We enhanced the uniformity of cell distributions in the lung vasculature by increasing the injection flow rate from 3.81 ml/min to 9.40 ml/min. As a result, the cell seeding efficiency increased in both the numerical and experimental results by 42 and 66%, respectively.

Negative Pressure Cell Delivery Augments Recellularization of Decellularized Lungs.

For end-stage lung disease, lung transplantation remains the only treatment but is limited by the availability of organs. Production of bioengineered lungs via recellularization is an alternative but is hindered by inadequate repopulation. We present a cell delivery method via the generation of negative pressure. Decellularized lungs were seeded with human bronchial epithelial cells using gravity-based perfusion or negative pressure (via air removal). After delivery, lungs were maintained in static conditions for 18 h, and cell surface coverage was qualitatively assessed using histology and analyzed by subjective scoring and an image analysis software. Negative pressure seeded lungs had higher cell surface coverage area, and this effect was maintained following 5 days of culture. Enhanced coverage via negative pressure cell delivery was also observed when vasculature seeded with endothelial cells. Our findings show that negative pressure cell delivery is a superior approach for the recellularization of the bioengineered lung. Impact statement New strategies are required to overcome the shortage of organ donors for lung transplantation. Recellularization of acellular biological scaffolds is an exciting potential alternative. Adequate recellularization, however, remains a significant challenge. This proof of concept study describes a novel cell delivery approach, which further enhances the recellularization of decellularized lungs. Organs seeded and cultured with this method possess higher cell surface coverage and number compared to those seeded via traditional gravity-based perfusion approaches.

Short-Term Preclinical Application of Functional Human Induced Pluripotent Stem Cell-Derived Airway Epithelial Patches.

Airway pathologies including cancer, trauma, and stenosis lack effective treatments, meanwhile airway transplantation and available tissue engineering approaches fail due to epithelial dysfunction. Autologous progenitors do not meet the clinical need for regeneration due to their insufficient expansion and differentiation, for which human induced pluripotent stem cells (hiPSCs) are promising alternatives. Airway epithelial patches are engineered by differentiating hiPSC-derived airway progenitors into physiological proportions of ciliated (73.9 ± 5.5%) and goblet (2.1 ± 1.4%) cells on a silk fibroin-collagen vitrigel membrane (SF-CVM) composite biomaterial for transplantation in porcine tracheal defects ex vivo and in vivo. Evaluation of ex vivo tracheal repair using hiPSC-derived SF-CVM patches demonstrate native-like tracheal epithelial metabolism and maintenance of mucociliary epithelium to day 3. In vivo studies demonstrate SF-CVM integration and maintenance of airway patency, showing 80.8 ± 3.6% graft coverage with an hiPSC-derived pseudostratified epithelium and 70.7 ± 2.3% coverage with viable cells, 3 days postoperatively. The utility of bioengineered, hiPSC-derived epithelial patches for airway repair is demonstrated in a short-term preclinical survival model, providing a significant leap for airway reconstruction approaches.

Computational fluid dynamics for enhanced tracheal bioreactor design and long-segment graft recellularization.

Successful re-epithelialization of de-epithelialized tracheal scaffolds remains a challenge for tracheal graft success. Currently, the lack of understanding of the bioreactor hydrodynamic environment, and its relation to cell seeding outcomes, serve as major obstacles to obtaining viable tracheal grafts. In this work, we used computational fluid dynamics to (a) re-design the fluid delivery system of a trachea bioreactor to promote a spatially uniform hydrodynamic environment, and (b) improve the perfusion cell seeding protocol to promote homogeneous cell deposition. Lagrangian particle-tracking simulations showed that low rates of rotation provide more uniform circumferential and longitudinal patterns of cell deposition, while higher rates of rotation only improve circumferential uniformity but bias cell deposition proximally. Validation experiments with human bronchial epithelial cells confirm that the model accurately predicts cell deposition in low shear stress environments. We used the acquired knowledge from our particle tracking model, as a guide for long-term tracheal repopulation studies. Cell repopulation using conditions resulting in low wall shear stress enabled enhanced re-epithelialization of long segment tracheal grafts. While our work focuses on tracheal regeneration, lessons learned in this study, can be applied to culturing of any tissue engineered tubular scaffold.

Use of High-Rate Ventilation Results in Enhanced Recellularization of Bioengineered Lung Scaffolds.

While transplantation is a viable treatment option for end-stage lung diseases, this option is highly constrained by the availability of organs and postoperative complications. A potential solution is the use of bioengineered lungs generated from repopulated acellular scaffolds. Effective recellularization, however, remains a challenge. In this proof-of-concept study, mice lung scaffolds were decellurized and recellurized using human bronchial epithelial cells (BEAS2B). We present a novel liquid ventilation protocol enabling control over tidal volume and high rates of ventilation. The use of a physiological tidal volume (300 µL) for mice and a higher ventilation rate (40 breaths per minute vs. 1 breath per minute) resulted in higher cell numbers and enhanced cell surface coverage in mouse lung scaffolds as determined via histological evaluation, genomic polymerase chain reaction (PCR) analysis, and immunohistochemistry. A biomimetic lung bioreactor system was designed to include the new ventilation protocol and allow for simultaneous vascular perfusion. We compared the lungs cultured in our dual system to lungs cultured with a bioreactor allowing vascular perfusion only and showed that our system significantly enhances cell numbers and surface coverage. In summary, our results demonstrate the importance of the physical environment and forces for lung recellularization. Impact statement New bioreactor systems are required to further enhance the regeneration process of bioengineered lungs. This proof-of-concept study describes a novel ventilation protocol that allows for control over ventilation parameters such as rate and tidal volume. Our data show that a higher rate of ventilation is correlated with higher cell numbers and increased surface coverage. We designed a new biomimetic bioreactor system that allows for ventilation and simultaneous perfusion. Compared to a traditional perfusion only system, recellularization was enhanced in lungs recellularized with our new biomimetic bioreactor.