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The development of biomimetic in vitro lung models as an alternative to animal studies is urgent to improve the predictability of the pharmacokinetics of potential new drugs. For pharmacokinetics studies, advanced in vitro lung models such as lung-chips should mimic a functional air-blood barrier. Unlike in vivo conditions, stem/primary cells and cell lines do not necessarily form a functional and tight barrier when cultured in vitro. Here, we explore the two gold standard techniques for monitoring barrier integrity: transepithelial electrical resistance (TEER) and permeability. We discuss the advantages and limitations of these methods, provide recommendations for methodological improvements, and we elude on possible future directions.

Background and Aim: Ganoderma lucidum is a traditional medicinal mushroom that has many therapeutic applications. However, the application of this beneficial fungus has been limited due to its secondary metabolite’s low production. Many investigations have been carried out to improve the production of G. lucidum as well as Ganoderic Acid (GA); however, prior studies suffer from the lack of sufficient monitoring platform. A real-time monitoring study could be useful to find more information through cultivation and evaluating secondary metabolite production. Materials and Methods: In the current study, aerated shaken flasks (AF) with different culture mediums were used for online measurement of the respiration activity of G. lucidum in small scale bioreactors. Then, to find more information through G. lucidum growth, four factors, including biomass formation, GA, residual sugar, and gene expression were evaluated on different days. Results: Online monitoring of cell proliferation demonstrated that GA starts to synthesize in the second growth phase as a partially growth-associated metabolite. High maximum biomass and GA production were obtained at an initial glucose concentration of 35 g/L with vitamin and KH2PO4. During fermentation, the YO2/GA and Y O2/X the rate of oxygen consumption rate per GA production and biomass formation, respectively, were introduced as beneficial parameters to scale-up the process. Conclusions: A novel monitoring strategy was suggested which can be beneficial for future investigations.

We describe the engineering design, computational modeling, and empirical performance of a moving air-liquid interface (MALI) bioreactor for the study of aerosol deposition on cells cultured on an elastic, porous membrane which mimics both air-liquid interface exposure conditions and mechanoelastic motion of lung tissue during breathing. The device consists of two chambers separated by a cell layer cultured on a porous, flexible membrane. The lower (basolateral) chamber is perfused with cell culture medium simulating blood circulation. The upper (apical) chamber representing the air compartment of the lung is interfaced to an aerosol generator and a pressure actuation system. By cycling the pressure in the apical chamber between 0 and 7 kPa, the membrane can mimic the periodic mechanical strain of the alveolar wall. Focusing on the engineering aspects of the system, we show that membrane strain can be monitored by measuring changes in pressure resulting from the movement of media in the basolateral chamber. Moreover, liquid aerosol deposition at a high dose delivery rate (>1 mu l cm(-2) min(-1)) is highly efficient (ca. 51.5%) and can be accurately modeled using finite element methods. Finally, we show that lung epithelial cells can be mechanically stimulated under air-liquid interface and stretch-conditions without loss of viability. The MALI bioreactor could be used to study the effects of aerosol on alveolar cells cultured at the air-liquid interface in a biodynamic environment or for toxicological or therapeutic applications.

Background and purpose: Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β-catenin signaling is reduced in emphysema and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β-catenin signaling and asses its potential to activate lung epithelial cells and repair. Experimental approach: We screened 1216 human-approved compounds for Wnt/β-catenin signaling activation using luciferase reporter cells, and selected candidates based on their computational predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell derived lung organoids and in vivo using a murine elastase-induced emphysema model. Key results: The primary screen identified 16 compounds that significantly induced Wnt/β-catenin-dependent luciferase activity. Selected compounds activated Wnt/β-catenin signaling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β-catenin inhibition, confirming the Wnt β-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signaling in emphysema. Conclusion and implications: Using a drug screen based on Wnt/β-catenin activity, organoid assays, and a murine emphysema model, amlexanox was identified as a novel potential therapeutic for emphysema.

Purpose Lung transplantation (LTx) is the final treatment option in end-stage lung diseases. Despite significant improvements, the long-term outcome remains uncertain with a 6 year survival of 53% LTx patients. Among risk factors, primary graft dysfunction (PGD) is the leading cause of early morbidity and mortality after LTx. PGD is a form of acute lung injury that results from ischemia, defined as a deficiency of oxygen, caused by the restriction of the blood flow, and blood reperfusion of the ischemic tissue, which may induces tissue injury reperfusion injury (IRI). This process is associated with increased permeability of capillaries, hypoxemia, cell death, and cytokine release. Our aim is to investigate the pathway involved in PGD and develop a therapeutic approach. Methods An orthotopic left lung murine model was used to simulate PGD. Lung grafts were stored at 4°C for 18h in Perfadex® solution, supplemented with and without cysteine-protease inhibitor, and perfused for 2h via ex vivo lung perfusion (EVLP) with and without inhibitor. The left lung was implanted for 4 hours in a recipient mouse, in order to simulate IRI. Gas exchange function, Apoptosis, bronchoalveolar lavage fluid (BALF), and perfusate were determined Results Lungs stored and perfused with cysteine-protease-inhibitor showed a better oxygen-exchange (93,9 % vs 74,47 % pO2) in the alveolar compartment, less total cell (30,4 vs 65,26 per field of view) and AT2 cell apoptosis after 4h of warm reperfusion compared with the control group. These findings correlate with reduced TNF-α release in perfusate after cold storage and EVLP, which promotes a better preservation of the organ. In-vitro experiments well simulated IRI injury features that further confirmed in-vivo findings. Cysteine-protease inhibitor, added to the cell medium prevents cysteine-protease activity and TNFα release after cold storage Conclusion Here, we showed that ischemia-reperfusion damage is associated with an increase of cysteine-proteases´s activity, in particular after warm-reperfusion of the graft, which results in enhanced maturation of TNF-α, and enhanced cell apoptosis of alveolar AT2 cells, that affect oxygen exchange of the implanted lung. These results provide a new therapeutic strategy for a safer organ preservation and reperfusion after LTx.

Chronic respiratory diseases are among the leading causes of death worldwide, but only symptomatic therapies are available for terminal illness. This in part reflects a lack of biomimetic in vitro models that can imitate the complex environment and physiology of the lung. Here, a copolymeric membrane consisting of poly(epsilon-)caprolactone and gelatin with tunable properties, resembling the main characteristics of the alveolar basement membrane is introduced. The thin bioinspired membrane (0.5 mu m) is stretchable (up to 25% linear strain) with appropriate surface wettability and porosity for culturing lung epithelial cells under air-liquid interface conditions. The unique biphasic concept of this membrane provides optimum characteristics for initial cell growth (phase I) and then switch to biomimetic properties for cyclic cell-stretch experiments (phase II). It is showed that physiologic cyclic mechanical stretch improves formation of F-actin cytoskeleton filaments and tight junctions while non-physiologic over-stretch induces cell apoptosis, activates inflammatory response (IL-8), and impairs epithelial barrier integrity. It is also demonstrated that cyclic physiologic stretch can enhance the cellular uptake of nanoparticles. Since this membrane offers considerable advantages over currently used membranes, it may lead the way to more biomimetic in vitro models of the lung for translation of in vitro response studies into clinical outcome.

In this study, we improved a perfusion-based bioreactor (PBB) system to recellularize acellular lung scaffolds. A method was used for the online monitoring of the recellularization process in order to find more data during a re-seeding procedure. Lungs were harvested from young adult (2-month-old) male rats. After preparation and pre-decellularization, the lungs were decellularized with liquid detergent in order to obtain an acellular scaffold. Acelluar tissues were stained by hematoxylin and eosin to verify efficient removal of cell components and debris. Furthermore, the maintenance of the extracellular matrix architecture was evaluated using scanning electron microscopy. After rinsing for detergent removal, human umbilical cord vein endothelial cells were cultured on acellular tissue using a PBB. Data analysis of the dissolved oxygen through culturing indicated a specific growth rate of 0.0199 h−1 (doubling time, td = 34 h). The average oxygen uptake rate per cell was measured as 14.3 nmol/min per 106 cells. This study demonstrates that recellularization on acellular tissue can be monitored online to provide insight into the growth behavior of cells. The results show the high performance design of PBBs for tissue-engineered lungs.

Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.

Nanoparticle‐based targeted drug delivery holds promise for treatment of cancers. However, most approaches fail to be translated into clinical success due to ineffective tumor targeting in vivo. Here, the delivery potential of mesoporous silica nanoparticles (MSN) functionalized with targeting ligands for epidermal growth factor receptor and C─C chemokine receptor type 2 is explored in lung tumors. The addition of active targeting ligands on MSNs enhances their uptake in vitro but fails to promote specific delivery to tumors in vivo, when administered systemically via the blood or locally to the lung into immunocompetent murine lung cancer models. Ineffective tumor targeting is due to efficient clearance of the MSNs by the phagocytic cells of the liver, spleen, and lung. These limitations, however, are successfully overcome using a novel organ‐restricted vascular delivery (ORVD) approach. ORVD in isolated and perfused mouse lungs of Kras‐mutant mice enables effective nanoparticle extravasation from the tumor vasculature into the core of solid lung tumors. In this study, ORVD promotes tumor cell‐specific uptake of nanoparticles at cellular resolution independent of their functionalization with targeting ligands. Organ‐restricted vascular delivery thus opens new avenues for optimized nanoparticles for lung cancer therapy and may have broad applications for other vascularized tumor types.

Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.

Background: Lung transplantation is the only curative approach for end stage lung disease, but tissue shortage remains a problem. Generating lung tissue ex vivo could overcome tissue shortages. Both synthetic and biologic scaffolds have been explored, yet both have limitations. Biologic scaffolds have low reproducibility while synthetic scaffolds lack biologically inductive properties. Hybrid materials, comprised of both, have been used for tissue engineering, but have been limited to simple shapes. Recent advances in 3D printing may permit for complex shapes, but potential bioinks are unknown. We hypothesized that a hybrid hydrogel generated by combining a synthetic polymer with lung derived extracellular matrix (ECM) could be a suitable bioink for 3D bioprinting lung tissue. Methods: We generated hybrid hydrogels from a synthetic polymer, alginate, and ECM derived from decellularised mouse lungs to make hybrid hydrogels. Viability and proliferation of distal lung epithelial cell lines (MLE12 and A549) encapsulated in the hydrogel were measured via immunofluorescence (Ki67+) and flow cytometry (EdU+) after 7 days. Results: Both MLE12 and A549 cells remained viable and were distributed homogenously within manufactured hydrogels of various shapes. MLE12s in ECM-alginate hybrid hydrogels were significantly more proliferative (30% increase) than MLE12 cells seeded in an alginate hydrogel as assessed by flow cytometry. Conclusions: A hybrid hydrogel generated from alginate and decellularised lung derived ECM supports proliferation of lung epithelial cell lines and can be manufactured in a variety of shapes. The hybrid alginate-ECM solution is thus a promising candidate as a bioink for 3D bioprinting.

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Lung disease is one of the major causes of death, and the rate of pulmonary diseases has been increasing for decades. Although lung transplantation is the only treatment for majority of patients, this method has been limited due to lack of donors. Therefore, recently, attentions have increased to some new strategies with the aid of tissue engineering and microfluidics techniques not only for the functional analysis, but also for drug screening. In fact, in tissue engineering, the engineered tissue is able to grow by using the patient's own cells without intervention in the immune system. On the other hand, microfluidics devices are applied in order to evaluate drug screenings, function analysis and toxicity. This article reviews new advances in lung tissue engineering and lung-on-a-chip. Furthermore, future directions, difficulties and drawbacks of pulmonary therapy in these areas are discussed.