3D organoids systems allow to re-create the architecture and physiology of human organs in remarkable detail. They also allow to better mimic the in vivo tissue by improving the structural complexity of cell arrangement in culture, including cell polarity. This make 3D organoids an ideal system to be dissected and interrogated for fundamental mechanistic studies on development, regeneration and repair in human tissues, and can also be used in diagnostics, disease modelling, drug discovery and personalized medicine (Kim et al., 2020; Zhao et al., 2022). Despite being powerful in vitro models, organoids present also some limitations, including low scalability, limited lifespan, difficulty in imposing the proper extracellular forces, incomplete matching of organ-level functionality, and lack of vascularization (Tang et al., 2022). To overcome these limitations engineering solutions are in constant development to gain control over self-organization and functionality of 3D organoids (Garreta et al., 2021).

Lung organoids represent a powerful in vitro model to study human lung development and disease modelling (Liberti and Morrisey, 2021). The human developing lung has a complex architecture that comprises up to 144 different cell types/states based on scRNA-seq, scATAC-seq, Visium Spatial Transcriptomics, and mRNA in situ hybridization using Hybridization Chain Reaction (HCR) (Trivedi et al., 2018; He et al., 2022). Various specialized cell types contribute to a normal functional lung for essential respiration. Perturbations of the extracellular environment during development can result in severe pulmonary diseases from birth to adulthood (H.-T. Kim et al., 2018). As high-throughput microscopic imaging is challenging due to the highly complex architecture of lungs, models to faithfully recapitulate lung development and disease are in urgent demand. While 2D cell culture will inevitably fail to fully mimic the spatial responses to the environmental biochemical and biophysical cues (Reviewed more thoroughly by Cunniff et al., 2021), airway epithelial cells-derived lung organoids emerge as a powerful in vitro 3D model that can faithfully recapitulate the pathophysiological aspects of human lungs with different extent of complexity in terms of structure and cell morphology, as well as interaction of different cell types (Huch and Koo, 2015; Sachs et al., 2019). Limitations of 3D organoids related to the difficulty for the inner cells to receive nutrients and exchange the waste with the extracellular environment while growing, could be overcome by culturing organoids in microfluidic devices to facilitate the nutrients and waste circulation and using engineered scaffolds to direct the morphological growth (De Santis et al., 2021). This highlights the importance of biophysical and biomechanical properties in lung organoids culture (Huch and Koo, 2015; Sachs et al., 2019).

Lung organoids can be classified as three major types: i) airway-organoids consisting of secretory club cells, goblet cells, ciliated cells, and bronchiolar epithelial cells; ii) alveolar organoids consisting of alveolar type one (AT1) and alveolar type two (AT2) cells; iii) complex organoids consisting of both proximal and distal airway epithelial cells (Demchenko et al., 2022). In addition, there are two main sources of lung organoids generation: primary lung epithelial progenitor-derived organoids and induced pluripotent stem cell (IPSCs)-derived organoids (Dye et al., 2015; Leeman et al., 2019; Rabata et al., 2020). The generation of primary lung progenitors-derived organoids requires biopsy sections which undergoes single cell suspension through enzymatic digestion. The obtained single cells can be selected against specific cell markers and seeded in Matrigel drops to be further cultured as a more homogenous cell population (Katsura et al., 2020). However, lung organoids generated from progenitors in primary tissues usually lack mesenchymal cells and stromal cells which supplies signalling and structural support (Schutgens and Clevers, 2020). IPSCs-derived organoids are generated from reprogrammed somatic cells, generally skin fibroblasts (Jacob et al., 2019). IPSCs-derived organoids have great advantages in terms of the involvement of both epithelial and mesenchymal cells in the system and retaining genetic information as they can be derived from patients (Choi et al., 2016; Chen et al., 2017). In fact, mesenchymal cells contribute to the maturation of organoids in alveolospheres (Barkauskas et al., 2013). By embedding the lung organoids in Matrigel and applying a cocktail of growth factors and adjusting extracellular matrix (ECM) compositions, different combinations of biochemical and biophysical signals can direct the lung organoids under self-expansion or further differentiation (Figure 1) (Barkauskas et al., 2013; Chen et al., 2017; Sachs et al., 2019).

Both human primary tissue and IPSCs-derived lung organoids allow studying cell-cell and cell-environment interactions during lung development and disease modelling due to their ability of mimicking in vivo pathophysiological conditions. Several studies used primary tissue and IPSCs-derived lung organoids to model pulmonary diseases, including Idiopathic pulmonary fibrosis (IPF), Chronic obstructive pulmonary disease (COPD), Cystic fibrosis (CF), and respiratory viral infections like COVID-19 (Y. Han et al., 2022; Mou et al., 2012; Strikoudis et al., 2019; Tan et al., 2017; Wilkinson et al., 2017; Wong et al., 2012). The addition of transforming growth factor beta 1 (TGF-β1) to 3D lung organoids containing mesenchymal cells resulted in morphological scarring typically found in IPF, but not in 2D cell culture (Wilkinson et al., 2017). In addition, alveolar organoids derived from human pluripotent stem cells have been used to study the pathogenesis of CF and drug screening. Treatment with TGF-β1 successfully induced fibrotic changes that is characterized in CF. This phenotype is greatly ameliorated by supressing ERK signalling, providing a potential target pathway for CF (Kim et al., 2021).

While the biochemical and genetic aspects of lung development and disease have been widely investigated, the role of mechanical aspects have been investigated in the last 20 years only. This review will shed light on the crucial role of different types of mechanical forces in regulating lung development and disease pathogenesis.

In the developing lungs, a ramified network of pulmonary epithelium arises from the protrusion of lung bud into the surrounding mesenchyme (Grobstein, 1953; Hogan, 1999). The reciprocal biochemical interactions between lung epithelium and mesenchymal cells are thought to mediate branching morphogenesis. Non-canonical Wnt signalling has been shown to play a role in determining the direction and position of early lung branching. Specifically, Wnt5a-null mutant lungs showed defective cytoskeleton organization and focal adhesion which resulted in perturbed lung branching (Zhang et al., 2022). Peak and others showed that Fgf10-loaded bead can induce ectopic budding of lung epithelium which does not affect the focal adhesion and myosin light chain phosphorylation expression in the protruding area. The ectopic buddings are disrupted when applying epithelium tension by mimicking luminal fluid flow using pharmaceutical treatment, which highlights the crucial role of mechanical forces in lung morphogenesis (Unbekandt et al., 2008; Peak et al., 2022).

Lung epithelial cells are constantly exposed to various mechanical forces throughout development, which have been shown to shape airway tree branching morphogenesis and regulate alveolar specifications. The biochemical signals secreted by the surrounding mesenchymal tissue guide lung branching morphogenesis. Interestingly, this can be overridden if mechanical forces are applied. For instance, the physical instability occurring between the epithelium and the extracellular microenvironment resulted in local morphological protrusion and branching of embryonic airway epithelium independent of the surrounding mesenchyme (Varner et al., 2015). Smooth muscle cells surrounding the airway epithelium during development also contribute to airway morphogenesis. Mouse embryonic airway branching can be altered by manipulating smooth muscle cells differentiation surrounding the airway, likely as a consequence of the high stiffness of the smooth muscle outer layers. By promoting smooth muscle differentiation, lung buddings are inhibited, whereas by inhibiting the smooth muscle differentiation, ectopic branching appears (Goodwin et al., 2019; H. Y; Kim et al., 2015). Bioengineered ‘microfluidic chest cavities’ have been used to generate different levels of pressure, and this allowed regulating the interval of smooth muscle contraction. This confirmed that the physical contraction of smooth muscle directs the lung branching morphogenesis (Nelson et al., 2017). Another study conducted by the Nan Tang group shed light on the key role of mechanical forces in lung branching morphogenesis during early lung development. Using Cre-Shh reporter mice, they distinguished epithelial cells with two types of spindle fibres orientations, i.e., parallel to the long axis and randomly oriented spindle fibres. By embedding epithelial cells in stretchable gels, they found the ratio of normally oriented and randomly oriented spindle fibres are biased when mechanical forces are applied (Tang et al., 2018).

The mechanical forces-regulated cell division pattern contributes to the normal branching morphogenesis. The ablation of the mechanical-responsive transcriptomic activator Yes-associated protein (Yap) in developing mouse lungs has also been shown to affect epithelial cells proliferation and affect branching morphogenesis. There is an uncovered feedback regulation between Yap and myosin light chain phosphorylation which represents the mechanical force responsive signalling mediator. Deletion of Yap results in downregulated myosin light chain phosphorylation (Lin et al., 2017). The relationship between mechanical forces and regulated cell proliferation greatly impacted on normal lung branching morphogenesis. Despite the role of mechanical forces in early lung branching process, they also regulate the alveolar specification at later sacculation stage. In vivo and ex vivo stretching of whole lungs induces activation of Yap/Taz signalling which promotes AT1 differentiation. By using ROCK inhibitor, Y27632, AT1 cells significantly decreased, which suggests the regulation of AT1 differentiation through ROCK-YAP/Taz signalling pathway (Nguyen et al., 2021). The Nan Tang group showed the shear stress, the force exerted by the flowing amniotic fluid within the epithelial air tree to the surface of epithelium, regulates AT1 and AT2 fate decision. They suggested progenitors protrude from the epithelium due to the attraction of Fgf10 signalling from the surrounding stromal cells, and myosin accumulates at the apical surface of the protruding epithelium. The inhalation of amniotic fluid exerts shear stress to the epithelium, where the protruding cells are protected from the mechanical force by the accumulated myosin and become AT2 cell which remains cuboidal shape. In contrast, cells subjected to shear stress differentiate into flattened AT1 cells (Li et al., 2018). While further studies are needed to uncover the underlying molecular mechanisms of mechanical forces-regulated branching morphogenesis and alveolar specification, it is evident that mechanical forces have a key role in lung development. The reciprocal interactions between mechanical forces and biochemical signals may be the key to further decipher the process of lung branching morphogenesis and further alveolar specification.

The major function of the lungs is gas exchange. As such, the mechanical forces exerted to the lung epithelium by the constant breathing movement play an important role in maintaining tissue homeostasis and epithelial cell fate decision (Shiraishi et al., 2023). Perturbation of the mechanical forces can greatly impact on lung function, leading to pathogenesis. AT1 are flattened, non-proliferative alveolar cells, which are mainly responsible for gas exchange and account for 95% of alveolar surface. AT2 are cuboidal proliferative epithelial cells that secret surfactant proteins to prevent the collapse of the alveolar units during breathing. The proliferative ability of AT2 cells and the secretion of surfactant proteins are promoted by the mechanical stress generated by ventilation, which maintains the integrity of the alveolar epithelium (Arold et al., 2009; B; Han, 2005). The mechanical forces generated by ventilation and exerted to the airway epithelium by various environmental stiffness regulate the activity of Yap by manipulating the shape of nuclei to control the nuclear influx rate of Yap in airway epithelium (Elosegui-Artola et al., 2017). The activity of Yap is crucial for maintaining the balanced differentiation of club cells and goblet cells. Ablation of Yap in the proximal epithelium directs the trans-differentiation of goblet cells into club cells, therefore altering the epithelial homeostasis (Hicks-Berthet et al., 2021). Recent studies have shown surprising postnatal AT1 plasticity in reprogramming into AT2 upon inhibition of Yap signalling. Nuclear Yap activity is required to maintain the AT1 identity (Penkala et al., 2021). Another study suggests the reprogramming of AT1 into AT2 in response to mechanical cues through extracellular matrix-cytoskeleton, mediated by Cdc42 and Ptk2. The loss of mechanical constraint in AT1 cells results in rapid AT1-AT2 trans-differentiation. These findings suggest the crucial role of mechanical forces exerted by breathing in maintaining AT1 and AT2 homeostasis in postnatal lung (Shiraishi et al., 2023). In addition, it has been suggested that myeloid cells are responsive to the cyclic hydrostatic pressure through mechanical-responsive ion channel PIEZO1 which, in turn, regulates the innate immune system of the lung via c-Jun activation (Solis et al., 2019).

As epithelial cells along the airways have been shown to be mechano-sensitive and mechano-responsive, the disruption of the mechanical forces can result in altered tissue homeostasis and lung disease, such as COPD and emphysema, characterized by the accumulation of ectopic levels of elastin and collagen fibres, resulting in alveolar destruction (Kononov et al., 2001). Lung fibrosis is caused by over-proliferation of fibroblasts and, consequently, the accumulation of extracellular matrix proteins. IPF is the most common type of pulmonary fibrosis, characterized by impaired alveolar functions, mainly due to AT2 cells loss (Plantier et al., 2011). Extracellular matrix-cytoskeleton interactions regulated by Cdc42 in AT2 cells play an important role in IPF progression. Cdc42 ablation in AT2 cells results in failure of AT2-AT1 transition which exposes alveolar type cells to the constant mechanical forces from the extracellular matrix. The accumulated mechanical stress, in turn, activates TGF-β signalling that mediates further peripheral-central IPF progression (Wu et al., 2020). These findings, that highlight the connection between mechanical forces and pulmonary pathogenesis, are determinant for building up a patho-physiologically relevant disease model.

Although numerous studies have successfully reported the generation of lung organoids derived from pluripotent stem cells comprising multiple differentiated epithelial cells, the differentiated cells retain embryonic rather than mature characteristics, as revealed by transcriptomic analysis (Barkauskas et al., 2013; Firth et al., 2014; Dye et al., 2015; Konishi et al., 2016).

Different types of ECM provide mechanical, biophysical and signalling cues in a variety of developing organisms and tissues. The ECM helps to regulate tissue morphology during embryonic development by integrating cell genetic programs with chemical and mechanical input from the local microenvironment (Walma and Yamada, 2020). This implies that epithelial cells continually remodel their intercellular contacts throughout morphogenesis, which significantly rearranges cells within the epithelial sheet (H. Y. Kim et al., 2013). Interestingly, epithelial cells themselves contribute to producing ECM components which finally determines their differentiation towards specific cell types (Mamidi et al., 2018; Fernandez Vallone et al., 2020; Michielin et al., 2020). Lung alveolarization requires precise coordination of cell growth and differentiation with extracellular matrix (ECM) synthesis and deposition to allow alveolar septation. Laminin, which is a key component of the basement matrix in the developing lungs by supporting first epithelial branching and then capillary network formation, displays a simplified network along with the thinning of the alveolar septa. Conversely, elastin density is continuously increasing during alveologenesis as recently evidenced by 3D volumetric analysis of mouse and human lung tissues (Luo et al., 2018). This allows for proper maturation of elastin fibres to ensure elastic recoil of the lung during breathing. ECM deposition defects and alveolar simplification underlie many pulmonary diseases such as emphysema that results from the enzymatic destruction of ECM components leading to destruction of alveolar walls and airspace enlargement. Interestingly, alterations of the actomyosin network during development, for example, due to mutation in genes encoding cytoskeletal proteins, has been shown to repress metalloproteinase activity leading to disrupted ECM remodelling (H.-T. Kim et al., 2018).

Scaffold biomaterials can, therefore, provide physical and biochemical properties to promote maturation of lung organoids (De Santis et al., 2021), as previously reported (Xinaris et al., 2012; Watson et al., 2014; Finkbeiner et al., 2015). To enhance lung organoids engraftment, Dye et al. introduced an alternative niche consisting in microporous poly (lactide-co-glycolide) (PLG) scaffolds for the lung organoids to grow, which was previously utilized for the survival, and functionalization of human islet upon transplantation (Gibly et al., 2013; Hlavaty et al., 2014; Dye et al., 2016). These scaffolds provide a rigid surface for the epithelial cells to adhere with porous structures that allows further growth and vasculature of the epithelium (Figure 2B). Thanks to the scaffolds support, transplanted human lung organoids can form pseudostratified layers structurally similar to the adult airway. In addition, CC10+ secretory club cells and MUC5AC + goblet cells have been detected, which were not or rarely detected in foetal lung airway but found within adult airway epithelium. The retrieved scaffold after in vivo culturing also showed high level of vasculature with various types of mesenchymal tissues. The structure and cellular maturation of lung organoids are promoted upon the introduction of PLG scaffolds, whereas the lack of alveolar cells suggests further improvement is required for a full maturation (Dye et al., 2016).

Another natural scaffold for lung epithelium culture consists in the decellularization of the whole lungs preserving its original acellular extracellular matrix (Gilbert et al., 2006; Tommasini et al., 2023) (Figure 2A). Recellularization of the decellularized lungs using lung progenitors derived from human IPSCs have shown successful expansion and alveolar specification of AT2 progenitors (Gilpin et al., 2014; Leiby et al., 2023). Of note, the seeding of human lung organoids on the decellularized lung airway showed minimal or no club cell and goblet cell differentiation (Dye et al., 2015). Further characterization of the acellular lungs in terms of mechanical properties and stiffness will provide better insight into the role of the mechanical properties in guiding the maintenance and differentiation of epithelial cells (Melo et al., 2014).

As organoids are usually spherical in shape with multiple layers of cells, the growth factors, nutrients and oxygen poorly diffuse into the deeper layers of the organoids. From this perspective, microfluidics-based organoid-on-chip devices provide a platform for organoids to grow with a fine control of chemical and physical parameters (Bhatia and Ingber, 2014; Giulitti et al., 2016). These devices are characterized by membranes-integrated multi-micro-channels and holes from multiple dimensions filled with fluids which allow continuous perfusion of cells in culture. The membranes can separate different compartments in the device with independent channels, thus allowing the co-culture of alveolar epithelial cells and endothelial cells (Huh et al., 2010). In addition, a “breathing” microfluidics-based model has been developed by the Guenat group to mimic the mechanical stretches applied to the alveolar epithelial cells and showed successful alveolar function (Stucki et al., 2018) (Figure 2C). The incorporation of such “breathing” lung model with scalable mechanical stimuli and adjustable architecture and the 3D lung organoids from different developmental stages has great potential for studying the role of mechanical forces in instructing lung architecture organization and epithelial differentiation.

In summary, the three types of scaffolds provide adhesion sites for lung organoids to grow and are capable of maintaining the survival of progenitors, as well as of leading to lineage specification with respective advantages and drawbacks (Table 1). This suggests the crucial role of physical environment in tissue organization and cellular differentiation.

The crucial role of mechanical forces in embryonic lung development and adult lung homeostasis have been elucidated in the previous section. Mechanical stretching generates tensional force against the limited range of motion of the material due to its elastic physical property such as stiffness. The implementation of various biophysical stimulations, such as mechanical stretching mimicking fluid or air flow and environmental stiffness, is, therefore, likely to enhance the maturation and functionality of lung organoids. Previous studies showed significant upregulation of Surfactant protein b (SPB) and c (SPC) in isolated rat embryonic AT2 cells at late developmental stages in response to mechanical cyclic stretch (Sanchez-Esteban et al., 2001). The Tang group also showed the role of shear stress in regulating alveolar differentiation, where tip progenitor epithelial cells exposed to mechanical stress become flattened and differentiate into AT1 cells, whereas those protected from the stress by myosin accumulation differentiate into AT2 cells (Li et al., 2018). In contrast, lung alveolar organoids derived from tumour-free tissue from patients undergoing lung resection surgery for lung cancer or from emphysematous tissue cultured in a chip with stretchable polydimethylsiloxane (PDMS) membrane showed no variation of mRNA levels in SPB and SPC upon stretching (van Riet et al., 2022). It is, therefore, possible that the alveolar cells from postnatal tissue or from adult lung disease patients’ tissue are less responsive to the mechanical stretch in terms of surfactant protein production. Of note, the conventional in vitro AT2 cell culture tends to trans-differentiate into AT1 cells over time (Mao et al., 2015), whereas the culture of AT2 organoids in the stretchable chip maintains the AT2 phenotype for longer time without trans-differentiating to AT1 cells (van Riet et al., 2022). This study suggested a potential role of mechanical stretch in alveolar type cells maintenance. Further validation of the role of mechanical stretch in enhancing embryonic alveolar maturation by growing embryonic lung tip epithelial progenitors-derived organoids on the breathing device is needed. By applying constant cyclic stretch to the organoids derived from postanal day5 mouse lungs with co-culture of fibroblast and CD326-positive cells, expression of mesenchymal genes is significantly upregulated in lung organoids. Upon lateral stretching, the population of α-smooth muscle actin (αSMA)- and platelet-derived growth factor receptor-α (PDGFRα)-positive cells increased with highly activated transforming growth factor-beta (TGF-β) signalling (Joshi et al., 2022). The PDGFRα-positive mesenchymal cells have been shown to provide physical support to maintain the proliferative ability of AT2 cells in adult mouse lungs. A reduced number of PDGFRα-positive cells resulted in depletion of proliferative AT2 population with impaired alveologenesis (Gao et al., 2022; Riccetti et al., 2022). As the only surfactant protein-associated gene detected in the lung organoids derived by Joshi and others is surfactant protein D, which is secreted by the proximal airway epithelial cells, it is believed no alveolar cells are included in their organoid system. It would be interesting to investigate further if cyclic stretch increases the population of mesenchymal cells. This may promote alveologenesis or contribute to alveolar maintenance in embryonic lung development and adult lung homeostasis. This investigation can be done using alveolar cells-integrated organoids derived from embryonic and adult lung tissue.

Despite the importance of mechanical stretch in embryonic lung development and postnatal lung homeostasis, the impact of environmental stiffness in proximal and distal airway epithelial differentiation and alveolar maintenance is still understudied. Various pulmonary diseases, such as COPD and IPF, are characterized by pathologic stiffening of the environmental ECM. By seeding primary rat alveolar epithelial cells in polyacrylamide gel substrates of varying stiffness, no influence of substrate stiffness in alveolar differentiation, specifically in AT2-AT1 transition was detected (Jones, 2011). However, 2D primary cell culture cannot faithfully recapitulate the interactions between ECM and epithelial cells in vivo. The combination of 3D lung organoids model and stiffness-tuneable 3D synthetic hydrogels (Urciuolo et al., 2023) may fill the gap of the impacts of environmental stiffness in epithelial specification, especially alveolar differentiation and maintenance.

Many pulmonary disorders are associated with various forms of mechanical stimuli perceived by cells in the lung. These stimuli derive from the pathogenic alteration of ECM stiffness, like in IPF and COPD, or from direct mechanical compression, like in Congenital Diaphragmatic Hernia (CDH), where the ectopic protrusion of abdominal organs hinders the developing lungs in the thoracic cavity (Edel et al., 2021; Nho et al., 2022). IPF is characterized by the failure of AT2-AT1 transitioning and bronchiolization of the distal epithelial cells, along with accumulation of fibroblast and differentiated myofibroblast in the distal ECM, which results in local stiffening of the extracellular environment. The local accumulation of fibroblasts is associated with increased migration and proliferation via the activation of multiple signalling pathways such as TGF-β signalling, and deposition of ECM proteins (Selman and Pardo, 2002; Selman, 2006). Schruf and others successfully established an iPSC-derived AT2 organoid that recapitulates airway epithelial-like expression signature in IPF through the treatment of 9 IPF-relevant molecules cocktails (Schruf et al., 2020). Strikoudis and others modelled embryonic stem cell-derived fibrotic organoids using CRISPR-Cas9 gene editing, which showed high similarities in gene expression compared to IPF patients. They documented the important role of interleukin-11 in fibrosis during IPF pathogenesis (Strikoudis et al., 2019). Despite the success in recapitulating the gene expression profiles observed in IPF patients by gene editing or applying biochemical signals, the role of biophysical signals in IPF pathogenesis are still underappreciated. As such, can the disease phenotype and pathogenesis be better recapitulated by integrating biophysical cues to lung organoids models? A recent study conducted by the Magin group revealed the key role of environmental stiffness in IPF pathogenesis through the crosstalk between fibroblasts and AT2 cells (Caracena et al., 2022). A 3D co-culture of fibroblasts and AT2 cells were embedded in PEG-based hydrogels with tuneable stiffness, that recapitulates the mechanical properties of healthy and fibrotic lungs. In stiff conditions, the upregulation of profibrotic genes is highly promoted with upregulated ECM remodelling genes that has been shown to contribute to pathogenic fibrogenesis condition in IPF. Surprisingly, AT2 cells are also stiffness-responsive and their crosstalk with fibroblasts contributes to the myofibroblast differentiation through TGF-β activation. The presence of AT2 cells synergistically promote fibrosis with the high-stiffness microenvironment (Caracena et al., 2022). By manipulating mechanical properties alone, the pathogenesis of IPF was promoted. The emergence of a transient population of epithelial cells in the 3D model was identified in IPF condition in vivo. It would be, therefore, interesting to perform RNA-sequencing on the transient population of cells to compare the transcriptional profiles with the epithelial cells in IPF tissue to evaluate the level of recapitulation of IPF pathogenesis in addition to evaluating the level of fibrogenesis. Moreover, additional biochemical signals can be integrated into the 3D model together with the biophysical cue to study the conditions of fibroblasts activation.

Despite the cause of CDH remain elusive, growing evidence has suggested the key role of mechanical compression exerted to the developing lungs since the pseudoglandular stage from the herniated abdominal content in causing pulmonary hypoplasia (Nelson et al., 2017). Mechanical compression is recapitulated in the nitrofen-exposed rat lung to create an ex vivo CDH model. The transcriptomic profile of these lungs showed downregulation of Spc and Pdpn suggesting impaired alveolar functionality (Fox et al., 2018). A human CDH foetus-derived iPSCs lung organoids model including mesenchymal cells and epithelial cells was also generated to study the effects of mechanical compression in CDH pathogenesis. Interestingly, comparing lung organoids generated from CDH patients and healthy embryonic lungs without mechanical compression, the expression levels of SPB, SPC, NKX2.1, SOX2, SOX9 and PDGFRα were significantly downregulated in CDH organoids. This suggests an inherently impairment of epithelial progenitors and alveolarization in CDH-organoids development. By applying mechanical compression ex vivo to the CDH-organoids, the expression of SOX2, SOX9, NKX2.1, PDPN and Ki67 are remarkably downregulated, which suggests further disturbed progenitor identity, alveolar function and proliferative ability (Kunisaki et al., 2021). Although the iPSCs-derived lung organoids model with integrated mechanical compression recapitulate multiple aspects of CDH pathogenesis, a functional evaluation of the alveolar epithelial cells is still missing. Due to the time course of iPSC generation and organoids culture, the mechanical compression is only exerted to the organoid system at one time point which may not faithfully mimic the prolonged mechanical compression to the developing lungs in vivo. Thus, a model that can provide constant mechanical compression to the lung organoid, for example, using gels with tuneable stiffness, may create a more reliable in vitro CDH model to study its pathogenesis.

Overall, by integrating biophysical signals, the phenotypes and pathogenesis of pulmonary diseases are better recapitulated in 3D organoid cultures.

Organoids have been shown to be a robust model to successfully recapitulate key transcriptional signatures of healthy and diseased tissue, although they often turn to be functionally immature. A better understanding of cellular mechanisms is required and could be achieved by integrating biophysical cues in culture to develop a more reliable organoids-based in vitro model.

Alveoli are the functional units for gas exchange which is the major function of the lungs. Alterations in their development cause malfunctions and represent the basis of many pulmonary distal diseases. It is well recognized that mesenchymal cells can sense the mechanical forces and influence epithelial cells behaviour. However, how the epithelial progenitors and alveolar epithelial cells receive and respond to the mechanical signals remain poorly studied (Zepp et al., 2021). Emerging studies revealed the astonishing plasticity of AT1 and AT2 cells in response to the mechanical stress from various sources. Different types of scaffolds mimicking physical properties of the in vivo environment and the integration of biophysical cues have been developed to promote the maturation of lung organoids and showed different levels of success (van Riet et al., 2022). This is supported by the culture of AT2 cells in decellularized lung scaffolds with co-culture of endothelial, epithelial cells and fibroblast. It was found that mechanical stretch alone is unable to affect AT1 differentiation when high levels of Wnt and FGF agonists are present. However, a Wnt- and FGF-low environment synergizes with tidal-level mechanical strain to promote AT2 differentiation to AT1s within the engineered lung tissues (Leiby et al., 2023). Although challenging, in the future, the combination of biophysical and biochemical cues at different stages in culturing lung organoids should be performed and may be crucial to further promote their maturity.

Despite different levels of organoids maturation promoted by PLG scaffolds and decellularized lung scaffolds, there is an obvious limitation in both methods due to the uncontrollable physical and biochemical parameters in culture. Microfluidic devices may overcome these disadvantages thanks to the integration of sensors that can detect the alterations of these parameters. Mechanical inputs such as through membrane stretching and shear stress exerted by air or fluid flow have also some limitations related to the local control of the stimuli. Synthetic hydrogel scaffolds possess physical properties that mimics in vivo environment. The combination of hydrogels and microfluidics may maximise their individual advantages, to further promote organoids maturation.

IPSCs-derived lung organoids have been favoured as the model to study embryonic development and pathogenesis due to their availability, scalability and plasticity that allows to differentiate them into the desired state. Adult cells-derived organoids, although more difficult to be established in vitro, are valuable in studying disease pathology as well. For example, primary human nasopharyngeal and bronchial organoids are derived from COPD patient, consisting of goblet, club and ciliated cells. The COPD lung organoids exhibit hallmarks of COPD pathologic conditions including hyperplasic goblet cells and lowered beating frequency ciliated cells. The SARS-CoV-2 infection are more severe in the bronchial region with greater pro-inflammation response in COPD organoids. The COPD lung organoids provided a model to study COPD pathology and the pathogen-host interaction (Chan et al., 2022). The pathological characteristics retained in the patients’ adult cells-derived organoids make them invaluable as the model for disease pathology study and drug screening. Dysregulation of the mechanical forces or physical properties may result in malfunctioning of alveolar cells and lead to severe pulmonary diseases. Thus, it is crucial to understand the role of mechanical forces in lung development and epithelial maintenance to create more mature in vitro lung organoids to model lung development and disease pathogenesis. Different tissue compositions surrounding the airway epithelium potentially create a differential gradient of environmental stiffness in the adult lungs. Specifically, there is a gradual loss of cartilage and smooth muscles surrounding the proximal-distal airway axis. Both cartilage and smooth muscle cells are completely missing in the alveolar region and the tissue is enriched with soft capillaries for gas exchange (Badri et al., 2008; Nikolić et al., 2018). This suggests a potential spatio-temporal stiffness gradient change along the airway during lung development, which may also contribute to the different epithelial cell specification. The potential spatio-temporal stiffness gradient can be measured using Atomic Force Microscopy (AFM), which will give insight into how differential stiffness relates to epithelial commitments. A high environmental stiffness has been suggested to promote pathogenesis of IPF through the regulation of myofibroblast differentiation and fibroblast-AT2 cells crosstalk (Caracena et al., 2022). Fibroblasts and lung epithelial cells are stiffness-responsive, which confirms the importance of the fine-established environmental stiffness along the airway. Alteration of mechanical stiffness displayed by the surrounding tissues and the resulted dysregulated signalling likely affects IPF pathogenesis. In addition, the prolonged mechanical compression exerted to the lungs will result in alterations of mechanical-responsive signalling in epithelial progenitors and alveolar epithelial cells which may lead to changes in their fate decisions. Impaired alveolarization will greatly impact the postnatal lung function and is associated with high mortality. Therefore, in the next future researches should focus on the underlying molecular mechanisms of different mechanical forces in regulating epithelial commitment and maintenance during lung development and adulthood homeostasis. By manipulating the molecular pathways in the lung disease organoid model, the pathogenesis and potential therapeutic targets of the pulmonary diseases can be better investigated.

In conclusion, lung organoids represent a robust in vitro model to study lung development and disease pathogenesis. Mechanical forces are crucial in lung development in terms of epithelial commitment and maintenance. As such, combining biochemical and biophysical cues will possibly lead to the next level of organoids-based disease modelling.