Basic Science Perspective on Engineering and Modeling the Large Airways

5.1. Introduction

The airways are critical physical and biochemical barriers protecting the lungs from a plethora of exogenous environmental insults during air inspiration, which can lead to inflammation and infection. Their structure is finely tuned to provide optimal tissue homeostasis and regulation of innate immunity. The effectiveness of the epithelium as a barrier is reliant upon its capacity for mucociliary clearance, immune surveillance, and regeneration subsequent to injury: a product of the functional cells that comprise the airway epithelium and the niche in which they reside. Cellular diversity during both homeostatic and diseased states in the adult lung is tightly regulated by interactions within their specific anatomical niche during the orchestration of epithelial regeneration. Niche components include the neighboring epithelial cells, infiltrating inflammatory cells, surrounding extracellular matrix, and supporting mesenchymal cells. Changes in the extracellular matrix (ECM) during disease can significantly change the functional dynamics of the airways including the airways’ ability to stretch. Functional dynamics including stretch and directional airflows should also be carefully considered when engineering models of human airway tissues. Effective recapitulation of physiological and pathological states of the proximal airways requires the generation of complex structures comprising the surface airway epithelium (SAE), submucosal gland epithelium (SMG), extracellular matrix, and niche cells, including airway smooth muscle cells (ASMCs), fibroblasts (FBs), and immune cells in addition to dynamic regulation of stretch and airflow. The field is moving toward more complex models as the tools and technology evolve to facilitate bioengineering and analytical approaches. This chapter will introduce the basic cellular biology that underlies a functional proximal airway epithelium, focusing on the relationships between structure and function in the airways and the challenges of developing complex models that accurately recapitulate the healthy and diseased human airway.

5.2. Proximal Airways: Composition and Function

The proximal airways of the respiratory system begin with the trachea, which bifurcates into the mainstem bronchi and then subsequently smaller branches of cartilaginous bronchi, which enter the pulmonary lobule becoming bronchioles. Bronchioles have a diameter less than 5 mm and branch into an additional 5–7 terminal bronchioles before transitioning to alveolar ducts. For this review, we refer to the proximal airways as branching generations with a diameter greater than 5 mm. In contrast to the distal airways, the proximal branches comprise a mucosal epithelium with cartilaginous rings, SMGs, and ciliated pseudostratified columnar epithelium. As the airways decrease in diameter, they simplify into a columnar epithelium, and cellular composition changes along the proximal-distal axis in accordance with the requirements for regional lung function [116, 177].

The principal role of the proximal airways is to facilitate airflow to and from the alveoli, the site of gas exchange and blood oxygenation [191]. As with other epithelia, the airway epithelium forms a protective barrier against environmental hazards. This barrier comprises two core components: mucociliary clearance and tight junctions. When either of these fail airway inflammation, mucus accumulation and infection all ensue. These are hallmarks of many obstructive lung diseases, including asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and primary ciliary dyskinesia (PCD), among others [242]. Mucociliary clearance relies on the synchronous beating of 100–300 cilia on specialized multiciliated cells, occupying most of the surface area, and creates a mucociliary escalator to carry debris to the pharynx. Here, fluid and mucus can be swallowed [261]. Successful mucociliary clearance relies upon maintenance of airway surface liquid (ASL) homeostasis [31]. ASL comprises a mucous layer to trap inhaled particles overlying a lower viscosity periciliary layer (PCL) that serves to lubricate the airway surface and facilitate ciliary beating [125]. The functional coordination of multiciliated cells can be restricted in situations of excess mucus accumulation in the airways [212, 242]. Mucus is a viscous mixture of large glycoproteins, known as mucins, combined with fluids, electrolytes, and other antimicrobial proteins, and is a critical component of airway homeostasis responsible for capture of particulates and pathogens entering the airways. Polymeric mucins, MUC5B and MUC5AC, are most common in the airway and are primarily secreted from specialized goblet cells [232]. Goblet cells comprise approximately 25% of the epithelial cells in the proximal airways [203]. In addition, the SMGs contribute to mucus secretion through the release of MUC5B strands coated with MUC5AC [142], slowing the transport of fluid up the airway [69, 242]. Increased occurrence of goblet cells, or goblet cell hyperplasia, results in the hypersecretion of mucus, associated with many lung diseases, including asthma, CF, COPD, and bronchitis [68, 86, 120, 122, 123, 133, 144, 146, 216].

As the epithelium transitions from the larger diameter bronchi to the smaller bronchioles, goblet cells become less frequent and club cells predominate [264]. This distribution can be significantly changed in inflammatory airway diseases [23, 173, 184, 206]. Club cells are another secretory cell type, largely responsible for secretion of surfactant proteins, including surfactant proteins A, B, and D (SPA/SPB/SPD) and club cell secretory protein (CCSP), into the airways [247]. The primary function of SPA and SPD are to regulate immune responses, while SPB primarily decreases surface tension at the air-liquid interface [16, 83, 135, 143, 209]. SPA is critical in the response to infection and allergens and for maintaining lung homeostasis, as it is responsible for pathogen opsonization [57]. Additionally, SPA appears to be linked to the modulation of eosinophils, and its expression protects against inflammation in asthmatics [40, 57, 73]. SPD also regulates inflammation and innate immunity in the airways and, thus, can be used as a marker for inflammatory lung damage [118, 262]. SPB has been shown to have anti-inflammatory and antioxidant effects, indicating that it may modulate oxidative stress response [57, 112, 151]. SPB, along with SPA and SPD, are important biomarkers for COPD [139, 250] and can be used to predict the early development of non-small cell lung cancer tumors and the development of lung cancer, in general [67, 219]. Altered ratios of goblet and club cells are characteristic of changes in the airway associated with chronic lung diseases, such as asthma and COPD. Goblet cell metaplasia, occurring at the expense of club cells, increases mucus production and is connected to increased airway inflammation [181, 214]. A reduction in club cells lowers the secretion of CCSP (aka CC10), which has important anti-inflammatory properties and protects against oxidative stress [107, 237, 247].

While secretory and ciliated cells are the main functional cells at the apical surface of the epithelium, basal cells (BCs) are stem cells that line the basement membrane [234]. In the larger bronchi, BCs form a continuous monolayer with decreasing numbers forming smaller clusters and eventually single cells in the terminal bronchioles. BCs are perhaps the best characterized stem cell in the airway epithelium capable of both self-renewal and differentiation into all the functional airway cells (Fig. 5.1). They are typically quiescent during homeostasis and primed to rapidly respond to injury restoring barrier function. Their stemness makes them ideal targets for gene therapy/gene editing and of particular interest in lung regeneration and cellular therapeutics. In addition, they play an important functional role in the anchorage of the epithelium to the basement membrane [164].

Fig. 5.1.

Stem cells of the proximal airway epithelium. The proximal airway has several cells capable of regeneration. Basal cells can self-renew and give rise to club, goblet, and ciliated cells. Club cells can also give rise to the same progeny and can dedifferentiate to basal cells. Pulmonary neuroendocrine cells have been shown to self-renew and differentiate under specific injury conditions. (This figure was created at Biorender.com)

There are also several, less common, cell types present in the airway epithelium: pulmonary neuroendocrine cells (PNECs), brush cells, and ionocytes [46, 177]. PNECs are rare, multifunctional epithelial cells, whose main function are as intrapulmonary chemosensors, capable of detecting changes in the composition of inhaled air and adapt to signal accordingly. Recently, three different types of PNECs were identified having differential expression of neuropeptide markers and changes in functional responses to injury and disease [161]. PNECs primarily perform chemosensory functions in the airway regulating responses to injury and disease [179, 224]. While their specific origins have been debated, a recent study showed that BCs are able to give rise to specific subpopulations of PNECs [161] (Fig. 5.1). Ionocytes became a specific cell of functional interest after the discovery that they highly express the cystic fibrosis transmembrane regulator (CFTR), the defective protein causative for CF disease [51, 156, 211]. The precise functional role of ionocytes in the airways is still an active area of research [95, 211].

5.3. Regeneration of the Airways

Engineering a functional airway, either as a model or for transplantation, will rely upon the presence of stem cells to both generate and regenerate the airway epithelium. While generally quiescent, the respiratory epithelium has an immense capacity for rapid regeneration to restore barrier function and physiological homeostasis in response to injury. As mentioned above, the predominant stem cells in the proximal airways are BCs [200, 201, 231]; alveolar type 2 (AT2) cells are stem cells of the alveoli and are reviewed in detail in the chapter focused on the developing lung [4, 5, 38, 43, 136]. These cells are precisely regulated by signals they receive from their immediate niche comprising the surrounding mesenchyme, neighboring epithelium, infiltrating immune cells, and interactions with the ECM. A valuable tissue-level model of the airways should consider all these factors. In this next section we will briefly describe airway BCs and the airway niche.

5.3.1. Endogenous Stem Cells

In the proximal airways, BCs are the main stem cell population that are capable of self-renewal and differentiation to mature airway progeny, including core functional secretory, ciliated, and goblet cells [13, 98, 200]. They are crucial for maintenance and regeneration of cells in the airway after injury and are known to be regulated by changes in Notch signaling [157, 200, 222]. In previous research BCs were considered a homogeneous population, but recent studies suggest there is more complexity [35, 252]. To study early origins in development process, lineage tracing of cytokeratin 5 (KRT5)-expressing BCs indicated that two BC populations existed in the upper airway, one acting as self-renewing stem cell and another population committed to differentiation [240]. More recently, our understanding of the identity of stem cells has been significantly facilitated through the application of single-cell RNA sequencing (scRNAseq) to evaluate cells of the human airways [37, 55, 81, 85, 101, 108, 114, 159, 194, 244, 246, 259, 263]. These studies have provided critical information on stem cell subtypes, transitional cellular states, and the complex pathways implicated in stem cell differentiation [156, 177, 182]. Despite being collectively well studied in their capacity to generate all the differentiated cells of the airway, we are only beginning to understand the functional relevance of the subtypes identified through transcriptomic analysis. Basal stem cells are primarily identified through their expression of core markers, KRT5 and tumor protein 63 (TP63). Small subsets (approx. 20%) also express KRT14, and under homeostatic environments KRT5/TP63/KRT14 populations are considered self-renewing and responsible for maintaining the basal cell stemness. Extensive lineage tracing experiments have been performed in mice, establishing the capacity of KRT14-expressing BCs to generate both secretory and ciliated cells of the airway epithelium [76].

Other putative progenitor cells in the proximal airways include the club cells and PNECs. Club cells, expressing secretoglobin family 1A, member A1 (SCGB1A1), act as facultative progenitor cells and have been shown to harbor the capacity to self-renew and dedifferentiate in cases of severe injury where all basal cells are ablated [12, 119, 128, 149, 195, 196, 231, 257]. While PNECs are less well characterized, they have been shown to act as a more classical unipotent stem cell, self-renewing in response to injury [176] (Fig. 5.1).

5.3.2. The Stem Cell Niche

Airway function and stem cell behavior are inherently influenced by changes in the cellular microenvironment. Despite this, models of the epithelium are often studied in the absence of niche factors. During lung development the temporal regulation of growth factors and cytokines secreted by the surrounding mesenchyme intricately directs lineage specification, generating contiguous branches of specialized epithelium. This has been extensively reviewed in Riccetti et al. [199], Yuan et al. [256], Volckaert and De Langhe [235], and Morrisey et al. [158]. In the adult lung, signals from the niche remain critical regulators of stem cell function and homeostasis [13]. During homeostasis the airway BCs are relatively quiescent but are rapidly mobilized in response to tissue injury [12, 13]. This epithelial injury is sensed by the adjoining epithelial cells, leading to the production of alarmins, and damage-induced phenotypic alterations of the BCs, together initiating the tissue repair. As a result of such events, immune cells are prompted to generate a suitable response mechanism for tissue homeostasis [204]. The ECM is also active in wound healing through the provision of signals to activate the mesenchymal cells to promote inflammation, cellular migration, and tissue remodeling [213]. The pulmonary mesenchyme consists of FBs, endothelial cells (ECs), ASMCs, and macrophages [39, 148].

Following injury, dynamic changes also occur in the cellular niche to coordinate epithelial recovery. ASMCs secrete fibroblast growth factor 10 (FGF10), stimulating airway regeneration through activation of BCs throughout the airways [256]. Further exploration of the ASMC niche in the airways recently identified a particular subset of ASMC, coined “repair-supportive mesenchymal cells” (RSMCs), which are distinct from conventional ASMCs and highly express platelet-derived growth factor alpha (PDGFRα). While RSMCs expressed typical ASMC markers at much lower levels, they were enriched FGF10 [155]. As a niche regulated signal FGF10 has been well studied in the context of regulating both airway development and repair. After a major lung injury, the surviving epithelial cells rapidly spread to sustain barrier function. Through recruitment of integrin-linked kinase signaling, the Hippo pathway is downregulated, increasing the nuclear translocation of YAP and ultimately the secretion of WNT7b from the epithelium. This Wnt signal stimulates cellular cross talk to the niche and induces Fgf10 expression in ASMC [236]. The functional importance of Wnt signaling in the lung was initially demonstrated during lung development, as early as at the stage of lung endoderm specification [109, 145]. In the adult lung, Wnt signaling influences the differentiation of regionally distinct populations of epithelial cells during homeostasis, regeneration, and disease [25, 78, 193, 201]. Wnt is crucial for lineage commitment of glandular stem cells within tracheal niches [140] and can inhibit differentiation of club cells into goblet cells through the RYK receptor [87, 130, 195, 231].

Similarly, interleukin-6 (IL-6) signals from the mesenchymal stromal cells in the niche, alongside neighboring epithelial cells, can regulate cell fate decisions, influencing ciliogenesis through STAT3-dependent pathways [41, 229]. Severe tracheal damage leading to complete loss of luminal epithelial cells differentially regulates p-STAT3, suggestive of influence over two different differentiation states. Firstly, in BCs and early undifferentiated progenitors, STAT3 inhibits differentiation into secretory cells and promotes ciliated cell differentiation through inhibition of Notch signaling. Secondly, in ciliated progenitors, STAT3 promotes differentiation by upregulation of FOXJ1, MCIDAS, and CDC20b/miR-449 [41, 229].

An important cellular niche unique to the proximal airways, and of specific relevance in humans, are the SMGs. The microenvironmental niches provided by the SMG contain progenitor cells capable of restoration of the surface airway epithelium [138, 140, 141, 230]. It still is unclear exactly how the surface airway signals to the niche to recruit the progenitors. Anomalous proliferation and differentiation SMGs have been associated with pathophysiology observed in hypersecretory lung diseases, including asthma, chronic bronchitis, and CF, highlighting their importance in formulating disease models [101, 248].

While resident lung cells provide a significant contribution to the epithelial niche in the proximal airways, inflammation is also a major player in disease pathogenesis and a critical component of lung health in chronic airway disease [63]. Inflammation involves the recruitment and activation of immune cells, including macrophages and neutrophils, to respond to infection and tissue injury. Recently, Puttur and colleagues reviewed how pulmonary macrophages (PMs) act as guardians of tissue repair in the lung environments, discussing how PMs respond to tissue inflammation and contribute to lung remodeling in response to infection and how this inflammatory response is sustained in chronic lung diseases, such as asthma, COPD, and CF [190]. In the mouse trachea three populations of macrophages have been identified: submucosal macrophages, interstitial macrophages, and intraepithelial macrophages. Injury stimulates the recruitment of neutrophils and submucosal macrophages, and successful airway regeneration is facilitated by c-c chemokine receptor type 2 (CCR2)-dependent recruitment of monocytes, which can interact with BCs and impact their regenerative behavior [63]. Macrophages have been more extensively studied for their roles in the alveolar niche, and further understanding of their impact on the proximal airways may shed new light on critical pathways to restore homeostasis.

The influence of the niche on epithelial cell behavior cannot be underestimated and is a critical component to consider in the engineering of new cellular models and the construction of lung tissues. Understanding the intimate regulation of airway homeostasis, disease, and regeneration through the coordination of ECM, mesenchymal cells and BCs will be critical as we strive to model and restore functional airways in disease and aging.

5.3.3. Stem Cell Attrition with Disease and Aging

Aging and disease both take their toll on the lungs. Stem cell phenotype and function has been shown to decline with age; however, we do not yet have a comprehensive understanding of how BCs are dynamically regulated by their niches, especially in the context of injury and aging. Some recent studies have highlighted the importance of spatiotemporal regulation of Wnt-secreting niches in both airway regeneration and aging [6]. PDGFRα-expressing cells, in the intercartilaginous zone (ICZ) of the tracheal and bronchial niche, transiently secrete Wnt ligand in response to injury to stimulate BC proliferation during regeneration [6]. Aging can also stimulate Wnt-ligand-dependent formation of glandular-like epithelial invaginations (GLEIs) generated from BCs in this ICZ. With aging comes accumulation of airway injury exposures, which can accelerate the “biological aging” of airway BCs, augmenting abnormal repair and disease initiation. Such stem cell aging and/or exhaustion is a pathological feature of many chronic lung diseases, which are typically associated with repeated airway injuries [6, 137, 172, 228]. Stem cell attrition is associated with repeated proliferation, leading to terminal differentiation of BCs [77]. Concomitant with stem cell attrition are changes in the ECM, which has perhaps been most widely studied in the context of aging, fibrosis, and COPD [1, 33, 66, 163, 165, 187, 218, 243]. Since many lung diseases develop over time and have a predominance in the aged population, it is important to consider such changes when constructing phenotypic models of disease. This may require models where matrix stiffness can be tuned over time or where airway models can be subject to long-term repeated insult/injury to understand stem cell attrition in the context of their cellular microenvironment.

5.4. Developing Cellular Therapies for Regeneration of Airway Tissues

While cellular therapeutics for airway disease are being actively pursued, how close these treatments are to availability in the clinic remains indeterminate for now. To develop cellular therapies or to engineer for engraftment or for disease modeling, an abundance of autologous cells is likely necessary. Current research efforts are focusing on the identity of the most suitable cells to be delivered to replace the damaged stem cells and the methods to generate sufficient cells, ex vivo, to be able to successfully regenerate the desired lung tissue. Unfortunately, ex vivo expansion of these cells is still plagued by rapid phenotypic changes induced by culture [132, 198]. The advances in induced pluripotent stem cell (iPSC) research have steered an exciting new age of regenerative medicine. Pluripotent stem cells are widely used in vitro by applying combinations of growth factors and cytokines, mimicking the mechanisms of respiratory development. Over the past decade, there had been significant progress in the generation of airway epithelium from iPSC [59, 72, 96, 97, 127, 150, 162]. Figure 5.2 summarizes the timeline for early specification of iPSC-derived lung progenitors, mimicking stages of lung development. While challenges in efficiency and purity of the differentiated progeny persist, RNAseq indicates that there is significant similarity between the derived cells and their endogenous counterparts [96]. The purification of iPSC-derived basal cells (iBCs) that recapitulate the biological and functional properties of basal cells, including mucociliary differentiation in tracheal xenografts, has facilitated a new wave of studies focused on gene editing and functional restoration of the airways [96]. These iBCs can be maintained in 3D spheroid cultures over several passages. Although challenges remain in the functional characterization of iBCs, these recent advances should facilitate the study of inherited or acquired diseases of the human airway and open the door for potential use of iBCs in regenerative medicine [96, 227]. Challenges in regenerating an iBC truly akin to its endogenous counterpart likely lie with the impact of the environment of the epigenetic manipulation of cellular state [249]. Epigenetic comparisons between differentiated iBCs and endogenous BCs may be important in generating an iBC that truly recapitulates the functional phenotype of primary cells. Patient- and/or mutation-specific models can be readily developed to model genetic airway diseases, including chloride channel dysfunction in CF and ciliary defects in PCD [34, 96, 177].

Fig. 5.2.

Early lung development from iPSC. Pluripotent stem cells are isolated and expanded in vitro from the inner cell mass of the blastocyst (embryonic stem cells or ESC) or reprogrammed from somatic cells, such as fibroblasts, from individuals (induced pluripotent stem cells or iPSC). To generate lung endoderm cells are differentiated using a combination of growth factors and small molecules to mimic lung development through the anterior primitive streak and definitive endoderm, where anteriorizing of the definitive endoderm will give rise to the anterior foregut endoderm. This will subsequently have the capacity to generate NKx2–1-expressing primordial lung progenitor cells. (This figure was created at Biorender.com)

The identity and selection of long-term repopulating BCs and the differentiation of iPSCs to bona fide basal cells are being actively pursued [10, 49, 71, 75, 80, 96, 117, 147, 160, 223, 227]. Once this is achieved, as technologies evolve and progress toward tissue regeneration and cellular therapeutics, it will be critical to first determine the prospect of primary or induced BCs engrafting and integrating in the airway epithelium. While delivery itself perhaps poses the biggest challenge in the success of cellular therapeutics, once this is achieved, consideration of the diseased niche is likely to be equally important in regulating the fate of those delivered cells or therapeutics. Unfortunately, there is a paucity of studies considering the niche in terms of successful stem cell regulation, cellular engraftment, and tissue regeneration.

5.5. In Vitro Models of the Human Airways

Over the past decade, there have been several advances in the development of more complex and physiologically/pathophysiologically relevant ex vivo lung models, enhancing our ability to model lung disease. Air-liquid interface culture of primary airway cells, established in Transwell, has long provided a gold standard for evaluation of airway injury and repair. More recently, three-dimensional (3D) lung spheroid and organoid structures have been used to create more spatially relevant structures, and lung-on-a-chip technology adds the complexities of airflow and stretch. Each of these models will be discussed below. Table 5.1 summarizes the core pros and cons of using each of these lung models in scientific research, and Fig. 5.3 summarizes each model system.

Table 5.1.

In vitro models of the airway system

Model	Application	Advantages	Drawbacks	References
Air-liquid interface	Disease models, drug delivery, toxicity, mucociliary clearance, cilia function, barrier function, CFTR activity	Differentiation of a pseudostratified airway epithelium Mucociliary flow measurements Cilia beat frequency measurements Mucus production Dose controlling for inhaled agents Useful for drug transport/delivery Wound-repair investigation TEER measurements and CFTR activity Inflammatory cell interactions Can be long-lived (>3 months)	Not all primary donor cells differentiate well at the ALI No directional flow (confined by Transwell size and shape) No mechanical stretch Lot-to-lot variability in Transwell 28-day differentiation	[25, 53, 77, 129, 171, 184, 185, 197, 200, 202, 210, 225, 229]
Organoid	Disease models, drug delivery, toxicity, lung development	Mimics lung development and branching morphogenesis Provides the structural architecture and maintains cellular interactions of the lung microenvironment It can be used for personalized medicine studies	Lack of breathing mechanics Lacks structures that can be useful for studying the effect of inhaled therapy on involving mouth-to-airway transit Long culture period and difficulty to reproduce at industrial level Lacks an air-liquid interface (more applicable to development than adult lung) Complexity limits analysis	[39, 54, 58, 93, 99, 115, 130, 152, 208]
Spheroids	Disease models, drug delivery, toxicity, CFTR activity	3D structure allows for more physiological ECM interactions and structure Swelling model for CFTR activity	Apical surface is limited (typically inside the spheroid) with no airflow Variation in spheroid size No niche cells present	[21, 32, 45, 52, 62, 91, 103, 110, 208, 221]
Lung-on-a-chip	Disease models, drug delivery, toxicity, CFTR activity, inflammation, mechanical breathing, airflow	Regulated mechanical stretch Regulated airflow Vascular channel for endothelial/inflammatory cell interactions Time-dependent drug treatment and monitoring	Challenging to assemble Donor-specific success in differentiation without migration Expensive chips and equipment	[106, 126, 258] [8, 15, 100, 183, 217, 166, 167]
Xenografts	Regeneration, gene editing/therapy, drug delivery, toxicity	Regeneration can be evaluated in context of the native niche	Implantation can be technically complicated Damage response generated during tracheal denudation	[9, 44, 48, 56, 82, 88, 89, 94, 121, 124, 239]

Fig. 5.3.

Cellular models of the proximal lung. This schematic summarizes the currently available in vitro model systems to study the proximal lung epithelium, including air-liquid interface in Transwell, microfluidic lung-on-a-chip, multicellular organoids, spheroids (tracheo/bronchospheres), and xenografts. (This figure was created at Biorender.com)

5.5.1. Transwell Air-Liquid Interface (ALI) Cultures

For decades, most pulmonary in vitro studies were performed using cell lines in submerged cell culture conditions, resulting in compromised physiological conditions [74, 192]. The transition to cell growth on Transwell, where epithelial BCs are grown to confluence on ECM-coated synthetic membranes and then polarized by exposing the apical surface to air, created a more physiologically applicable air-liquid interface model (ALI) [74, 102]. Over approximately 28 days, cells polarize and mature into a differentiated mucociliary airway epithelium, comprising BCs, club and goblet secretory cells, and multiciliated cells with scattered ionocytes present [207]. This model rapidly became a gold standard for recapitulating functional properties of the epithelium, including ciliary flow and mucus production. Such ALI cultures are widely used as an effective tool for cell-cell interaction studies, disease models, and drug efficacy/toxicity testing in the lung [3, 14, 19, 22, 26, 42, 192, 233]. They have been instrumental in the validation of small molecules that are now widely used for the treatment of CF [11, 92, 226, 251]. While extremely effective, they do have their limitations. Despite featuring all the major cell types of the proximal airways, these ALI cultures are static; they have no epithelial supporting niche; they are not designed to drive epithelial differentiation toward different branching generations; and choice of media, donor cells, and protocol can significantly impact phenotypic, transcriptomic, and physiological features of the differentiated epithelia [207, 245]. These limitations constrain the capacity of the models to completely recapitulate disease pathophysiology.

5.5.2. Airway Spheroids: Tracheo/Bronchospheres

Moving from a 2D to a 3D culture system allows for a closer replication of cell behaviors during tissue assembly. In vivo cells are exposed to circulating molecules, neighboring cells, and ECM, which are factors more effectively recapitulated in 3D models. The first airway spheroids, known as tracheo- or bronchospheres, were generated from murine basal cells [201], and, akin to ALI, matured bronchospheres comprise the major functional cells in the proximal airways. Spheroid models have been applied to model disease phenotypes, such as IL-13-induced goblet cell metaplasia in asthma [178], and to investigate pathogen-associated molecular patterns (PAMPs) in airway differentiation, replicating mucus hypersecretion observed in COPD [221]. Spheroids have been particularly useful in the higher-throughput evaluation of effective CFTR modulators through investigation of forskolin-induced spheroid swelling [36, 50, 84, 205]. Bronchospheres can also be grown in suspension in the absence of ECM, a model which can be expanded to 384-well format for studying the function of the human airway epithelium in higher-throughput assays [103]. While having more high-throughput application and a greater capacity for tissue organization and swelling as an experimental readout, these epithelial-only spheroids are limited by the accessibility of the apical surface, commonly inside the spheroids and niche influence other than ECM.

5.5.3. Organoids

Organoids are more complex 3D tissues that mimic the structural organization of an organ, capable of simultaneously representing multiple different cell lines and germ layers. Lung organoids can be applied to study spatial cellular organization, cellular interactions, and, using pluripotent cells, pathways regulating human lung development [168]. Organoids, generated from lung bud progenitor cells of human fetal lungs, have been used to study early airway and alveolar specification [169]. However, there are limitations to tissue availability, in addition to ethical and regulatory hurdles associated with research on human fetal tissue. iPSC-derived lung bud tip progenitors have emerged as a promising alternative developmental model [99, 152–154]. While transcriptomic differences exist between the primary bud tip and iPSC-derived cells, even if the cells are taken from the same source, the longer the iPSC-derived organoids were cultured, the closer the cells became transcriptomically similar, indicating that a spatial and temporal maturation of the iPSC-derived cells occurs [99, 152–154]. Developmental models have unique application to the evaluation of respiratory virus infections in the developing lung and allow for study of infection in both proximal and distal lung tissues [186]. There is an enormous success in culturing relevant organoids under desired physiological conditions; however, there are several challenges associated with its real-life application. With the organoid system, variability of self-organizing growth has been observed, and there is limited experimental and analytical feasibility [105]. iPSC-derived lung bud tip organoids have not been documented to mature past an equivalent of the second trimester and thus not reach terminal maturation [186]. Additionally, branching morphogenesis is seemingly random, and how closely the branching morphogenesis and transitional zones mimic those of adult human lungs remains uncertain; similarly, the nature and pattern of the mesenchyme is unclear [39, 59]. Beyond genetic diseases, iPSC-derived airway organoids have been applied to predict drug responses, to study patient-specific host response to respiratory pathogens [238] and viral infection, including SARS-CoV-2 [134, 180, 220].

5.5.4. Lung-on-a-Chip

While ALI, spheroid and organoid models all have advantages for studying functional human lung epithelium, they also have their limitations. Lung-on-a-chip systems have evolved over the past decade that overcome some of the shortcomings of other model systems [8, 15, 17, 166]. These engineered cell-based systems facilitate the evaluation of cells in an environment that mimics an in vivo tissue environment [2, 20, 106]. Most models of the human lung do not account for dynamic cues, such as blood flow and breathing cycles, that can impact airway biology. Recently, second-generation chips have been optimized for studying the effects of physiological airflow and stretch on differentiation, cellular composition, and mucociliary clearance in the proximal and distal airways [167]. Chip perfusion with air in the apical compartment accelerates maturation and polarization of mucociliary clearance, when compared to traditional ALI Transwell cultures, and the additional application of airflow and stretch influences cellular composition and the inflammatory status of the epithelium [167]. Commercially available microfluidic chips (Chip-S1^®, Emulate Inc.) feature adjacent perfusable microchannels, where BCs can be cultured in the apical channel, which is separated from a vascular channel by a flexible and highly porous poly(dimethylsiloxane) (PDMS) membrane [167]. This specific membrane can be dynamically stretched in the presence of airflow, compared to previously developed protocols for airway chips using cell-culture optimized, but rigid, membranes [15, 113, 166].

5.5.5. Xenografts

Tracheal xenograft models were first developed over two decades ago for the analysis of airway regeneration and the identification of progenitor cell subpopulations mediating the process [61, 215, 260]. In these models, human epithelial cells were seeded into an epithelium-denuded rat trachea, which was subsequently grafted into a xenon-tolerant/athymic mouse. These models have the benefit of having the entire cellular niche present, including tracheal cartilaginous rings [32, 64, 70, 131, 189]. Some of the earliest studies in xenografts enabled the dynamics of cell turnover, lineage, and differentiation to be evaluated. Patterns of clonal expansion of transgene-expressing cells allowed for the identification of airway progenitor populations, giving rise to clones of different sizes, and were instrumental in the initial identification of stem cells in the human airway epithelium capable of self-renewal. Such studies led to a conclusion that several stem cell subpopulations of cells are involved in the regeneration process of airway epithelium [60, 189, 260]. In an approach to establish intrapulmonary genetic reconstruction, CF xenografts were infected with CFTR recombinant adenovirus, reconstituting cAMP-driven Cl transport [82]. Though this correction was partial and variable, it established the applicability of this approach for disease correction. The presence of an intact epithelial niche in xenografts has been capitalized upon to evaluate epithelial-mesenchymal trophic units generated through the seeding of control and asthmatic epithelial cells [89]. Interestingly, this system demonstrated the importance of cellular cross talk between the niche and epithelium; in these studies the presence of an asthmatic epithelium alone was sufficient to drive mesenchymal remodeling [89]. Advances in bioengineering have enabled the evolution of airway models beyond the static, 2D ALI airway models, adding physiological and mechanical cues to mimic tissue dynamics. Each model system has its own qualities and limitations and should be carefully considered for their applicability to specific research questions. For the successful preclinical evaluation of a novel inhaled therapy, for example, a human model capable of mimicking the physiological parameters that facilitate the in vivo transport of drug molecules is critical. Engineered complete lung tissue models with 3D lung architecture, cellular composition, and mechanical and physiological cues would be ideal, enabling evaluation of drug efficacy, transport kinetics, toxicity, and therapeutic potential [42]. Continued collaboration between bioengineers and basic cell biologists will be instrumental to bring new innovations to this space.

5.6. Cell-Matrix Interactions

An important component of developing more advanced models relies on the precise understanding of the role of the ECM in regulating airway stem cell function during both homeostasis and disease. The ECM forms a major component of the multicellular 3D architecture of the human lungs, providing structural, mechanical, and biochemical support. The importance of the lung ECM in regulating BC stemness and function was recently reviewed in Busch, Lorenzana, and Ryan [30]. Lung ECM is composed of fibrous proteins (elastin and collagen), glycoproteins (fibronectin, laminin), glycosaminoglycans, and proteoglycans, which are all important for normal lung development. All the major components of the lung (conducting airways, respiratory airways, lymphatics, and the pulmonary vasculature) require their unique ECM environments for specialized cell survival, proliferation, and differentiation [27]. Apart from providing structural integrity, ECM regulates cell behavior due to changes in its molecular composition and stiffness [79, 253] and acts as a reservoir for growth factors and cytokines. Several growth factors (FGF-10, TGF-β1, etc.) and cytokines are reported to regulate the breakdown of the ECM, in addition to regulating lung development and basal cell phenotype [188, 204]. However, precise implications of compartment-specific ECM composition, substrate stiffness, and other mechanical properties, as well as biochemical signaling, remain an active area of research [7, 29, 79, 254].

ECM remodeling is a core feature of many chronic lung diseases. In idiopathic pulmonary fibrosis (IPF), changes in the interstitial matrix within airways (conducting and respiratory) are indicative of disease progression [90]. Activated fibroblasts and myofibroblasts within fibroblast foci are considered as the key producers of ECM. In IPF tissues, type I procollagen, versican, hyaluronan, and tenascin C are highly expressed within the fibroblastic foci, while decorin and biglycan are present at relatively low level of expression, indicative of enhanced synthesis of ECM and remodeling [18, 65]. In COPD, ECM remodeling changes the composition of the basement membranes and the interstitial matrix at different anatomical niches of the lung and is also considered to be associated with progression of disease [90].

Modeling the lung ECM can provide insight into the interactions between the ECM and functional elements of lung airway. While 2D models of cells grown on tissue culture plastic are relatively simple by design, they are typically significantly stiffer than normal lung tissue [24, 104, 241]. In vitro 3D tissue-engineered model systems (lung-on-a-chip models, organoids, lung slices, ECM gels, and coculture systems) have been used in recent years for modeling the unique spatial geometry of the lung and understanding the complexity of cell-cell-ECM interactions [174, 175]. Generation of complex ECM scaffolds, which can facilitate cell growth and differentiation, is required to continue development of these complex models. Recently, a protocol was devised for generation of decellularized human lung bioink for downstream process of 2D and 3D lung cell culture [47]. While the importance of the ECM in organizing niche architecture and regulating cellular function has been extensively studied, there still is a significant knowledge gap in terms of the ECM-mediated regulation of cell behavior and phenotype. The complexity, including cross-linking, and insoluble nature of the ECM has posed a persistent obstacle to determining the generation and function of the ECM [28, 111]. Now, engineered biomaterials are being applied to mimic the in vivo characteristics of stem cell niches, which facilitate a desired in vitro tool for investigating the different roles exercised by the ECM on the stem cell phenotype. Recently, Yu et al. developed a natural ECM biomaterial enriched with miR-29-loaded exosomes for the treatment of pulmonary fibrosis, which promises gene therapy options for treatment of different diseases using ECM [255]. In the lung physiology, there is a strong relationship between ECM proteins and leukocyte migration to the locations of injury [170]. A detailed understanding on the complex ECM-cell interactions will advance the design and development of functional models and engineering tissues for lung regeneration.

5.7. Conclusion

The ECM is not simply a scaffold. It is an integral component of lung physiology and pathophysiology that changes with disease progression. Abnormal ECM, niche cells, and BC phenotype are closely related in the context of chronic lung disease. Increasing our understanding of the functional interactions between the ECM, niche cells, and stem cells in the epithelium will have a substantial impact of the recapitulation of lung homeostasis and disease in model systems. Perhaps the most challenging application of ECM-BC research is the potential to repopulate a decellularized ECM scaffold to create functional lung tissues. Techniques for tissue regeneration are reliant upon the creation of a receptive cellular microenvironment that can be applied to both ex vivo expansion and in vivo engraftment. Tunable models that can reflect disease progression and be integrated with niche cells would allow for evaluation of mechanical and biochemical factors in disease pathogenesis. The subsequent chapters will focus on bioengineering approaches for the generation of models for lung homeostasis and disease.