Air-liquid interface culture (ALI) allow scientists to generate stable and functional in vitro 3D human airway cell models that closely mimic respiratory tract epithelia. With such models, researchers can investigate physiological and pathological processes of the respiratory tract. They can study interactions between epithelial cells and respiratory pathogens or air pollutants, and observe how cells respond to aerosolized drugs.
We do not think about it. We almost do not notice it, but every minute we inhale and exhale seven to eight liters of air. This air contains the oxygen we need to live. But it also can introduce dust, pollutants and potentially harmful pathogens into our bodies. It is the job of the epithelial cells in our airways to build a barrier and make sure that those agents are quickly removed so as to avoid damage to our lungs. However, pulmonary diseases such as asthma or chronic obstructive pulmonary diseases (COPD) impair this “cleaning” ability of the airway epithelium, and the consequences can be very severe. In order to get a closer look into what is happening inside our airways, researchers need to replicate the native environment in vitro as closely as possible. With the current strict regulations and the emphasis on the so-called 3R principle aimed at replace, reduce and refine the use of animal testing, there is a growing need for physiologically relevant models of the respiratory epithelium. Air-liquid interface (ALI) systems closely mimic the in vivo characteristics of the pulmonary epithelial cells and therefore represent an optimal alternative to animal studies. Overall, ALI systems can provide valuable information on physiological and pathological processes in the respiratory tract, with their potential applications ranging from the study of pulmonary diseases to the fight against infectious agents to the investigations of the effects of air pollution on the respiratory system. Yet, as with every in vitro system, there are several challenges scientists need to overcome to ensure that the ALI system used is validated, standardized and offers relevant and reproducible results.A major challenge: replicating the complex anatomy of human lungs in vitro
With approximately 80 m², the surface of our lungs is the size of a tennis court and is paved with not just one but with multiple cell types. There are goblet cells, basal cells and ciliated epithelial cells, among others (Miller and Spence, 2017). The reason for this variety of cell types is the multiple tasks the airway epithelium has to accomplish. First, every hour it has to deal with up to 25 million inhaled particles and prevent them from entering and damaging the body. Second, goblet cells and sero-mucous glands produce mucus, a very effective defensive tool. This viscoelastic gel forms a protective coating which captures inhaled microorganisms. Then a mechanism called mucociliary clearance transports any foreign particles to the mouth to clear them away (Rogers, 2007). Last, epithelial cells produce several cytokines that play a role in innate and adaptive immune response mechanisms (Mertens et al., 2017). Because epithelial cells perform so many tasks, reestablishing a well-functioning respiratory epithelium in vitro is challenging. Two-dimensional cell cultures present several disadvantages, such as the loss of tissue-specific architecture, the absence of cellular differentiation and damage of barrier integrity. All of these features are very important when investigating respiratory diseases. In fact, the ability to undergo differentiation, develop cilia and produce mucus are key features of human bronchial epithelial cells. Often these features are lost in 2D in vitro cultures, where cells show fast dedifferentiation, high senescence and low proliferation (Ramirez et al., 2004). Moreover, monolayers do not exhibit the typical pseudostratified morphology and do not form tight junctions, so they are more sensitive to disruptions. The use of airway derived cell-lines also presents some limitations as those are often cancer-derived, and their physiological processes do not always resemble the in vivo characteristics of normal epithelial cells (Ryner et al, 2019). Due to these reasons, 3D primary human airway epithelial cultures have gained more and more interest recently and are increasingly used for in vitro scientific studies. ALI culture systems manage to overcome many of the challenges of 2D cultures and have many advantages when compared to submerged culture systems. ALI cultures exhibit a pseudostratified morphology, undergo mucociliary differentiation, secrete mucin and form tight junctions. Also, the extracellular environment is similar to the in vivo conditions (Lacroix et al., 2018).
ALI cultures: powerful models of the human airways
Cell cultures performed at the air-liquid interface facilitate the establishment of stable and functional 3D models of the respiratory tract. In these models, the basal side of the cells is in contact with the culture medium, and the apical side is in contact with the air. Therefore, the created in vitro scenario is close to what occurs in vivo (Lacroix et al., 2018). To establish ALI cultures, primary epithelial cells are first seeded on plastic culture vessels, where they can expand until they reach 70-80% confluence. After this first 2D expansion phase, cells are transferred to compartmentalized culture systems on porous membranes. Here, cells are submerged in culture medium and start proliferating in 3D until reaching confluence. Finally, they are ‘air-lifted,’ i.e. exposed to the surrounding air, and nutritive supply is provided only at the basolateral cell pole. This system allows morphological and functional cell differentiation and the formation of a pseudostratified epithelium with full basoapical polarity. The reconstituted epithelium shows many features observed in vivo – such as the production of cilia and mucus (Chen and Schoen, 2019). ALI systems also support the development of tight junctions which are critical for the epithelial barrier function. The measurement of the transepithelial electrical resistance (TEER) allows the assessment of the barrier integrity. The measurement of TEER is an important strategy to confirm epithelial polarization and therefore the maintenance of normal function of airway epithelium. It can be used routinely without damaging the cells and allows the study of the same cells in different setups (Papazian et al., 2016). For this purpose, an alternating current is applied to the cell layer, and the electrical resistance is then calculated (Knowles et al., 1982). Due to their physiological relevance, ALI systems are widely used and are now an essential tool in respiratory research. However, the lack of validation and standardization in the field hinders the comparison of different studies. In fact, the use of heterogeneous cultivation protocols, cells, scaffolds and media by different research groups complicates the inter-laboratory reproducibility of the results (Zscheppang et al., 2017).
Standardization: a key requirement for creating relevant culture systems
Standardization and validation of methods based on original human material become even more important when investigation results have clinical relevance. As animal models do not exactly mirror human physiology, standardized experimental approaches in human models significantly contribute to expediting translational processes while meeting, at the same time, ethical concerns regarding the usage of animal testing. Robust procedures are also a prerequisite for acceptance by regulatory authorities and pharmaceutical industries (Zscheppang et al., 2017). Using defined culture media and pre-screened cells is a concrete step forward towards the establishment of such standardized cultures. The use of serum and bovine pituitary extract (BPE)-free culture media allows the generation of predictive and reproducible 3D airway model systems and offers several advantages, such as:- Defined and controlled culture conditions
- Reduced variability in qualitative and quantitative culture medium composition
- Reduced risks of microbial contamination
- Higher resemblance of in vivo physiological conditions
- Reduction or even avoidance of the suffering of fetuses and animals
Using airway epithelial cell systems to better understand pulmonary diseases
Pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma, pneumonia and lung cancer have a huge global impact, affecting millions of people worldwide. As well, billions of people are exposed to indoor toxic smoke, outdoor pollutant air and tobacco smoke. These data make it clear why there is a high need for establishing reliable in vitro research models to study both pathological processes in the airway epithelium and the influence of infectious agents and toxic substances on epithelial cells. Further, dysfunctions of epithelial cells are often the cause of chronic lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). ALI systems use primary human cells to model such diseases and allow a better understanding of the underlying pathological changes. Moreover, cultured cells can be exposed to pathogens, aerosolized medications and toxic substances, and air pollution can be used to model phases of acute exacerbation or observe the effects of therapeutic drugs (Mertens et al., 2017). In fact, when studying the effects of drugs used in inhalation therapy, ALI systems offer a more physiological model than submerged cultures. When drugs are dissolved in the medium, they cover the whole cell and do not mimic the in vivo scenario where drugs, e.g. applied as an aerosol, only come in contact with the air-facing side of the epithelial cells (Lenz et al., 2014). ALI cell culture models can also be used to assess the effect of infectious pathogens such as viruses and bacteria on airway epithelial cells. As animal models are not always natural hosts of human pathogens, it is crucial to develop standardized systems which closely resemble human airways. During the current SARS-CoV-2 pandemic, for example, researchers have used ALI culture systems to study host responses to the infections and to screen therapeutic drugs. In a recent study, Mulay et al. isolated human epithelial cells from trachea and upper bronchi and differentiated them at ALI for 16–20 days until a pseudostratified mucociliary epithelium was formed. The team then infected these ALI cultures with SARS-CoV-2 and observed virus replication and gene expression in infected cells. The proximal airway ALI cultures, together with 3D alveolar organoids, were also used to study the effect of a selected panel of drugs such as IFNB1, Remdesivir and Hydroxychloroquine on viral infection and replication. Toxicology research also greatly benefits from the use of ALI exposure systems. Scientists can investigate cellular reactions and cell-cell interactions following exposure to toxic substances, air pollutants or drugs. Moreover, they can use these systems to better understand how aerosolized or gaseous substances contribute to the development of lung diseases (Upadhyay et al., 2018). The advantage of ALI systems is that they provide a method to challenge your cultured epithelium with any agent of interest. Either air pollutants, toxic substances, viruses, bacteria or drugs can be used to observe the reactions of the cells to these agents. The system is a valid alternative to in vivo experiments or classic in vitro methods. Further development of ALI-models is aimed at bringing the systems even closer to human physiology, increasing standardization and establishing validated methods. The use of ALI systems for 3D organoid culture could also significantly improve human relevance of the results obtained in in vitro studies and provide important insights into the dynamic processes in our airways. Such powerful tools will allow researchers to obtain more detailed insights in the highly specialized barrier mechanisms of the lung epithelia.
Frequently asked questions about ALI
What are ALI pre-screened cells?
The pre-screening of primary human bronchial epithelial cells for ALI cultures guarantees an optimal barrier function and minimizes lot-to-lot variations. Cells are collected from surgical samples after obtaining the consent of the patients. Patients are aware that their consent to donate cells contributes to the reduction of animal experiments. Screening is then performed by analyzing the transepithelial electrical resistance (TEER) of the cells over a time period of at least 14 days. TEER values of over 500 Ω*cm² are accepted. Moreover, a strict quality control process ensures that the cells meet high quality standards. PromoCell human bronchial epithelial cells are low in passages and have very good proliferation and barrier forming function capacities, with a minimum of 15 population doubling.What are the advantages of using a standardized ALI culture medium?
Standardized media display less lot-to-lot variations and help to maximize your specific test reproducibility. Moreover, BPE- and serum-free media provide more test-to-test consistency and eliminate possible contaminations. The PromoCell ALI-airway medium is a valuable tool for basic respiratory research, toxicity studies, infectious disease research and drug development. It is easy to use, optimized for 3D cell cultures and allows the formation of tight junctions and, therefore, a long-lasting barrier function.When do you recommend starting ‘air-lifting’ the cells?
We recommend a seeding density of 150.000 living cells/cm2 on the Transwell inserts in Airway Epithelial Cell Growth Medium. Twenty-four hours after seeding, a medium change has to be performed. At this point you should check the cell layer under a microscope, which should be nearly confluent. After three more days of culture, the cell layer should form a monolayer with nearly 100% confluence. When you are sure that the cell layer is confluent, you can change the medium in the basal chamber and keep the apical chamber empty to airlift the cells and expose them to the air.Air-liquid interface (ALI) culture is a cell culture method in which the apical surface of airway epithelial cells is exposed to air while the basolateral surface remains in contact with culture medium. This setup supports epithelial polarization and differentiation, allowing researchers to establish human airway models with features such as cilia, mucus production, tight junctions, and epithelial barrier function.1,2
We rarely think about breathing. At rest, however, we inhale and exhale several liters of air every minute. This air contains the oxygen we need, but it can also introduce dust, pollutants, and potentially harmful pathogens into the respiratory tract.
The epithelial cells lining our airways form an important protective barrier. They help capture and remove inhaled agents before they can damage the underlying tissue. In respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD), these defense mechanisms can become impaired.3
To investigate what happens inside the airways, researchers need in vitro models that reproduce relevant features of the respiratory epithelium. Air-liquid interface (ALI) cultures provide a human-relevant system for studying airway physiology and disease, epithelial interactions with respiratory pathogens or pollutants, and responses to aerosolized drugs.
ALI systems can also contribute to the Replace, Reduce, and Refine principles, commonly known as the 3Rs, by supporting research questions that can be investigated using human airway epithelial models. However, like every in vitro system, ALI cultures require appropriate validation and controlled culture conditions to generate interpretable and reproducible results.2,4
Why is the human airway difficult to model in vitro?
The respiratory epithelium contains several specialized cell types, including basal cells, goblet cells, and ciliated epithelial cells. This cellular diversity reflects the different functions that the airway lining must perform.5
Goblet cells and seromucous glands produce mucus, which forms a protective layer and captures inhaled microorganisms and particles. Ciliated cells then move this material toward the throat through mucociliary clearance. Airway epithelial cells also produce cytokines and participate in innate and adaptive immune responses.3,6
Reproducing these functions in vitro is challenging. Conventional two-dimensional cultures often lose tissue-specific architecture and cellular differentiation. Primary bronchial epithelial cells may lose their ability to form cilia or produce mucus, while the barrier properties of the epithelium can also become altered.7
Airway-derived cell lines are useful for many applications, but some are cancer-derived or immortalized and may not fully represent the behavior of normal primary airway epithelial cells. Primary human airway epithelial cultures have therefore become increasingly important for studying differentiated respiratory tissue. Culture conditions used during initial expansion can also influence whether primary normal human bronchial epithelial cells retain the characteristics required for subsequent three-dimensional differentiation.8
ALI culture helps overcome some of the limitations of submerged monolayers. Under suitable conditions, the cells can develop a pseudostratified structure, undergo mucociliary differentiation, produce mucins, and form tight junctions.2
Figure 1: Different levels of complexity in respiratory research models. In vitro systems range from two-dimensional cultures to mixed cultures, three-dimensional cultures, and complex co-cultures. Increasing model complexity may improve physiological relevance, but it can also increase cost and experimental requirements.
What is air-liquid interface culture?
In an ALI culture, airway epithelial cells are grown on a permeable membrane that separates an apical and a basolateral compartment. During the differentiation phase, the apical surface is exposed to air while the basolateral surface continues to receive nutrients from the culture medium.1,2
This configuration reflects an important feature of the respiratory tract. In vivo, the apical surface of airway epithelial cells faces inhaled air, while nutrients are supplied from the underlying tissue.
Exposure to these different environments supports basoapical polarity and epithelial differentiation. Under appropriate culture conditions, the cells can form a pseudostratified epithelium with ciliated and mucus-producing cells, as well as tight junctions that contribute to barrier function.1,2
Figure 2: Differentiated human bronchial epithelial cells cultured at the air-liquid interface. The image shows a multilayered columnar epithelium with apical cilia growing on a permeable Transwell membrane.
How does air-liquid interface culture work?
An airway ALI workflow generally includes an initial expansion phase, transfer to a permeable insert, submerged culture, and differentiation after air-lifting.1
- Cell expansion: Primary airway epithelial cells are first expanded in conventional culture vessels. In the PromoCell workflow, cells are passaged once they reach approximately 70–80% confluence.
- Transfer to an insert: The expanded cells are seeded onto a porous membrane, commonly coated with an extracellular matrix component such as collagen.
- Submerged culture: Culture medium is initially present in both the apical and basolateral compartments. The cells proliferate until they form a confluent epithelial layer.
- Air-lifting: Once the layer is confluent, the apical medium is removed. Nutrients are then supplied only from the basolateral compartment, while the apical side is exposed to air.
- Differentiation: Air exposure promotes polarization and differentiation. The cells can develop cilia, produce mucus, and establish a functional epithelial barrier.
Exact culture times and handling steps depend on the selected cell type, donor, medium, insert, and protocol. In the PromoCell workflow, the differentiation phase continues for at least 14 days after air-lifting.
For detailed culture instructions, see the application note Air-Liquid Interface Culture System for Standardized Respiratory Research.
Figure 3: Main stages of an airway epithelial ALI workflow. Primary bronchial epithelial cells are expanded in two-dimensional culture, seeded onto collagen-coated permeable inserts, maintained under submerged conditions, and then air-lifted. The post-airlift phase supports epithelial polarization and barrier formation.
How is epithelial barrier integrity assessed?
Transepithelial electrical resistance (TEER) is a non-destructive measurement commonly used to assess epithelial barrier integrity. It measures the electrical resistance across the cell layer and can indicate the formation and maintenance of tight junctions.9
During the measurement, electrodes are positioned on either side of the epithelial layer and electrical resistance is recorded. Because the procedure does not destroy the culture, TEER can be measured repeatedly to monitor barrier development over time or following experimental exposure.
Earlier in vivo studies also measured transepithelial electric potential differences across the human tracheal and bronchial epithelium, helping to establish airway electrophysiology as a functional epithelial readout.10
TEER values should be interpreted in the context of the cell source, donor, insert, culture conditions, and measurement system. A single threshold should not be treated as a universal definition of a successful ALI model. Where possible, TEER should be considered together with other readouts, such as morphology, permeability, viability, tight-junction markers, cilia formation, or mucus production.9
Why are standardization and validation important in ALI culture?
ALI cultures can vary between laboratories because researchers may use different cells, donors, passages, media, inserts, coatings, seeding densities, and air-lifting schedules. These differences can make it difficult to compare results between studies.2,4
The issue is particularly important because ALI experiments commonly run for several weeks. Incomplete attachment, poor differentiation, or weak barrier formation may only become apparent after considerable time and material have already been invested.
Standardization does not mean that every laboratory must use an identical protocol. It means that the relevant culture parameters are documented, controlled, and validated for the intended research question.2,4
| Factor | Why it can affect the model | What researchers should document or control |
|---|---|---|
| Cell and donor source | Donor characteristics can influence proliferation, differentiation, and barrier formation | Tissue source, donor status, disease information, medication history where available |
| Passage and confluence | Over-confluence or extensive passaging may alter proliferation and differentiation | Passage number and confluence at passaging, seeding, and air-lifting |
| Insert and coating | Membrane properties and coating can affect attachment and barrier development | Insert material, pore size, surface area, and coating method |
| Seeding density | Uneven or insufficient seeding may delay formation of a confluent layer | Living cells seeded per cm2 and cell distribution |
| Culture medium | Medium composition affects expansion, differentiation, and barrier stability | Medium used during each phase, supplements, and lot information |
| Air-lifting and feeding schedule | Timing and handling influence differentiation | Confluence at air-lifting, medium-change schedule, and apical washing |
| Quality-control readouts | Different endpoints may capture different features of the model | TEER, morphology, viability, mucus, cilia, permeability, and marker expression |
Table 1: Factors that can influence ALI culture performance
How do donor variability and cell selection affect ALI culture?
Variation between primary-cell donors is expected. Donor characteristics, disease status, and previous medication may influence epithelial proliferation, differentiation, or barrier formation.
This biological variability can be valuable when the objective is to compare different donors or disease states. However, it may complicate studies that require a stable reference culture or positive control.
Human Bronchial Epithelial Cells that have been pre-screened for ALI culture have been evaluated for specified barrier-forming performance under defined conditions. Pre-screening can reduce the risk of selecting a donor lot with unsuitable performance for the planned workflow, but it does not eliminate biological variability or guarantee identical results in every laboratory.
Human leukocyte antigen (HLA) typing may be relevant where antigen presentation, immune recognition, or disease-associated HLA variation forms part of the research question. Bronchial epithelial cells from patients with asthma have been reported to display altered expression of functional HLA-G isoforms.11 HLA typing is not, however, a general requirement for every airway ALI study.
Why does ALI medium composition matter?
Culture medium can influence epithelial-cell expansion, differentiation, and barrier development. Researchers should distinguish between the medium used during the initial expansion phase and the medium used after air-lifting to support differentiation.2,4
Serum- and bovine pituitary extract (BPE)-free formulations can reduce variability associated with incompletely characterized supplements. They do not, however, eliminate variability arising from donors, handling, inserts, or other protocol parameters.
For a serum- and BPE-free workflow, Airway Epithelial Cell Growth Medium 2 can be used for the expansion of epithelial cells from large air passages and combined with Air-Liquid Interface Medium during the differentiation phase.
To explore this variable in more detail, read our technical resources on how Air-Liquid Interface media compositions can influence epithelial barrier integrity.
What are ALI cultures used for in respiratory research?
ALI systems are used to investigate processes that depend on differentiated airway epithelium, epithelial barrier function, or exposure at the air-facing surface. Applications include disease modeling, infection research, inhaled-drug studies, and toxicology.2,3
You can explore additional cell types and resources on our respiratory research overview page.
Respiratory disease models
Airway epithelial cells from healthy donors or donors with respiratory conditions can be cultured at the air-liquid interface to investigate disease-associated changes in epithelial function.
ALI cultures have been used in research into asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, and other respiratory conditions. Depending on the model, researchers can evaluate endpoints such as barrier integrity, mucus production, ciliary function, cytokine release, epithelial repair, or gene expression.3
Inhalation and drug-delivery studies
ALI systems are particularly relevant when studying substances delivered to the air-facing surface of the respiratory epithelium.
In submerged culture, a compound added to the medium may contact the entire cell surface. In an ALI model, aerosols or droplets can be applied to the apical side, more closely reflecting the route by which inhaled drugs reach airway epithelial cells.
Researchers can use these systems to investigate epithelial uptake, barrier transport, biological responses, and local toxicity. The deposited dose and exposure conditions still need to be characterized carefully.12
Respiratory infection research
Viruses and bacteria can be applied to the apical surface of differentiated airway epithelial cultures. This enables researchers to investigate pathogen entry, replication, epithelial damage, and host responses in a human airway model.
ALI cultures have been used to study respiratory pathogens including influenza viruses, respiratory syncytial virus, and SARS-CoV-2. Variation in host immune responses is also an important consideration in COVID-19, where differences in disease severity cannot be explained by viral exposure alone.13
For example, Mulay et al. differentiated primary human proximal airway epithelial cells at the air-liquid interface and used the resulting mucociliary cultures to investigate SARS-CoV-2 infection, epithelial host responses, and candidate drug effects.14
Toxicology and air-pollution research
ALI exposure systems allow researchers to study epithelial responses to particles, gases, tobacco smoke, air pollutants, and other inhaled substances.
Because the material can be applied directly to the air-facing surface, researchers can investigate effects on barrier integrity, viability, inflammatory signaling, mucus production, and ciliary function. ALI models can therefore complement conventional in vitro and in vivo approaches in respiratory toxicology.2,15
No single ALI model can reproduce the complete respiratory system. Standard epithelial models generally lack the full immune, vascular, stromal, and mechanical environment of the lung unless these components are added through co-culture or more complex systems.2,15
ALI culture provides a closer look at airway epithelial function
Air-liquid interface culture enables primary human airway epithelial cells to develop structural and functional properties that are difficult to maintain in conventional submerged cultures. These models can support research into airway physiology, respiratory disease, infection, inhaled substances, and epithelial barrier function.
Reliable results depend on the complete experimental system. Cell source, donor, passage, insert, medium, air-lifting, and quality-control readouts should therefore be selected and documented according to the research question.
At PromoCell, we offer high-quality primary human airway epithelial cells and specialized media for different stages of airway culture, from initial cell expansion to differentiation at the air-liquid interface.
Serum-free cell culture medium for epithelial cells from large air passages.
Primary Human Bronchial Epithelial Cells isolated from the surface epithelium of human bronchi.
Serum-free and BPE-free cell culture medium for an optimal and standardized culture of human bronchial epithelial cells at the air-liquid interface (ALI).
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