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Conventional submerged culture systems do not adequately capture the complex morphological and functional characteristics of the in vivo human airway, and animal primary cell models can have limited experimental windows and lack perfect homology to human systems. Addressing these limitations, air-liquid interface (ALI) culture of human primary airway epithelial cells is being increasingly adopted as a model system for the in vivo human airway epithelium.

For the study of respiratory viruses, such as the novel SARS-CoV-2 virus that causes COVID-19, ALI cultures are advantageous because they preserve key characteristics of the in vivo airway epithelium that viruses target. Viruses evolve in parallel to their host cells and, consequently, show pronounced specificity for both host species and cell type. Infection models for a given virus must therefore be specific to and closely representative of the native cells it targets. To date, the ALI culture system has been used to study a wide range of viruses, including the novel coronavirus.

Webinar: Studying Respiratory Viruses with ALI Cultures

ALI as a Model System to Study Viruses, Including SARS-CoV-2

The ability of ALI cultures to model the key features of the in vivo airway highlights the greater physiological relevance of this system over other conventional cell culture models. Human airway ALI cultures allow for efficient infection of cultured cells and have been used to model various mechanisms of viral pathogenesis and human disease (see publication highlights below). This model provides a platform for researchers to study how specific viral characteristics influence pathogenesis during infection. This includes investigating protein interactions during intracellular replication,¹ factors impacting viral entry into cells,² and infection kinetics.³ The ALI infection model has also been used to interrogate the mechanisms of tissue damage and cell death⁴ as well as aspects of host response, such as interferon response to infection.⁵ The applications of ALI cultures, which can be generated from pediatric⁶ and adult human cells, are wide ranging. ALI cultures have been co-infected with bacteria and viruses, and cultures generated from zoonotic vectors (e.g. pig) have also been studied in the context of human disease dynamics.⁷ The utility of ALI cultures for studying viral infection represents a valuable tool for developing antiviral therapeutic approaches;⁵ viral neutralization by monoclonal antibodies has also been tested in this model.⁸

Coronaviruses (CoV) are a large family of viruses that cause illnesses ranging from the common cold to severe respiratory diseases, including COVID-19 caused by the SARS-CoV-2 virus. The cell entry receptor for SARS-CoV-2 has recently been confirmed by independent research groups to be angiotensin-converting enzyme 2 (ACE2)—the same entry receptor for the SARS-CoV virus that caused the 2003 SARS outbreak^9,10. Human large and small airway cultures grown at the ALI show abundant ACE2 expression (Figure 1) and provide a physiologically relevant model for investigating ACE2 as a target for antiviral intervention against COVID-19.

Figure 1. Presence of ACE2 in HBECs Cultured in �ʲԱ�ܳ�����ܱ���™-������ Medium and HSAECs Cultured in �ʲԱ�ܳ�����ܱ���™-������-S Medium

Confocal images of whole mount immunostained ALI cultures of (A) human bronchial epithelial cells (HBECs) cultured in �ʲԱ�ܳ�����ܱ���™-������ Medium and (B) human small airway epithelial cells (HSAECs) cultured in �ʲԱ�ܳ�����ܱ���™-������-S Medium, at P2. The ALI cultures were fixed and stained with antibodies for ciliated cells (AC-tubulin; green), SARS-CoV-2 receptor (ACE2; magenta), and tight junctions (ZO1; red). The nuclei were counterstained with DAPI (blue). ACE2 was detected in both (A) large and (B) small airway culture systems during early passages (≤P6, HBECs; ≤P4, HSAECs). Both HBECs and HSAECs were expanded in PneumaCult™-Ex Plus Medium prior to differentiation at the ALI.

Publication Highlights

Why Is ALI Culture Physiologically Relevant?

Primary human bronchial epithelial cells (HBECs) cultured at the ALI undergo extensive mucociliary differentiation that recapitulates what is observed in vivo. As a result, ALI culture of HBECs is increasingly being recognized as the most physiologically relevant in vitro model system for respiratory research. While the use of immortalized cell lines and primary cells from animals is common, data generated using these models are not directly applicable to the human system.¹¹ Submerged culture of primary HBECs is possible; however, cells in this system fail to undergo mucociliary differentiation and thus do not accurately represent the in vivo airway.

The physiological relevance of the ALI culture system is supported by phenotypical and histological evidence. Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining show that HBECs cultured at the ALI are (Figure 2A and 2C) pseudostratified in morphology and make up a heterogeneous cell population that includes ciliated and mucus-secreting (PAS-positive) cells, similar to the in vivo bronchial epithelium (Figure 2B and 2D). An important function of the tracheobronchial epithelium in vivo is to act as a protective barrier against inhaled insults. The same epithelial barrier function has been confirmed in ALI cultures by the expression of tight junction proteins and the development of high transepithelial electrical resistance.¹² The suitability of ALI cultures for modeling the airway has been further confirmed through transcriptome analyses¹³ and a wide range of experiments demonstrating physiological responses to insults such as toxicants and pathogens.^14-16 Importantly, ALI cultures of primary cells from donors with respiratory disease (e.g. asthma, cystic fibrosis, COPD) recapitulate in vivo disease characteristics, forming robust in vitro models of these conditions.^17-18

Webinar: In Vitro Human Airway Modeling Using Primary Airway Epithelial Cells

How Are Cells Grown at the ALI?

The defining feature of ALI culture is that the basal surface of the cells is in contact with liquid culture medium, whereas the apical surface is exposed to air. A common approach is to seed cells onto the permeable membrane of a cell culture insert, which is initially supplied with culture medium to both the apical and basal compartments (Figure 3A). Once confluence is reached, the cells are subjected to ‘air-lift’, where the medium is supplied only to the basal chamber (Figure 3B). This configuration mimics the conditions found in the human airway and drives differentiation towards a mucociliary phenotype.

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