Air-liquid interface (ALI) culture is a widely used method to study respiratory tract epithelia and is also known to support differentiation of multi-layered skin models. By seeding cells on to a permeable support suspended in growth media and allowing an initial period of propagation before removing the media from the apical compartment, only the basolateral side of the cell monolayer remains submerged. ALI systems therefore allow researchers to more accurately mimic in vivo conditions compared to using conventional cell culture models, making them ideal to perform mechanistic studies of respiratory epithelial cells as drug permeation barriers; to model respiratory diseases like cystic fibrosis and asthma; to investigate essential dermal processes such as wound healing; and to advance understanding of respiratory epithelium infections including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This article highlights some of these applications and includes tips for using permeable supports to enhance ALI research.
Permeable supports for culturing cells are available in various formats. These include inserts suitable for 6–24 well plates, HTS-compatible 24-well permeable supports, and large format inserts that fit into a rectangular tray or cell culture dish. For researchers wishing to reduce the frequency of cell feeding when culturing cells at the ALI for extended periods, a 6-well deep well companion plate designed for use with Falcon® 6-well cell culture inserts holds up to seven times more medium in the basolateral compartment than a regular 6-well plate. ALI studies are typically performed using collagen I or collagen IV-coated inserts with a 0.4 µM pore size, although other pore size inserts may be used depending on the application. Cells can be cultured in ALI on permeable supports for up to 28 days, or longer if handled appropriately.
Primary and immortalized keratinocyte cells are valuable models for investigating the biology of the epidermis. However, for cultured keratinocytes to differentiate into organotypic skin equivalents and display similar architecture to the skin in vivo, they require an environment representative of the ALI. This can be produced by casting dermal fibroblasts on to a collagen-coated permeable support that is then submerged in suitable growth media. After seeding keratinocytes on to the fibroblasts and allowing them to establish, the media is removed from the chamber to promote keratinocyte growth under simulated ALI conditions (Figure 1).
Figure 1. Co-culture of keratinocytes grown on a collagen raft embedded with dermal fibroblasts.
Two-dimensional cell monolayers are of limited use to study the mammalian airway due to the inability of airway epithelial cells to differentiate well in submerged culture. Using permeable supports, researchers have successfully differentiated primary human bronchial epithelial cells (HBECs) into mature airway tissue for improved functional studies and have demonstrated that culturing primary HBECs at the ALI promotes the formation of a pseudostratified epithelium that can be used to model the human airway (Figure 2). This approach has been employed to compare primary airway epithelial cells from healthy human donors to those of donors with cystic fibrosis.
Figure 2. Primary bronchial epithelial cells cultured at the ALI form pseudostratified epithelia comprising ciliated cells (Beta-IV tubulin), mucus-producing goblet cells (Muc5AC), and basal cells (p63), with the ciliated cells appearing on the apical (top) side and the basal cells appearing on the basolateral (bottom) side. Cells from the healthy donor (left) are distinct from those of the donor with cystic fibrosis (right).
As a result of the COVID-19 pandemic, demand to study cellular behaviors at the ALI has increased dramatically. Recent publications have seen permeable supports used for culturing human airway epithelial cells at the ALI to study the cytopathic effects of the virus and to evaluate the therapeutic potential of a broad-spectrum antiviral drug, while using permeable supports to form differentiated organoid-derived gut cultures has underpinned the finding that SARS-CoV-2 also targets human gut enterocytes.
For further advice on using permeable supports, see this tips and techniques page.
Corning offers a complete range of permeable supports to accelerate your ALI research, including Transwell®, Falcon® and BioCoat® products. To find out more visit corning.com.