Small Airway Epithelial Cell Model

Air-liquid interface (ALI) culture holds enormous potential for respiratory research, with a wide range of applications including:

• Modelling of respiratory diseases
• Toxicity testing of therapeutic compounds

Our team have over 10 years of experience working with lung epithelial cells across a wide range of assays and would be happy to discuss your project needs

Formation of a 3D Pseudostratified Epithelium

Immortalised cell lines and primary cells derived from animals models are commonly used, however, the data they generate lack relevance to human physiology. In order to recapitulate the pseudostratified mucociliary phenotype observed in vivo, human lung small airway epithelial cells need be cultured at the air-liquid interface.

For our ALI-SAEC model, primary human small airway basal cells are expanded in culture flasks before being seeded onto permeable Thincert supports containing culture medium in both the apical and basal compartments. The basal cells are then expanded until confluent prior to being subjected to “air-lift”, i.e. with medium only supplied to the basal chamber to create the air-liquid interface. This configuration mimics the conditions found in human and drives differentiation towards a mucociliary phenotype, forming a 3D pseudostratified epithelium containing club cells in greater abundance than goblet cells. Full differentiation takes 21-28 days and cultures are used from 21-days onwards.

Expression of Junctional Genes & Proteins

This model is further characterised by the development of a functional epithelial barrier, as indicated by the expression of tight junction proteins and the development of high transepithelial electrical resistance (TEER) as shown by staining of the junctional proteins ZO1 and E-Cadherin. ZO1 and E-Cadherin protein expression, in conjunction with TEER data, confirms cells are polarised and successfully differentiated. Gene expression levels of junctional genes (TJP1, JAM-A, CDH1) can also be tracked throughout the differentiation period.

Cell-Specific Gene Expression

As the basal cells differentiate, we quantify the expression of cell-specific genes as an indirect assessment of basal cell differentiation by qPCR. This also confirm the presence of all relevant cell types following 21 days in culture.

Cell-Specific Protein Expression

MUC5B and MUC5AC are mucin proteins expressed by goblet cells in both the upper and small airway, however their ratio differs depending on the location. MUC5B is predominantly expressed in the small airways. Validation data collated from the SAEC model suggests a higher expression of MUC5B than MUC5AC,  similar to the expression pattern observed in vivo.

Validation of Increase in Ciliary Beating Frequency during SAEC differentiation

Newcells’ SAEC model cultured in ALI conditions has successfully demonstrated increase in cilia expression during differentiation. Data from the CiliaBeat software analysis confirms an increase in ciliary beat frequency  and area of the apical cell surface covered by active cilia from day 14 post-airlift onwards aligning with physiological characteristics.