Culturing epithelial cells using advanced 3D culture systems for airway modeling


The ability to appropriately model the human airway has been greatly impacted by the development of specialized in vitro cell culture techniques that promote the formation of 3D structures recapitulating the morphological and functional characteristics of the airway.

Last week’s post Cool Tools – Producing 3D Tissue Models of the Airway Epithelium with PneumaCult detailed the ways in which airway tissue models contribute to the respiratory research field, and how these advanced culture systems are supported by the defined and bovine pituitary extract-free PneumaCult™ culture system. This article discusses the features of the air-liquid-interface (ALI) culture system, which supports extensive cellular differentiation to form a pseudostratified mucociliary epithelium featuring mucus-producing cells, ciliated cells with coordinated cilia movement, and physiological epithelial barrier function. Sphere cultures of airway epithelial cells incorporating a differentiated epithelial cell layer with an open lumen lined with the apical cell surfaces were also discussed in the context of an airway culture model that is easily adaptable to high-throughput investigations.

Have questions about modeling the human airway?

This week, Dr. Juan Hou will be answering your questions about modeling the human airway, including how to use PneumaCult™-Ex and PneumaCult™-ALI to generate high-quality ALI and sphere cultures. If you have any questions about culturing airway cells using advanced, 3D culture systems, Dr. Hou would be happy to address these, along with any inquiries on how these culture techniques can be applied to various research topics in the respiratory field.

Question 1

What type of cells can be used to initiate ALI cultures and how long can the ALI cultures be maintained?

Commercially available human airway epithelial cells are compatible with air-liquid interface (ALI) culture in PneumaCult-Ex™ and PneumaCult-ALI™ when they are seeded onto cell culture inserts no later than Passage 4. Using cells at later passages will result in a significant decrease in quality or failure of ALI culture. PneumaCult media also work well with primary human nasal and bronchial epithelial biopsies. In addition to human airway epithelial cells, PneumaCult media also work well with ferret and mouse airway epithelial cells.

ALI cultures can be maintained up to a year if there are no issues with culture contamination.

Question 2

Are sphere cultures of airway epithelial cells compatible with ICC analysis? What other downstream assays and analysis methods work well for this culture system?

Yes, airway epithelial cells grown in sphere cultures are compatible with ICC analysis. Contact our Product and Scientific Support department to request the protocol for isolating spheres from Matrigel® ( Regarding downstream assays, there are many possibilities, depending on the specific research questions you are using the sphere cultures to address. For example, the spheres from each well can be isolated and assessed for the presence of specific markers by subjecting cells to either qPCR analysis, to quantify the level of specific RNA transcripts present in the cells, or western blot analysis, to assess the expression levels of specific proteins.

Question 3

Can this ALI system also be used for murine airway epithelial cells? And if yes do you have to change the protocol (as compared to human cells)? How many cells do you need per transwell?

Yes, the PneumaCult ALI system is compatible with murine airway epithelial cells. The protocol was optimised using commercially available human airway epithelial cells, so you might need to optimize the seeding density when you use murine airway epithelial cells. Starting with a range between 1X10^5 and 5X10^5 cells/cm^2 will likely provide a positive result. The cells should reach confluence after 1-4 days post-seeding at optimized seeding density. Optimised seeding density should also result in ALI differentiation exhibiting robust cilia beating and mucus secretion. Epithelium integrity and health can also be measured by transepithelial electrical resistance (TEER), which should be within 150 - 500 Ω∙cm^2 for high-quality ALI cultures.The seeding density also depends on the passage number, with cells at later passages typically resulting in lower-quality cultures. Given the anatomy differences between the human airway and the murine airway, you should expect a smaller proportion of goblet cells in the differentiated ALI cultures when you use murine airway epithelial cells.

Question 4

I am interested in freezing primary nasal epithelial cells for subsequent functional analyses. Do you have any suggestions on at which step to freeze cells (passage#), what the best thawing procedure is, whether complete ALI differentiation is still possible after freeze-thawing and what number of cells you need to freeze to be able to regrow and differentiate successfully?

We recommend freezing the cells at the end of P0 (The cells will be at P1 when they are cultured after thaw). The cells should be frozen at approximately 1 X 10^6 cells/mL in PneumaCult™-Ex with 10% DMSO. The best thawing procedure is to quickly thaw the cells in a 37℃ water bath and seed 1 mL directly into a T75 flask containing 20 mL warm PneumaCult™-Ex. After an overnight incubation (~16 hours) at 37℃, exchange the medium with fresh PneumaCult™-Ex to remove the DMSO. The freeze-thaw procedure will affect downstream ALI differentiation quality; however, if you follow the proper protocol, good ALI differentiation should be achievable for P2 and P3 cells.

Question 5

For your 3D models of the airway epithelium air-liquid interface (ALI) cultures, is it possible to grow epithelial cells on fibroblasts? Or do you have other methods to generate stromal cells?

Yes, you can co-culture the airway epithelial cells with human airway fibroblasts. The human airway fibroblasts can be seeded onto opposite side of a transwell insert membrane so the fibroblasts are submerged in the PneumaCult™-ALI medium within the bottom chamber and epithelial cells in the apical chamber are at air-liquid interface.

It should first be noted that stromal cells are not required for achieving a well-differentiated pseudostratified airway epithelium at air-liquid interface when using PneumaCult™ media. This defined media system can be used to generate a high-integrity epithelial layer at the air-liquid interface in the absence of other cell types. If desired, however, you can co-culture airway epithelial cells with human airway fibroblasts, in order to investigate the interactions and signalling between these cell types. In this case, the human airway fibroblasts can be seeded onto the basal side of a cell culture insert, such that the fibroblasts are submerged in PneumaCult™-ALI Medium in the basal chamber after air-lift while the epithelial cells are exposed to the air in the apical chamber. There are also several other methods for co-culture of airway epithelial cells with other cells types, such as mesenchymal stem cells (Carbone et al.), to enable interrogation of the signalling interaction between those tissue types.

Question 6

I am interested in imaging the ALI epithelial cells at the lateral aspect, vs. a top view for immunofluorescence. Can you please advise how one would go about embedding and sectioning the cells/membrane (PET 0.4 um) for this application. A technical protocol would be greatly appreciated.

There are two histology methods that can be used for your purpose. One is paraffin sectioning,  and the other is cryosectioning. The choice of method depends largely on what equipment you have in your facility and what downstream experiments you want to to. The paraffin sections generate better samples for assessing morphology and are preferable if your  downstream assay involves HE and PAS staining. Cryosections, on the other hand, are preferable for downstream experiments such as immunofluorescence. Please contact our Product and Scientific Support department (  to request the specific protocol for your needs and equipment.

Question 7

With regards to mucus production, do you have any procedures to examine the mucus and periciliary fluid proteins in ALI cultures that lend to western analysis. E.g. collection and processing for protein. What would you suggest would be a good reference gene for loading control (MUC5?) in western analysis.

If you are interested in the secreted mucin, you can add warm PBS to the apical chamber,  incubate for 20 minutes and then collect the PBS with mucin. Acetone can be used to precipitate the protein, followed by standard western blotting procedure. You can also collect the whole culture using protein lysis buffer followed by standard western blotting  procedure. For the secreted mucin, Muc5AC and Muc5B are good markers to use. For the membrane-associated mucin, Muc1 is a good marker to use.

Question 8

What are the pros and cons for using ALI culture vs sphere culture?

ALI culture allows us to manipulate the environmental variables in the assay independently for the apical side and basal side of the epithelium.  Traditionally, sphere culture was carried out on inserts and supported clonal bronchosphere generation to facilitate enumeration of stem cells. The insert-dependent nature of this assay, however, makes adaptation of this system to a high-throughput format exceedingly challenging. Recently, insert-independent sphere culture techniques have been developed. This method is described in the recent Cool Tools post describing 3D airway epithelial models also published by Danahay et al.. This insert-independent sphere culture method is amenable for high-throughput studies.

Question 9

Can you use your system for culturing diseased ALI cells as well?

Yes, the PneumaCult culture system can be used for diseased cells as well as cells from healthy donors. Our collaborators and customers have used PneumaCult media for nasal epithelial cells isolated from asthmatic patients (Reeves et al., 2015; Xu et al., 2015) and airway epithelial cells from cystic fibrosis patients (Reeves et al., 2014)  Air-liquid-interface and sphere cultures of human airway cells provide excellent model systems to interrogate the cellular characteristics inherent in these and other respiratory disease states in vitro.

Question 10

Have you or anyone you know automated this process for high throughput screens?

We haven’t performed high-throughput assays in-house at this time. Regarding the sphere culture system, Danahay et al. used a 384-well format for bronchospheroid culture as the basis of a high-throughput screening system. Regarding the ALI culture system, there are 96-well format cell culture inserts that are commercially available, but not an off-the-shelf automated system. There is, however, an automated ALI culture system that uses PneumaCult medium published by Aufderheide et al.

Question 11

Can you use this product for epithelial corneal cells? Or can you direct me to another similar product?

We have not, as of yet, tried PneumaCult media on corneal epithelial cells in-house, nor are there any published accounts of other researchers performing these experiments. I do think that it would be an interesting experiment to try, however.

Question 12

What are the most important factors in promoting cilia development? Are there specific supplements or growth factors included in the PneumaCult-ALI media or added separately that support ciliogenesis?

Several factors contribute to robust and efficient mucociliagenesis, including asymmetric nutrition delivery, airlifting in ALI culture and matrices in sphere culture. In all cases, specialized media such as PneumaCult-ALI is pivotal for robust mucociliagenesis. The PneumaCult media formulations are proprietary, so I am unable to share information about the specific components that support efficient differentiation of airway epithelial cells.

Question 13

What is the typical or expected % cilia in an ALI airway culture?

The ciliated cells can make up ~80% of the total apical cells in the in vivo human airway. In our fully differentiated ALI cultures using PneumaCult media, the percentage of cultures that are composed of ciliated cells ranges from 80% for cultures initiated with passage 2 cells to 30% for cultures initiated with passage 4 cells

Question 14

Are the ALI and sphere culture methods compatible with hPSC-derived airway cells?

Yes. We have several customers who have been using PneumaCult media for expansion and maturation of their hPSC-derived lung progenitors using sphere and ALI culture methods. Most interestingly, a recent publication by  Konishi et al. demonstrated that combination of a 3D sphere culture methodology with PneumaCult-ALI medium promotes differentiation of hPSC-derived lung progenitors to proximal multi-ciliated airway cells.

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