We recently finished our Ask the Expert discussion on “Culturing epithelial cells using advanced 3D culture systems for airway modeling.” During this Ask the Expert session, we discussed the use of 3D epithelial cell culture models in airway modeling, cell types that can be used in this system, disease modeling, high throughput options, imaging solutions, and ALI culture vs. sphere culture. In addition specific questions regarding epithelial cell culture included freeze and thaw techniques, cilla development, and examining mucus production and associated proteins.

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. A previous 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.

In this Ask the Expert session, Dr. Juan Hou answered your questions about modeling the human airway, including how to use PneumaCult™-Ex and PneumaCult™-ALI to generate high-quality ALI and sphere cultures.

Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Culturing epithelial cells using advanced 3D culture systems for airway modeling.

Question:

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?

The Answer:

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:

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?

The Answer:

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 signaling 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 signaling interaction between those tissue types.

Question:

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

The Answer:

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.