Sponsored by: STEMCELL Technologies
Session ends: April 15th, 2016, 3:00pm MST
Answers by: Dr. Juan Hou, STEMCELL Technologies
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.
Questions & Answers
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 […]» Read MoreThe 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 […]» Read MoreSeveral 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 […]» Read MoreWe 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.» Read MoreWe 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 […]» Read MoreYes, 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 […]» Read MoreALI 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 […]» Read MoreIf 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 […]» Read MoreThere 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 […]» Read MoreYes, 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 […]» Read MoreWe 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 […]» Read MoreYes, 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 […]» Read MoreYes, 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® (firstname.lastname@example.org). 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 […]» Read MoreCommercially 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 […]» Read More