In using the ALI model I have read that people have trouble achieving population doublings. What kind of doublings should be expected in an optimized culture and do you have any suggestions for reaching these numbers?
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Population doubling is something that would be cell type dependent, as each cell type have their own growth rate pattern and this may vary on a permeable support, particularly if conditions are not optimized. As ALI is a long term (up to weeks or months) culture one should optimize initial cell seeding density and culture time for their specific cell type before performing the actual ALI culture. Typically, cells are seeded in the apical chamber, with media in both the apical and basolateral chambers, and maintained in growth media until they achieve the desired cell confluence.
The desired cell confluence is the confluence required to obtain a monolayer of cells that have formed tight junctions. The amount of time to achieve desired confluence is dependent on cell type and seeding density, and some primary cells types may require higher cell seeding densities or longer culture times. The amount and type of the coating (eg. Collagen Type I Rat Tail) can also impact the ability for cells to form a monolayer and may require optimization if the desired confluence is not being achieved. There are several methods that can be used to determine when a cell monolayer has been achieved, but the most commonly used method is to take transepithelial electrical resistance (TEER) measurements. Typical TEER values vary by cell type and can be found in ALI literature publications. Another less common method is to stain the cells for tight junction marker proteins like ZO-1.
When the desired cell confluence is reached, the cells are air-lifted, which means the growth media is removed from both chambers and the basolateral chamber is filled with differentiation media. An example of this protocol is highlighted in this application note, “Development of an Air-Liquid Interface Model using Primary Human Bronchial Epithelial Cells and HTS Transwell®-24 Permeable Supports from Corning”.
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