What Is Air-Liquid Interface Culture?

Long gone are the days where scientists had to rely on 2D cultures of immortalized cell lines to learn principles of human biology. Today, we have a variety of cell culture systems that come closer than ever before to mimicking the structure and function of our body’s organs. One example of these ‘organoid’ models are 3D cell cultures at an air-liquid interface (ALI). This system is especially useful for cells that like to be exposed to the air – like lung cells! Often, lung cells are collected from tissues leftover from lung transplants. The cells grow and differentiate into layers of multiple cell types on top of a porous membrane submerged in media that contains all the nutrients and materials that the cells need to grow (see Fig. 1). The top of the cells, or apical surface (remember those biology 101 terms!), is exposed to the air just like the surface of the inside of our lungs. Once the cells are cultured, they develop into all the cell types found on the airway surface, including ciliated cells that beat and goblet cells that make mucus. To learn more about how these cell cultures are made, check out the reference below. 1

Staying Alive: Tips for Air-Liquid Interface Cultures
Figure 1

Okay, but What Are Air-Liquid Interface Cultures Good For?

Air-liquid interface (ALI) cultures of human lung cells are a specialized system for studying the biology of the human airway. ALI cultures contain the cells found in the human airway, including ciliated cells that can be seen moving under the microscope and mucus-producing goblet cells. This system is really important for understanding diseases that affect how we breathe. For example, cystic fibrosis is a genetic disease that affects the entire body, but especially the lungs. People with cystic fibrosis have difficulty clearing mucus out of their lungs and slowly lose lung function as they age, resulting in the need for transplanted lungs. ALI cultures from cystic fibrosis patients help scientists understand how their lungs work, and how to design better treatments for this disease. ALI cultures are also a great system to study respiratory pathogens. ALI cells can be infected with bacteria and viruses (or both!) that commonly infect lungs and cause disease. One great example is flu. For more information on growing flu viruses in ALI cells, check out this reference.2

Neat! Now How Do I Keep Them Alive?

Often, researchers specializing in preparing ALI cell cultures can share extra cells with scientists in other labs. If you are lucky enough to receive ALI cell cultures, you’ll need to know how to keep them alive to use for your experiments.

Change the Media

Just like all cell cultures, the cells will use up the nutrients in their media after a few days, and so the media will need to be changed. After the cells have been established at ALI, they cannot be passaged further. But the good news is that, with proper care, ALI cells can live in culture for a long time – up to weeks or months! The cells are grown on a membrane that is part of a plastic insert that fits snugly into the wells of a 24-well plate. Plates should be kept in a 37ºC incubator with 5% CO2 to keep the cells happy. To feed the cells properly, all of the old media must be removed from the well below the insert. To remove the media, either transfer the inserts to a new 24-well plate or use an aspirator to vacuum away every last drop of the old media. Add back 0.5mL of warm fresh media into the well to feed the cells. Human lung ALI cells can live off a strict diet of supplemented DMEM (Dulbecco’s Modified Eagle Media),1 which can be a pain to get a hold of. If you only need a small amount of media, consider asking colleague if they have some to spare.

The key to keeping ALI cells alive is to keep the surface of the cells exposed to air. The cells can be submerged for a few hours, for example, if you are infecting them with your favorite bug. But cells left in liquid for a long period of time will die (trust me, I know). If you accidentally get some liquid on top of the cells, remove it all before putting them back into the incubator. An easy way to remove excess liquid is to aim your pipette tip at the edge of the membrane (don’t poke the cells!) and carefully aspirate the liquid. ALI cells can make a lot of mucus, so might find you need to change tips between each well to make the job easier. An easy way to remember to feed ALI cells is to schedule feedings each Monday, Wednesday, and Friday. If you are ever unsure, never be afraid to ask for advice from your local ALI expert!

Know When to Use Your Air-Liquid Interface Cultures

Now that you’ve put all this work into keeping your cells alive you might want to consider using them for an experiment! ALI cells that are happy and healthy will grow as a visible ‘slimy’ layer on top of the membrane. By taking a look at these membranes under the light microscope in your tissue culture room, you’ll probably notice lots of crud, cell debris, and mucus on the surface of the cells. Don’t be alarmed – they’re probably just fine! If you look closer you’ll just be able to make out the beating motion of the ciliated cells at the apical surface. Movement of the ciliated cells almost looks like your eyes are playing tricks on you, but a hint is to look for the mucus swirling and moving around on top of the cells. You’ll notice the mucus swirls a bit faster if you play them music from your favorite 80s hair band.

Another trick for assessing how your cells are doing is to use a media containing a phenol red pH indicator dye. As the cells eat up the nutrients in their wells you’ll notice the media changes from pink to slightly yellow over time. You should notice this change with each feeding, and if you don’t, they might no longer be viable.

Finally, the integrity of an ALI culture can be directly quantified by measuring the resistance to electrical current between electrodes placed in media above and below the cell layers. This technique, called TEER (transepithelial electrical resistance),3 is also a great tool for tracking the progression of an infection or any other manipulation you think might affect your cells’ barrier function.

To the traditional tissue culturist, working with ALI cells may seem a bit strange at first, but they’re really a wonderful system that can provide a lot of information and opportunities for cool new experiments.


1. Myerburg MM, Harvey PR, Heidrich EM, Pilewski JM, Butterworth MB. Acute regulation of the epithelial sodium channel in airway epithelia by proteases and trafficking. Am J Respir Cell Mol Biol.43(6): 712-9 (2010). doi: 10.1165/rcmb.2009-0348OC. PubMed PMID: 20097829; PMCID: PMC2993091.

2. Nturibi E, Bhagwat AR, Coburn S, Myerburg MM, Lakdawala SS. Intracellular Colocalization of Influenza Viral RNA and Rab11A Is Dependent upon Microtubule Filaments. J Virol. 91(19) (2017). doi: 10.1128/JVI.01179-17. PubMed PMID: 28724771; PMCID: PMC5599730.

3. Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER measurement techniques for in vitro barrier model systems. J Lab Autom. 20(2):107-26 (2015). doi: 10.1177/2211068214561025. PubMed PMID: 25586998; PMCID: PMC4652793.

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