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An in vitro scaffold‐free epithelial–fibroblast coculture model for the larynx



Physiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1.

Study design

In vitro.


Cell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor β1 (TGFβ1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin.


Fibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFβ1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture.


These data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.

Level of evidence

NA Laryngoscope, 127:E185-E192, 2017.

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