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Angela A Pitenis, Tristan T Hormel, Tapamoy Bhatacharjee, Juan Manuel Urueña, W Gregory Sawyer, Thomas E Angelini; In Vitro Characterization of Mucin Growth Dynamics by Confocal Fluorescence Microscopy . Invest. Ophthalmol. Vis. Sci. 2017;58(8):3096.
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© ARVO (1962-2015); The Authors (2016-present)
Entangled networks of mucin in the tear film are largely responsible for lubrication, ocular hydration, and health, but the growth dynamics of mucin have not been thoroughly characterized (in vitro or in vivo). This study directly observed corneal epithelial cells over 2 days and quantitatively measured mucin production and growth in vitro through confocal microscopy.
Human telomerase-immortalized corneal epithelial cells (hTCEpi) were incubated in a culture dish in KGM-Gold media and grown to confluence. Cell bodies were stained with CellTrace™ Calcein Red-Orange, and hTCEpi mucins were stained with Concanavalin A Alexa Fluor® 488 Conjugate (which binds to oligosaccharides in mucin glycoproteins). Time-lapse confocal microscopy monitored cell viability and mucin production over 2 days every 30 minutes during growth under steady conditions of 37±0.2°C, 5% CO2, and 80% RH. Increased fluorescence intensity indicated increased mucin abundance; 3D fluorescence intensity distribution determined mucin layer thickness.
A mucin layer was observed to grow from the apical side of the hTCEpi monolayer. Over 48 hours, the mucin layer increased by more than 2 µm and grew non-monotonically, exhibiting a long plateau between 10-20 hours. By contrast, the total amount of mucin produced over 2 days increased 10-fold and grew steadily, estimated from the integrated fluorescence intensity. To verify that results were not simply passive fluorescent dye accumulation, dead cells were imaged over a similar period and no corresponding increase in fluorescence intensity was observed. Mucin accumulation was nearly proportional to the square root of time. This time of growth kinetics is consistent with a diffusion-limited process, possibly linked to hTCEpi cell nutrient uptake, or diffusion-limited transport within the cell involved in mucin production. The power-law exponent obtained by fitting these data is 0.58+/-0.004.
This technique quantifies the spatial distribution and total amount of mucin growing from a living cell layer, and allows potential changes in mucin concentration and compaction to be determined. Mucin concentration increases during the plateau in layer thickening; the diffusion-limited kinetics of mucin production exhibit no slowing down during this period, suggesting the accumulation of mucin within a fixed volume in apical space.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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