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Nicole Qiaozhi Lu, Hongbo Yin, Osama Al-Sheikh, Jennifer Elisseeff, Michael Peter Grant; Engineering of a tear-secreting system by coculturing lacrimal gland and conjunctival epithelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2263.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye is a multi-factorial disease, and an in vitro ocular surface model is in need for dry eye study and therapeutic screening purposes. In our study, a coculture system composed of lacrimal gland (LG) and conjunctival epithelial cells (CECs) was established.
Rabbit LGs were digested with collagenase I, hyaluronidase and DNase I at 37 °C. Cell spheroids were formed in a shaking flask on an orbital shaker. CECs were isolated by dispase II. Coculture was established in a transwell insert system as illustrated in figure 1a. The morphology of LG cell spheroids was visualized by phalloidin staining. Cells under coculture conditions were harvested and compared to non-coculture samples. Gene expression was analyzed by qPCR. Mucin secreted by conjunctival goblet cells was stained with texas-red conjugated dextran, and imaged under a confocal microscope. LG secretion was quantified by β-hexosaminidase assay after carbachol stimulation.
LG cells aggregated into spheroids within 2 days, with an average diameter of 108 μm. The spheroids formed 3-dimensional organoids inside Matrigel, as revealed by F-actin staining (FITC, figure 1b). Gene expression analysis of LG spheroids showed that cocultured cells expressed significant higher level of E-cadherin, EpCAM and lactoferrin compared to non-cultured samples. After carbachol stimulation, cocultured LG cell spheroids had more hexosaminidase in both supernatants and cell lysates. For CECs, qPCR results indicated that mucin 5AC and cytokeratin 4 was upregulated, while cytokeratin 7 was downregulated in cocultured cells. After 10 days, cocultured CECs secreted a thinker, more continuous mucin layer than non-cocultured ones (figure 1c).
LG cell spheroids have the ability to develop into lacrimal organoids when embedded in Matrigel, which highly resembles the structure of LG acini. Coculture is beneficial for both LG cell spheroids and CECs, as proven by gene expression and protein secretion results. This coculture system could be further engineered into an in vitro model for dry eye study and therapeutic screenings.
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