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Daniela Marcano, Ghanashyam Acharya, Stephen C Pflugfelder; Characterization of Adult Mouse Lacrimal Gland Explant Cultures. Invest. Ophthalmol. Vis. Sci. 2014;55(13):60.
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Dry eye syndrome is caused by the dysfunction of the lacrimal gland. One of the proposed strategies to treat this pathology is based on organ transplant from an ex-vivo regenerated organ. Decellularization, germ method, culture on 3D Magtrigel systems and culture on nano fibers are techniques that have been used to mimic the cellular matrix in order to regenerate the organ by promoting cell proliferation and differentiation. However, most of these procedures use embryonic or purified acinar cells, which might be difficult to reproduce in humans. The purpose of the study was to establish lacrimal gland explant cultures from adult mice and to characterize the cellular outgrowth. This information will be beneficial for organ regeneration.
Lacrimal glands from 6-8 week old C57BL/6 mice were aseptically removed, washed in Ham’s media, minced, and digested in liberase. Tissue pieces were plated on plastic dishes and cultured for 8 to 14 days with constant monitoring of morphology and cell differentiation. Subsequently, cultures were washed, fixed with cold methanol, and analyzed by immunofluorescent (IF) microscopy. For comparison, lacrimal gland paraffin sections were also stained by IF. Cytokeratins 5, 7, 8, 14, and 18, vimetin and AQP5 and AQP11 were used as markers. Also, the secretory proteins epidermal growth factor (EGF), lactoferrin, and MUC5AC were evaluated.
Phase contrast images of the cellular outgrowth showed a mixture of elongated cells and polygonal cells. Some binucleated spindle-shape and large flattened cells were also detected. IF showed the elongated cells were vimetin positive. Moreover, the polygonal cells were positive for K5, K8, and K14. Interestingly, lacrimal cultures were repeatedly negative (at least three times) for K7 or K18. However; positive immunstaining for these markers was observed in lacrimal gland paraffin sections. Both explant cultures and paraffin sections were positive for AQP5, AQP11, EGF, lactoferrin and MUC5AC.
A mixture of epithelial (polygonal cells; keratin positive, producing secretory proteins) and mesenchymal cells (elongated cells; vimetin positive) was obtained from murine lacrimal gland explants cultures.
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