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Shubha Tiwari, Javed Ali, Murali M. Balla, Milind N. Naik, Santosh G. Honavar, VijayAnand P. Reddy, Geeta K. Vemuganti; Establishing Human Lacrimal Gland Cultures and Evaluating their Ex-vivo Secretory Function. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3711.
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Dry eye syndrome is a multifactorial chronic disabling disease affecting the function of the lacrimal gland. The treatment involves temporary palliative therapies like ocular surface lubrication and rehydration. Cell therapy involving the replacement of lacrimal gland cells is a promising alternative for providing long-term relief to the patients. This study aimed to explore the presence of stem cells and establish human lacrimal gland cultures with retained secretory functions under ex-vivo conditions.
Fresh human lacrimal gland tissues from patients undergoing exenteration were harvested for cultures after IRB approval. The stem cell compartment in the fresh tissue was investigated by flow cytometry using ABCG2, c-kit and ALDH1 expression. Following enzymatic digestion, cultures were established on Matrigel, collagen and HAM using supplemented HepatoSTIM media. The cultured cells were evaluated for the presence of stem cells markers (ABCG2, c-kit, ALDH 1) and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100µM) stimulation by ELISA.
Native human lacrimal gland cells expressed ABCG2 (5.3±3.5%), c-kit (10.06±3.6%) and ALDH1 (3.7±2.1%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem cells and differentiated cells. The epithelial cells formed a monolayer and ‘spherules’ with duct like connections, suggestive of ductal origin. The levels of scIgA (mean±SEM: 63.2±8.2ng/ml), lysozyme (88.0±60.8ng/ml) and lactoferrin (36.7±3.4ng/ml) in the conditioned media were statistically significantly higher than the negative controls (p <0.05 for all comparisons).
The study suggests the presence of stem cells in the fresh and ex-vivo cultured human lacrimal gland cells with preserved secretory function. This finding is likely to pave way for development of a functionally competent lacrimal gland replacement therapy.
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