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Zeeshan Pasha, Marwan Ali, Dhara Shah, Vinay Kumar Aakalu; Inhibition of Epithelial-Mesenchymal Transition in Human Lacrimal Epithelial Culture. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Study of human lacrimal epithelial cell biology is limited by poor access to tissue and difficulty with maintaining primary cultures. In addition, epithelial cultures often undergo epithelial-mesenchymal transition (EMT) with changes in morphology. We developed a method to maintain epithelial morphology in primary cultures of human lacrimal epithelium.
Methods: Primary human lacrimal glands were enzymatically digested using a cocktail of collagenase (130 units/ml) and hyaluronidase (300 units/ml). Cell suspensions were filtered using a 70 um sieve and then seeded on petri dishes for 2 hours to separate fibroblasts from epithelial cells. The supernatant containing lacrimal epithelial cells was removed and plated on Matrigel coated dishes in HepatoStim culture media supplemented with 2mM L-glutamine, 0.5% FBS, 2% antibiotics, EGF, Insulin and TGFB inhibitor (SB431542). Bright field images of cells were taken over a period of two weeks. Gene expression and cell marker expression were analyzed using RT-PCR and immunofluorescence. TGF beta supplemented cultures were compared with cells without supplementation using morphological, gene expression and immunofluorescence assessments.
Results: Rounded lacrimal epithelial cells with characteristic morphology were maintained and successfully cultured for 28 days. TGFB inhibition showed superior maintenance of epithelial morphology compared with standard culture media. Gene expression and immunofluorescence analysis corroborated maintenance of lacrimal epithelial gene expression and reduced expression of mesenchymal markers.
Conclusion: TGFB inhibitor supplementation of human lacrimal epithelial cell culture prevented EMT in vitro and may be useful for the development of lacrimal epithelial cell lines.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Rounded Human Lacrimal Epithelial Cells Passage 3 with TGFB (10mM)
Human Epithelial Mesenchymal Transition Passage 3 without TGFB
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