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SM Richards, F Schirra, DA Sullivan; Development Of A Defined, Serum-Free Culture System For The Maintenance Of Epithelial Cells From The Mouse Meibomian Gland . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3150.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our research has demonstrated that androgens regulate the meibomian gland, which is the primary tissue involved in promoting tear film stability and preventing tear film evaporation. Our studies also indicate that epithelial cells are the target for androgen action. To begin to explore the mechanisms underlying this hormonal control, we sought to establish a defined culture system for the maintenance of epithelial cells from the mouse meibomian gland Methods: Lid segments containing meibomian glands were removed from young adult BALB/c mice and dissociated by exposure to dispase II and collagenase. Meibomian glands were then isolated and digested in EDTA and trypsin. Following trypsin inactivation, glandular cells were dispersed by pipetting, passed through Nytex mesh, pelleted, resuspended in calcium- and phenol red-free Medium 154 (containing 11 supplements) and cultured on collagen type 1. After experimentation, cells were processed for the analysis of selected mRNAs by real-time PCR Results: Our results demonstrate that: [a] the isolation procedures yield over 60,000 viable meibomian gland cells/mouse; [b] maintenance of cells in culture is enhanced (and/or dependent) upon the inclusion of defined concentrations of insulin, transferrin, epidermal growth factor, keratinocyte growth factor, endothelial cell growth supplement, cholera toxin, hydrocortisone, gentamicin, amphotericin B, bovine serum albumin and calcium; [c] cells proliferate and become 40-60% confluent approximately 2 weeks after initial culture, and following the 1st passage reach ∼90% confluence in 4-5 days. To date, cells have been maintained through a 3rd passage; and [d] 95% confluent cells contain the mRNAs for fatty acid synthase (key enzyme in epithelial cell lipid metabolism), 17beta-hydroxysteroid dehydrogenase 7 (an essential enzyme involved in sex steroid synthesis) and sterol regulatory element binding proteins 1 & 2 (transcription factors that regulate lipid synthesis). Conclusion: Our studies show that it is possible to isolate and culture cells from the mouse meibomian gland. Supported by NIH grant EY05612, Allergan and the German Research Society DFG
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