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S. Liu, M. Hatton, D. A. Sullivan; Isolation, Culture and Immortalization of Human Meibomian Gland Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):88.
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We have discovered that sex and sex steroid hormones exert a significant influence on meibomian gland, which is a critically important tissue for promoting the health and integrity of the ocular surface. Our studies also indicate that meibomian gland epithelial cells are the primary target for sex steroid action. To help understand the mechanisms underlying this hormonal control, we sought to: [a] establish a defined culture system for the maintenance of responsive epithelial cells from human meibomian glands; and [b] immortalize these cells, with the goal of developing cell cultures that may be passaged multiple times in serum-free conditions and respond to stimulatory treatment with increased proliferation and lipogenesis.
Human meibomian glands were removed from lid segments after surgery, enzymatically digested and then dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, cells were exposed to retroviral pBABE-puro-hTERT and/or pBABE-neo-large T cDNA vectors and antibiotic resistant cells were selected, expanded and subcultured. Cell proliferation and differentiation were evaluated by using 5-bromo-2-deoxyuridine and Oil red O.
Our studies demonstrate that: [a] it is possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free media with or without serum or feeder layers. These cells proliferate, survive through at least a 5th passage and contain neutral lipids; and [b] infection with hTERT has generated epithelial cells with a normal karyotype that have continued to proliferate in serum-free media for the past 6.5 months, are currently at passage 35, and appear to accumulate neutral lipids during serum-induced differentiation.
Our results show that human meibomian gland epithelial cells may be isolated and cultured. In addition, our data indicate that these cells may be immortalized by retroviral exposure and maintained in a proliferative state in serum-free conditions.
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