Culture, immortalization, and characterization of human meibomian gland epithelial cells

• Published in: Investigative ophthalmology & visual science Vol. 51; no. 8; pp. 3993 - 4005
• Main Authors: Liu, Shaohui, Hatton, Mark P, Khandelwal, Payal, Sullivan, David A
• Format: Journal Article
• Language: English
• Published: United States Association for Research in Vision and Ophthalmology, Inc 01.08.2010
• Subjects: Adult; Aged; Aged, 80 and over; Antigens, Polyomavirus Transforming - genetics; Antigens, Polyomavirus Transforming - metabolism; Cell Culture Techniques; Cell Division; Cell Line; Cell Proliferation; Epithelial Cells - cytology; Epithelial Cells - metabolism; Female; Fluorescent Antibody Technique, Indirect; Genetic Vectors; Humans; Karyotyping; Lipid Metabolism; Male; Meibomian Glands - cytology; Meibomian Glands - metabolism; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; Telomerase - genetics; Telomerase - metabolism
• DOI: 10.1167/iovs.09-5108
• ISSN: 1552-5783; 0146-0404

Meibomian gland epithelial cells are essential in maintaining the health and integrity of the ocular surface. However, very little is known about their physiological regulation. In this study, the cellular control mechanisms were explored, first to establish a defined culture system for the maintenance of primary epithelial cells from human meibomian glands and, second, to immortalize these cells, thereby developing a preclinical model that could be used to identify factors that regulate cell activity. Human meibomian glands were removed from lid segments after surgery, enzymatically digested, and dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, the cells were exposed to retroviral human telomerase reverse transcriptase (hTERT) and/or SV40 large T antigen cDNA vectors, and antibiotic-resistant cells were selected, expanded, and subcultured. Analyses for possible biomarkers, cell proliferation and differentiation, lipid-related enzyme gene expression, and the cellular response to androgen were performed with biochemical, histologic, and molecular biological techniques. It was possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free medium. These cells proliferated, survived through at least the fifth passage, and contained neutral lipids. Infection with hTERT immortalized these cells, which accumulated neutral lipids during differentiation, expressed multiple genes for lipogenic enzymes, responded to androgen, and continued to proliferate. The results show that human meibomian gland epithelial cells may be isolated, cultured, and immortalized.