Trabecular cell monolayer culture
Originally described in 1979 and more recently modified by Stamer et al primary trabecular monolayer cell culture has been a cornerstone for investigation of the molecular and biochemical functions in this distinct cell type. The trabecular meshwork accounts for most aqueous outflow resistance in the anterior chamber of the human eye.1 It is believed that the interplay between the endothelial-like cells of this tissue (trabecular cells) and the surrounding extracellular matrix are responsible for maintaining the resistance necessary for preservation of the aqueous outflow pathway. The trabecular cells are phagocytic cells, actively removing debris such as pigment granules, erythrocytes, and pseudoexfoliation material from the aqueous outflow system. They also are involved in the production and turnover of extracellular matrix.
1. The trabecular meshwork was dissected from human donor eyes and subjected to extracellular matrix digestion with solution of primary cell isolation.
2. The cells were pelleted and resuspended in primary cell culture system containing penicillin-streptomycin (100 U/mL) and 10% FBS (DMEM-FBS).
3. Cells were placed in a T25 flask and grown to confluence at 37°C in 5% CO2 in primary cell culture system (passages 3–7).
4. The confluence of cell cultures was determined by observation of cell density at 100× magnification with an inverted light microscope.
5. Cultures were considered confluent when cell expansion had reached a point where cells touched each other on all sides and no intercellular gaps were present.
6. Once cells appeared confluent, trabecular cells were maintained in primary cell culture system for an additional 3 to 7 days to ensure a confluent state.
7. After incubation in primary cell culture system, cells were washed twice with phosphate-buffered saline (PBS) and grown in primary cell culture system and 50% human aqueous for up to 21 days.
Morphologic appearance of primary trabecular cell cultures. Trabecular cells incubated for 21 days. Cells displayed a broad, flat appearance when compared with the spindle shape of cells grown. Magnification, ×200.
Reference
1. Michael P. Fautsch, Kyle G. Howell, Anne M. Vrabel, M. Cristine Charlesworth, David C. Muddiman and Douglas H. Johnson. Primary Trabecular Meshwork Cells Incubated in Human Aqueous Humor Differ from Cells Incubated in Serum Supplements. Invest. Ophthalmol. Vis. Sci. 2005: 46: 2848-2856.
2. Polansky JR, Weinreb RN, Baxter JD, Alvarado J. Human trabecular cells. I. Establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci. 1979; 18: 1043–1049.
3. Stamer WD, Seftor REB, Williams SK, Samaha HAM, Snyder RW. Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion. Curr Eye Res. 1995;14:611–617.
