Trabecular meshwork-derived mesenchymal stem cells were isolated from anterior eye segments. Cells express mesenchymal markers and have osteogenic, chondrogenic, and adipogenic differentiation capacity.
Corneoscleral rims were isolated from anterior eye segments, rinsed in wash buffer (50% phosphate-buffered saline (PBS)) supplemented with calcium- and magnesium-containing Opti-MEM 1(25 %), human endothelial-SFM (serum-free medium, 25%), gentamycin reagent solution (25 mg/ml), penicillin–streptomycin (X1), and amphotericin B (X1). To visualize the trabecular meshwork (TM) tissue, the rims were stained with trypan blue (0.4 %), and then washed with PBS. The TM tissue was isolated under a stereomicroscope, and was digested overnight in type 1 collagenase (2 mg/ml) dissolved in Dulbecco’s Modiﬁed Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS, 10%). After a second digestion step with accutase or trypsin (0.05%), the cells were transferred to uncoated tissue culture plates and cultured in low-glucose DMEM supplemented with FBS (10%), Glutamax (4 mM), sodium pyruvate (1 mM), nonessential amino acids (1 %), and penicillin–streptomycin (1%).