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Limbal stem cells (LSCs) are undifferentiated, slow-cycling cells that self-renew and produce transient amplifying cells which migrate to the corneal epithelium. LSCs ensure continuous self-renewal of the corneal epithelium, and are also responsible for epithelial tissue repair and regeneration throughout the life of the adult cornea.
Limbal stem cells were obtained by excising a small area of the conjunctiva at the limbus. The limbal tissue was exposed, for 5–10 min, to Dispase II (1.2 U/mL in Mg2+ and Ca2+-free HBSS) at 37°C in a humidified environment containing 5% CO2. The tissue was rinsed with DMEM/F12 containing FBS (10%) and cut into cubes of approximately 1-2 mm2. The limbal epithelial cells were cultured on the basement membrane side of denuded human amniotic membranes (AMs) and cultured in DMEM/Hams F-12 (1:1) supplemented with FBS (5%), DMSO (0.5%), hEGF (2 ng/mL), insulin (5 μg/mL), transferrin (5 μg/mL), selenium (5 ng/mL), hydrocortisone (0.5 μg/mL), cholera toxin (30 ng/mL), gentamicin (50 μg/mL), and amphotericin B (1.25 μg/mL). Cultures were incubated in a humidified incubator with 5% CO2. The cultures were maintained for 3 weeks and the medium was replaced every 2 days.