Limbal stem cells (Singapore National Eye Centre)

Limbal biopsy was performed on the normal contralateral eye and a piece of limbal tissue (1mm x 2 mm) was excised from the superficial corneal stroma by lamellar keratectomy. The tissue was placed in a 35 mm dish containing 1.5 ml of supplemental hormonal epithelial medium in 5% fetal-calf serum. Medium consisted of DMEM:F12, supplemented with DMSO (0.5%), mouse epidermal growth factor (2 μg/ml), bovine insulin (1 μg/ml) and  cholera toxin (0.1 μg/ml). The medium was changed every 2 days and culture was maintained for 2-3 weeks, until epithelial cells had grown to form a layer of 2-3 cm in diameter.