Limbal stem cells (Antwerp University Hospital)

Biopsy: The limbal biopsy is taken from either the superior or inferior limbus, since these anatomic sites offer some protection by the eyelid and have been shown to contain a great number of limbal epithelial crypts. The edges of the biopsy are marked 24 mm along the length of the limbus, and tunneled to a depth of approximately 100 mm.  The limbal biopsy is lifted off and immediately placed in corneal epithelial medium (CnT-20). 

Cultivation: A fixed amniotic membrane (AM) is placed in a 55 mm culture dish, containing 3.5 mL CnT-20, supplemented with human serum (1%) and placed at 37°C with 5% CO₂ for 48 h.  Before the limbal biopsy is placed in culture, it is rinsed six times in CnT-20 medium containing antibiotics and then placed at the center of the fixed AM, epithelial side faced down. The biopsy is cultured at the air-liquid interface, placing 100 μL of medium on the fixed AM and 3.5 mL in the culture dish, until an outgrowth of approximately 6mm in diameter is observed. The culture is then shifted to a semisubmerged state, by increasing the volume of the medium on the fixed AM to 500 μL. These conditions are maintained for the duration of the culture period, with the medium changed every 2 to 3 days. When an outgrowth of about 12 mm is obtained (usually around days 11 to 15), the cell-AM composite is ready for transplantation.