Limbal corneal epithelial stem cells (LSCs) are responsible for maintaining the corneal epithelial cell mass. These cells undergo proliferation, migration and differentiation under both normal conditions as well as following injury. They can be expanded ex-vivo for intracorneal autologous transplantation.
Superficial limbal tissue sections of 2×2 or 3×1 mm were obtained, under local anesthesia, from the contralateral healthy eye (for autografting) or from a cadaveric limbal donor (for allografting), 2 to 3 weeks before a planned cultivated corneal limbal epithelial cell transplantation. The limbal tissue was washed in sterile calcium- and magnesium-free phosphate-buffered saline. The explants were then treated with 2 IU/mL dispase at 37°C for 20 minutes.
The denuded amniotic membrane (AM) was carefully separated from its nitrocellulose membrane carrier and pulled down to adhere to the bottom of the insert disc from which the membrane was previously removed. The AM was obtained during elective cesarean sections with negative tests for infectious diseases and kept at −70°C before use. The limbal epithelium was seeded onto the prepared, denuded AM fixed on the culture insert disc, which was put on a sterile culture plate.
The culture was submerged in keratinocyte growth medium for 2 to 3 weeks and then exposed to air by lowering the level of the medium (air lifting) for 1 to 2 days. The cultures were incubated at 37°C in a 5% carbon dioxide and 95% air incubator, and the medium was changed every other day.