Figure 5 Photomicrograph of cultivated limbal epithelial cells stained for keratin 3 and 12 (A) showing the corneal epithelial phenotype and basement membrane (green) secreted by the growing cells (B). membrane was thawed, washed 3 times with sterile PBS, and cut into pieces of approximately 2.5 cm2 and denuded. Conﬂuent 3T3 ﬁbroblasts were inactivated by incubation in 4 μg/ml mitomycin C (MMC) for 2 hours at 37 °C under 5% CO2, and then trypsinized and plated onto plastic dishes (Pellegrini et al 1997; Schwab 1999; Koizumi et al 2000a; Schwab et al 2000). Cell suspension The whole limbal ring was cut into two to three peices, and these were incubated at 37 °C for one hour with 1.2 IU dispase. The corneal limbal epithelium including the limbal Clinical Ophthalmology 2008:2(3) Cultivated limbal stem cell transplantation stem cells were suspended in 3 ml medium, seeded onto three pieces of denuded AM spread on the bottom of the culture inserts, and co-cultured with MMC-inactivated 3T3 ﬁbroblasts. The culture was submerged in the medium for 2 weeks and then exposed to air by lowering the medium level (airlifting) for 2 weeks to promote corneal epithelial differentiation. The culture medium used was Dulbecco’s modiﬁed Eagle’s medium and Ham’s F12 (1:1 mixture) (Koizumi et al 2002). Surgical technique The surgery is performed 10–14 days after the limbal biopsy. Following strict aseptic precautions, under local or general anesthesia, a drop of 1:1000 epinephrine is instilled into the conjunctival cul de sac to ensure hemostasis. The ﬁbrovas- cular pannus is dissected off the corneal surface starting 2–3 mm beyond the limbus using a conjunctival spring scissors until the limbus and beyond. A plane of dissection is usually noted in patients who have undergone previous ocular surface reconstruction with AM, facilitating the excision. Symblepharon release is carried out at the same time and AM is used to reconstruct the ocular surface. Fornix recon- struction is also performed where appropriate. The AM, with its monolayer of cultured limbal epithelial cells, is then transferred to the ocular surface and anchored in place at the limbus with interrupted 10–0 monoﬁlament ny- lon sutures placed circumferentially. The knots are trimmed and buried. The peripheral skirt of the AM is anchored to the conjunctiva with 8–0 vicryl sutures. Subconjunctival dexamethasone is given at the end of surgery. Some authors recommend mitomycin C subconjunctivally to prevent re- currence of symblepharon and a bandage contact lens may be inserted. In some patients with chemical injury, corneal scarring, and who may have undergone prior attempts at surface reconstruction, difﬁculties may be encountered in suturing the donor corneal tissue to the thin recipient bed. In such cases, cultured LSC transplantation may be combined with either a lamellar keratoplasty or deep anterior lamellar keratoplasty. Preoperative anterior segment interferometry or intraoperative pachymetry after pannus resection may indicate the residual stromal thickness and aid the decision for lamellar keratoplasty (LK) or deep anterior lamellar keratoplasty (DALK). In cases with anterior to mid stromal scarring and thin- ning, a lamellar keratoplasty may be done using a crescent blade or an automated keratome. The lamellar donor cor- neal tissue is sutured to the recipient bed with interrupted 497