In patients with unilateral total stem cell deﬁciency a limbal autograft transplantation is recommended. Partial removal of the limbus from the fellow eye is believed to be relatively safe, although some cases may have compromised donor surface after partial removal of the limbal zone. The risk of epithelial problems in the donor eye is low when less than four to six clock hours of limbal tissue and a moderate amount of conjunctiva are removed. When patients have bilateral total ocular surface dis- ease, allograft transplantation becomes necessary. If living relatives are potential donors, an HLA-matched tissue is preferred. When cadaver donor tissue is used, “fresh” eyes are preferred because the success of the procedure depends on the transplantation of healthy limbal stem cells. Whole eyes are convenient because they provide better stabilisation during dissection of the limbal sclerocorneal rim. In limbal allografts the surface disorder can recur if there is immunological destruction of the transplanted limbal stem cells. A high rate of immune reactions can be expected because of the high immunogenic stimulus of the limbal transplant, related to the relative abundance of Langerhans cells and HLA-DR antigens. They play an important role in the afferent arm of allograft rejection, and effective immu- nosuppression is considered essential for at least 12 months after surgery when non-HLA matched limbal allografts are used. In some instances permanent systemic immunosup- pression may be needed. Oral cyclosporine A is the most commonly used agent (Tseng et al 1995). In addition to oral cyclosporine, Tsubota et al also used topical cyclosporine (0.05%) and high dose intravenous dexamethasone in their patients (Tsubota et al 1995). The use of FK 506 (tacrolimus; Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan), a new immunosuppressive agent from the fermentation broth of Streptomyces tsukabaensis, for immunosuppression in limbal or corneal allografts has been recently reported by Dua and Blanco (1999). Limbal rejection can be suspected with the development of inﬂammation and/ or acute or chronic severe surface abnormalities. Daya and colleagues (1999) and Dua and Blanco (1999) have reported the clinical features of limbal allograft rejection. In their study, the clinical features of limbal allograft rejection varied according to the presenta- tion, whether acute or chronic, of the rejection episode. Acute rejection was associated with intense sector injection at the limbus, edema, and inﬁltration of the lenticule, punctate keratopathy and epithelial defects. In low-grade rejection there was mild diffuse or perilimbal injection, elevated per- ilimbal area, punctate epithelial keratopathy and epithelial irregularity (Daya et al 1999). Clinical Ophthalmology 2008:2(3) Cultivated limbal stem cell transplantation Cultivated limbal stem cell transplantation Kenyon and Tseng (1989) proposed the procedure of conjunctival limbal autograft taken from the healthy fellow eye in patients with unilateral total LSCD. The procedure involves the transplantation of two large free grafts, each spanning from 5–7 mm in limbal arc length, removed from a healthy eye (Kenyon and Tseng 1989). Reported complica- tions in donor eyes have included localized haze in a patient with contact lens-induced keratopathy, pseudopterygium, ﬁlamentary keratitis, microperforation during surgery, ab- normal epithelium, and corneal depression. Such a concern preludes one from obtaining two such grafts from a donor eye with undiagnosed partial LSCD. Ex vivo expansion of limbal epithelial stem cells has been developed to circumvent po- tential complications related to conjunctival limbal autograft transplantation. The aim of differentiating these epithelial stem cells ex vivo is to help develop an equivalent of the human cornea using tissue culture techniques. Transplanta- tion of cultivated limbal epithelium is currently the most successful alternative to surface reconstruction in patients with unilateral disease and offers a therapeutic chance to patients with severe bilateral disease (Pellegrini et al 1997; Koizumi et al 2001b; Sangwan et al 2006). The various protocols for cultivation of limbal epithe- lium differ in the use of intact versus epithelially denuded amniotic membrane, suspension of epithelial cells rather than explants, co-cultivation of 3T3 ﬁbroblast feeder layers, and air-lifting prior to transplantation. Currently no study has been conducted to compare these cultivation variables in order to determine which one is vital in achieving effec- tive expansion of limbal epithelial progenitor cells (Schwab 1999; Grueterich and Tseng 2002a; Grueterich et al 2002b, 2002c; Koizumi et al 2001a, 2002; Meller et al 2002; Espana et al 2003). Pellegrini and colleagues (1997) ﬁrst reported successful reconstruction of the ocular surface in LSCD. A 1 mm2 biopsy sample from the healthy fellow eye was treated with trypsin for 3 hrs. The cells were plated on lethally irradiated 3T3-J2 cells and cultured in 5% carbon dioxide in Dulbecco-Vogt Eagle’s and Ham’s F12 media. Grafts were prepared from conﬂuent secondary cultures which were released from the plastic dish by neutral protease Dispase II and either mounted on a petrolatum gauze or a soft contact lens (Pellegrini et al 1997). The next advancement in this technique was the use of human amniotic membrane as a substrate for in vitro epithelial cell culture. Koizumi and colleagues (2000a) ﬁrst cultivated 493