The AM stroma also contains a number of growth factors, various antiangiogenic and anti-inﬂammatory proteins, and natural inhibitors to various proteases (Sato et al 1998). In view of all of the above-mentioned properties of the AM, there is little doubt that it provides an ideal stromal niche desirable for stem cell expansion. Limbal cell culture: Explant culture tech- nique Many investigators have adopted the explant culture tech- nique with variations in substrate, media, feeder cells, and airlifting (Koizumi et al 2000c, 2001b; Tsai et al 2000; Grueterich et al 2002c; Shimazaki et al 2002). To elaborately describe the details of each protocol is beyond the scope of this article. Therefore the modiﬁcations that the authors have incorporated in their procedure have been described with respect to reports from previous investigators. Limbal biopsy After satisfying the Institutional Review Board, informed consent is obtained from the patients or guardians. Limbal biopsy is performed on the healthy contralateral eye or a healthy area of the ipsilateral eye. The procedure includes careful dissection of a 1 × 2 mm2 piece of limbal epithelium with 1 mm into clear corneal stromal tissue at the limbus under strict aseptic conditions. A conjunctival peritomy is made 2–3 mm from the limbus and dissection of the con- junctiva is carried forwards 1 mm beyond the limbal arcade. The harvested tissue should exclude the Tenon’s capsule and should include the palisades of Vogt. Post-operatively topi- cal antibiotic is used four times day for two weeks. Topical steroids are used in tapering doses for 4 to 6 weeks or until complete healing of donor site. AMG procurement, processing, and preservation Amniotic membrane is obtained from prospective donors undergoing caesarean section, who are negative for com- municable diseases including HIV, hepatitis, and syphilis. Different protocols exist for the processing and storage (Kim and Tseng 1995; Shimazaki et al 1997). According to Kim and Tseng (1995), the amnion is separated from the chorion by blunt dissection, and washed with balanced salt solution containing a cocktail of antibiotics (50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml of neomycin, as well as 2.5 μg/ml of amphotericin B) under sterile conditions. The separated membranes are cut in different sizes (3 × 4 mm2) and placed on nitrocellulose paper strips with the epithelial Clinical Ophthalmology 2008:2(3) Cultivated limbal stem cell transplantation side up. Dulbecco Modiﬁed Eagles’ Medium/glycerol (1:1) is used for cryopreservation and the tissues are frozen at −80 °C degrees until further use (Kruse et al 2000). Just before use, the amniotic membrane is thawed at 37 °C for 30 min. and placed on a sterile cut glass slide. The amniotic epithelium is removed by digestion with 0.1% trypsin-EDTA at 37 °C, followed by scraping. Under sterile conditions, the membrane is inspected under the microscope for complete removal of cells. The de-epithelized membranes are then spread on a glass slide (used as culture inserts) in a Petri plate tucking the edges for a uniform surface (Koizumi et al 2000c, 2001b; Sangwan et al 2006; Shimazaki et al 2002). Tsai and colleagues (2000) and Grueterich and colleagues (2002c) have cultivated explants on intact AM. Human corneal epithelial cell growth medium We use a modiﬁed human corneal epithelial cell (HCE) cul- ture medium, prepared using 9.7 g/l Modiﬁed Eagle Medium (MEM) with addition of 16.2 g/l Ham F12 serum, 0.01mg/l epidermal growth factor, 0.25 mg/l insulin, 0.1 mg/l cholera toxin, and hydrocortisone. The medium is ﬁltered with 0.22 mm membrane ﬁlters using a vacuum pump. This is supplemented with autologous serum or 10% fetal calf serum (FCS) at the time of use. Explant culture technique The authors follow a submerged explant culture system without the use of any feeder cell layer or airlifting (Tsai et al 2000; Grueterich et al 2002c; Shimazaki et al 2002; Sangwan et al 2006). Koizumi and colleagues (2000c, 2001b) have used 3T3 feeder cells along with airlifting for epithelial stratiﬁcation. Limbal tissue procured by biopsy is shredded into 4–6 fragments, and placed on the de-epithelized mem- branes separately and allowed to settle down by overnight incubation at 37 °C with 5% CO2 and 95% air (Figure 4). The medium is changed on alternate days for 10–14 days with daily monitoring of cell growth under phase-contrast microscope. In four days polygonal cells could be seen grow- ing from the edges of the explant. Whole mount preparation After conﬁrming conﬂuent growth of a monolayer from the explanted tissue over 1–2 weeks, the growth is terminated by replacing the medium with 10% buffered formalin. The whole mount preparation, stained with hematoxylin and eosin, could be seen as bluish rings of stained areas around 495