Burman and Sangwan Figure 4 Schematic diagram showing the steps in cultivation of limbal epithelial stem cells. A: Technique of limbal biopsy (See text for details). B: Processing of tissue in the laboratory and making the explant culture. C: Petridish, glass slide (white) with de-epithelialized human amniotic membrane with explants (red dots) with growing cells around it (blue dots). D: Monolayer of cells (10–14 days old) under phase contrast microscope. the original explanted tissue. When observed under the mi- croscope, the cultured cells appear as a monolayer of large polygonal cells with an epithelial appearance. The membrane with the cultured limbal tissue and the cultured cells is ﬁxed in formalin and processed for routine histopathology with parafﬁn embedding. The sections are cut at 4–5 cm and after deparafﬁnization, stained with hematoxylin and eosin stain and periodic acid-Schiffs stain. In contrast to the cuboidal epi- thelium of the normal amniotic membrane the cultured cells form an epithelium of 1–2 layered cells over the amniotic membrane. Immunostaining on the formalin-ﬁxed, parafﬁn- embedded sections can be done using prediluted antibodies to cytokeratin 3 (K3) and cytokeratin 19 (K19) from DAKO (Copenhagen, Denmark) to conﬁrm the corneal phenotype of the cultured cells (Figure 5) (Vemuganti et al 2004). Cell suspension culture of limbal epithelial cells Preparation of AM and 3T3 ﬁbroblast cells As described in detail in previous reports, AMs obtained at the time of caesarean section were washed with sterile phosphate-buffered solution (PBS) containing antibiotics and stored at –80 °C in Dulbecco’s modiﬁed Eagle medium and glycerol at a ratio of 1:1. Immediately before use the 496 Clinical Ophthalmology 2008:2(3)