PMC4588357_01
Limbal biopsy, limbal epithelial cultivation, and cultivated limbal epithelial transplantation (CLET). (a) Healthy donor eye 24 hr. after a 2 x 2 mm limbal biopsy; (b) the biopsy tissue was placed in an Eppendorf tube with culture medium (b1); (c) the biopsy was processed in a good manufacturing practice-cell processing unit within the next 4 hr. and for the next 4-5 weeks; and (d) the explant was placed on denuded human amniotic membrane, as viewed by contrast phase microscopy. Limbal epithelial cells began outgrowth from the explant at 1-2 weeks (d1). The explant was then removed, and the outgrowth was maintained until reaching confluence at which time it contained approximately 250,000 cells. The cell product was then sent to the medical center for CLET (d2). (e) Superficial keratectomy in the diseased contralateral eye (Case 1); (e1) human amniotic membrane with epithelial limbal cells confluent on top is removed from culture dish; (e2) the complex of amniotic membrane-limbal stem cells is placed on top of the previously denuded corneal and sclerolimbal surface; the amniotic membrane limit is observed (black arrow) with cells facing down and sutured (white arrows). A scleral lens is then applied.
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Allogeneic limbal rings were obtained within 7 days of death from corneal donors who were under 60 years old. Autologous limbal biopsies were marked by the surgeon with a stitch in the upper right corner to ensure consistency when plating the explant to ensure the same way up; they were sent from the operating room to the cell production unit at 4-10 C in culture medium (Figure 1). Limbal tissues were processed during the 4 hr. following arrival (Figure 1). When cultivated cells were ready for CLET, the final cellular products were sent to the medical institution within 4 hr. of the surgery (Figure 1). Biopsies measuring 2 x 2 mm were obtained from regions of the corneoscleral limbus (Figure 1) where the palisades of Vogt were better defined by SLE and by IVCM at the initial visit. The graft was then sutured to the perilimbal episclera, 2-4 mm posterior to the limbus, with 8 interrupted 10-0 nylon stitches (Figure 1).