Technique of cultivating limbal derived corneal epithelium on human amniotic membrane for clinical transplantation

Background : The technique of transplantation of cultivated limbal epithelium rather than direct limbal tissue isa novel method of "cell therapy" involved in reconstructing the ocular surface in severe limbal stem celldeficiency [LSCD], caused by chemical burns. Aim : To describe a simple feeder-cell free technique of cultivating limbal epithelium on human amniotic membrane[HAM]. Materials and Methods : The limbal tissues (2 mm) were harvested from patients with LSCD. These tissueswere proliferated in vitro on HAM supplemented by human corneal epithelial cell medium and autologousserum. Cultures covering more ≥50% area of 2.5x5 cm HAM were considered adequate for clinical use. Thecultured epithelium was characterized by histopathology and immunophenotyping.Results: A total of 542 cultures out of 250 limbal tissues were cultivated in the laboratory from January 2001through July 2005. The culture explants showed that clusters of cells emerging from the edge of the explantsin one-three days formed a complete monolayer within 10-14 days. In 86% of cultures (464 of 542), thegrowth was observed within one-two days. Successful explant cultures were observed in 98.5% (534 of 542cultures) with 91% explant cultures showing an area of ≥6.25 cm2 (6.25 - 12.5 cm2 range). The cultivatedepithelium was terminated between 10-14 days for clinical transplantation. The problems encountered wereinadequate growth (2 of 542) and contamination (2 of 542). Conclusions : We demonstrate a simple technique of generating a sheet of corneal epithelium from a limbalbiopsy. This new technique could pave the way for a novel form of cell therapy.