Limbal epithelial stem cells obtained from patients (autologous treatment) or from donors (allogeneic), can be cultured ex-vivo on human amniotic membrane with no feeders.
Full-thickness limbal explants were prepared under a laminar flow. Descemet’s membrane was removed using a scalpel and full-thickness limbal rims were isolated by removing the sclera with scissors. Superficial limbal explants were processed in the operating room under an operating microscope.
Limbal explants were cultured in cholera toxin-free Green medium. The medium was composed of a 3:1 mixture of calcium-free DMEM and Ham-F12 medium, supplemented with fetal bovine serum (10%), HEPES buffer (1 mM/ml), human recombinant insulin (5 μg/ml), hydrocortisone (0.4 μg/ml), L-glutamine (4 μM/ml), tri-iodothyronine (2 pM/ml), adenine (200 nM/ml), penicillin (100 IU/ml), streptomycin (100 μg/ml), amphotericin B (0.25 μg/ml) and human recombinant epithelial growth factor (10 ng/ml). The limbal explants were sutured onto a plastic lamella (one explant per lamella) with the epithelial side up and were cultured in 6-well or 24-well plates with 2 ml of medium.
Single-cell suspensions were cultured as follows: the whole limbal rim was incubated with 1.2 U/ml dispase II at 37 °C for 1 h. The epithelial sheets were then collected and treated with 0.125% trypsin at 37 °C for 15 min to isolate single cells.
No feeders were used to grow cells. The medium was changed three times a week and cells were cultured for three weeks at 37 °C with 5% CO2.