DNA damage in Human Limbal Epithelial Cells expanded ex vivo

Limbal stem cell deficiency, secondary to insults and diseases, may be treated by transplantation of ex vivo engineered epithelial grafts. We here present preliminary data on levels of cellular DNA damage in grafts produced in two different types of culture medium. Cultures were initiated using corneo-limbal donor tissue after removal of the central area for transplant purposes. Explants (approx. 2x2 mm) were positioned epithelial side down on tissue culture treated polyester membranes and expanded for four weeks in Dulbecco’s Modified Eagle Medium F12 Nutrient Mixture (Ham) [DMEM/F12 (1:1)] with either (1) H. medium; 10% human serum or (2) COM; 5% fetal bovine serum (FBS), Epidermal Growth Factor (EGF), insulin-transferrin-sodiumselenzine (ITS) , cholera toxin-A, dimethyl sulfoxide (DMSO) and hydrocortisone. Cells were dissociated using Trypsin-EDTA (0.05%) for 30 min., the enzyme activity was inhibited by medium and serum. The cell suspension was transferred to tubes on ice and processed using the Comet Assay. Duplicate samples from each culture were analyzed in each assay by visual scoring. Using a fluorescence microscope, 100 comets (50 from each gel) were classified into five categories, 0-4, representing increasing relative tail intensities. Summing the scores (0-4) of 100 comets therefore gives an overall score of between 0 and 400 arbitrary units. Preliminary data show some levels of DNA damage in cells dissociated from the grafts regardless of the type of culture medium used. Anyway more experiments with other donors have to be done to have some conclusions. Recent studies have shown that medium with human serum equally support production of grafts containing differentiated as well as undifferentiated cells suitable for clinical transplantation. Preliminary data from our experiments indicate that levels of molecular damage to the DNA do not increase in cells cultured in H. medium despite its lacks of complexity.