Premium
DNA damage in human llmbal epithelial cells expanded ex vivo
Author(s) -
Lorenzo Corrales Y.,
Haug Berg K.,
Noer A.,
Collins A.R.,
Nicolaissen B.
Publication year - 2016
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2016.0576
Subject(s) - ex vivo , in vivo , comet assay , transplantation , dna damage , microbiology and biotechnology , cell culture , in vitro , chemistry , explant culture , pathology , biology , dna , andrology , biochemistry , medicine , surgery , genetics
Purpose 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. Methods Cultures were initiated using corneo‐limbal donor tissue after removal of the central area for transplant purposes. Explants (approx. 2 × 2 mm) were positioned epithelial side down on tissue culture treated polyester membranes and expanded for 4 weeks in complex medium or in medium with human serum. Cells were dissociated using Trypsin‐EDTA (0.05%) for 30 min, the enzyme activity was further 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. Results 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. 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 human medium despite its lacks of complexity.