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Purpose  To examine levels of oxidative  DNA  base damage and expression of selected genes and proteins related to  DNA  damage repair in human limbal epithelium engineered  ex vivo .    Methods  Cells were expanded from limbal tissue on cell culture‐treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin ( COM ) and in medium with human serum as the single growth‐promoting additive ( HS ). Cells were analysed after two, three and four weeks in culture for  DNA  strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine  DNA  glycosylase, Fpg) and for expression of  DNA  repair enzymes  APE 1,  OGG 1 and Pol β  by  in situ  hybridization ( ISH ) and by immunohistochemistry ( IHC ).    Results  Levels of strand breaks were substantial while levels of net Fpg‐sensitive sites (8‐oxoguanine and ring‐opened FaPy bases) were relatively low in cells engineered in  COM  and in  HS . Both types of medium were found to support expression of base excision repair ( BER ) enzymes  APE 1,  OGG 1 and Pol β  at the gene level. At the protein level, expression of  APE 1 and  OGG 1 was noticeable in both conditions while expression of Pol β  was low.    Conclusion  Our findings indicate low levels of oxidative stress and/or efficient  DNA  purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth‐promoting additive as well as in traditional medium with xenobiotics.

Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue