Purchase this article with an account.
Yolanda Lorenzo Corrales, Bjørn Otto Nicolaissen, Giang Nguyen, Kahsai Beraki, Morten C. Moe, Goran Petrovski, Bjørn Nicolaissen; Effect of trypsin-EDTA on expression of DNA damage repair enzyme APE1 in human limbal epithelial cells.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):927.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Enzymatic cell dissociation may provide cell-cultures for experimental purposes as well as for tissue engineering. We hereby examine limbal morphology and expression of the DNA base-excision repair (BER) enzyme APE1 during incubation in trypsin-EDTA, an agent in common use for cell dissociation.
Experiments were conducted in accordance with the Declaration of Helsinki and Local Committees for Medical Research Ethics. Post mortem samples of human limbus were incubated in trypsin-EDTA for 0 (control), 1 or 3 hours, fixed, embedded, sectioned and stained with H&E or processed for immuno-histochemistry (IHC) for APE1 detection. Control samples were examined for 8-OHdG (marker for oxidative DNA-base damage) and for gene expression of APE1 by in situ hybridization (ISH).
Limbal epithelium in control samples was regularly bi- or multilayered and cells were moderately positive for 8-OHdG, while slight expression of APE1 could be detected by ISH. Incubation in trypsin-EDTA for 1 hour induced slit-like spaces between epithelial cell layers and promoted dissociation of epithelium into single cells and clusters of cells. These patterns were still recognizable in samples after 3 hours of incubation. Semi-quantitative evaluation of APE1 by IHC showed no detectable alteration in its expression during incubation in trypsin-EDTA.
Damage to the DNA may, when unrepaired, interfere with cell function, differentiation, proliferation and viability. Dissociation of cells by trypsin-EDTA may induce molecular stress and damage. In vivo, oxidized DNA bases are normally repaired by BER enzymes including APE1. Noticeable changes in the protein levels of this particular repair enzyme could not be observed during incubation in trypsin-EDTA in the present study.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only