July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Effect of trypsin-EDTA on expression of DNA damage repair enzyme APE1 in human limbal epithelial cells.
Author Affiliations & Notes
  • Yolanda Lorenzo Corrales
    Center for Eye Research, Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
  • Bjørn Otto Nicolaissen
    Department of Ophthalmology, Vestre Viken Hospital Trust, Drammen, Norway
  • Giang Nguyen
    University Of Oslo, Oslo, Norway
  • Kahsai Beraki
    University Of Oslo, Oslo, Norway
  • Morten C. Moe
    Center for Eye Research, Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    University Of Oslo, Oslo, Norway
  • Goran Petrovski
    Center for Eye Research, Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    University Of Oslo, Oslo, Norway
  • Bjørn Nicolaissen
    Center for Eye Research, Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    University Of Oslo, Oslo, Norway
  • Footnotes
    Commercial Relationships   Yolanda Lorenzo Corrales, None; Bjørn Otto Nicolaissen, None; Giang Nguyen, None; Kahsai Beraki, None; Morten C. Moe, None; Goran Petrovski, None; Bjørn Nicolaissen, None
  • Footnotes
    Support  Oslo University Hospital, University of Oslo, The Blindmission IL
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 927. doi:
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      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.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : 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.

Methods : 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).

Results : 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.

Conclusions : 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.

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