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Yolanda Lorenzo Corrales, Giang Nguyen, Morten C Moe, Goran Petrovski, Bjørn Nicolaissen; Ex vivo engineered human corneo-limbal epithelium express DNA damage-repair enzymes.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3867.
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© ARVO (1962-2015); The Authors (2016-present)
Human limbal epithelial cultures for engineering of transplantable tissue may be initiated by different protocols. By explant culture techniques, tissues are positioned directly onto a substrate. By suspension culture techniques, cells are dissociated in enzyme solutions and seeded in cultures. In studies, DNA strand breaks may be observed in dissociated cells using trypsin-edta. We here examine limbal samples subjected to enzymatic cell dissociation and further expression of central DNA repair enzymes in cultures initiated by these techniques.
Human tissue was obtained after penetrating keratoplasty. Experiments were conducted in accordance with the Declaration of Helsinki, all tissue harvesting was approved by the Local Committees for Medical Research Ethics. Tissues were divided into samples and were either placed directly in culture dishes or subjected to incubation in trypsin-edta (0.05%) for 1 or 3 hrs and dissociated cells were subsequently seeded into dishes. Samples of enzyme digested tissues and controls were embedded, and sections examined by light microscopy. Culture medium was DMEM/F12 supplemented with EGF, ITS, hydrocortisone, cholera toxin A, DMSO, Penicillin/Streptomycin, Amphotericin B and 5% FBS. Cultures were monitored by phase contrast microscopy. At three weeks, cells were assayed for expression of OGG1 and APE1 by immunocytochemistry (ICC).
Areas with high cell densities were observed in cultures expanded for 2 and 3 weeks regardless of procedure for initiation of the cultures. In samples subjected to cell dissociation, incubation in trypsin-edta for 1 and 3 hrs regularly detached all of the corneal epithelial cells and most of the cells in limbal areas. Using ICC, expression of APE1 and of OGG1 was detected in cells in cultures expanded for 3 weeks, regardless of procedure for initiation.
For patients with limbal stem cell deficiency, developments within the field of tissue engineering have provided new treatment options and protocols for engineering of tissue, which differ among clinics. By the explant culture technique, a step involving enzymatic cell dissociation with potential DNA damage is omitted. Our findings show that cultures with high cell density and where cells express central DNA repair enzymes may be generated by both techniques in combination with the traditional medium designed for ex vivo production of epithelial tissue.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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