Donor cornea transfer from Optisol GS to organ culture storage: a two‐step procedure to increase donor tissue lifespan

Abstract

PurposeStorage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC.MethodsMorphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay.ResultsA prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin+, while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased.ConclusionsThis dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders.

Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells

“…Although the culture conditions may contribute to some levels of DNA damage [29], the findings in previous experimental studies, and in the present, underline the need for a continuous critical examination of the effect of the various dissociation protocols on essential molecular constituents of the harvested cells.…”

“…Scant information is available about DNA damage in human corneal and limbal epithelium. In a recent study we found the levels of DNA strand breaks to be very low in corneo-limbal epithelial cells after storage in Optisol GS close to the upper recommended limit [29]. Transfer of such samples to culture was associated with proliferative activity and expression of markers characterizing differentiated as well as undifferentiated cells in the limbal epithelium, but also with an increase in the levels of DNA strand breaks.…”

Ocular Cell Lines and Genotoxicity Assessment

“…Haug et al stored the corneas in Optisol GS at 4°C prior to transplantation, collecting the remaining corneoscleral rims [63]. In this study, 10 rims were needed for the comet assay.…”

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

“…This protocol has been shown to support engineering of grafts with a high density of stem cells (Pellegrini et al 1997;Rama et al 2010;Yu et al 2016). Previous studies indicate that levels of DNA strand breaks and, in particular, oxidative damage to DNA purine bases may provide information regarding the quality of a culture system (Lorenzo et al 2009;Haug et al 2013;Osnes-Ringen et al 2013). Long-term renewal of the epithelium after transplant surgery depends critically on the presence of a pool of wellfunctioning stem cells.…”

“…The ex vivo systems represent a foreign microenvironment; wherein, oxidative reactions and other stressors may alter cell function temporarily or permanently by distortion of the integrity of cellular molecules, and microarray analysis could indicate that the levels of reactive oxygen species are higher in tissues generated in medium with human serum versus in the traditional complex medium with xenobiotics (Pathak et al 2016). Previous studies indicate that levels of DNA strand breaks and, in particular, oxidative damage to DNA purine bases may provide information regarding the quality of a culture system (Lorenzo et al 2009;Haug et al 2013;Osnes-Ringen et al 2013). In vivo, oxidative damage to DNA bases is generally efficiently repaired by the base excision repair pathway (BER) involving the action of a number of enzymes including APE1, OGG1 and Polb (Collins et al 2003).…”