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Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment

Parekh, M; Peh, G; Mehta, JS; Ramos, T; Ponzin, D; Ahmad, S; Ferrari, S; (2019) Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment. Experimental Eye Research , 189 , Article 107814. 10.1016/j.exer.2019.107814. Green open access

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Abstract

In a recent report, we showed that it is possible to establish the culture of Human Corneal Endothelial Cells (HCEnCs) from older donor corneas (usually over 65 year olds) when left to attach in the presence of a viscoelastic solution, potentially increasing the donor pool for culturing HCEnCs. Therefore, we set out to evaluate the outcome of using a viscoelastic solution (Viscoat) to accelerate the attachment of passaged cultured human corneal endothelial cells (HCEnCs). The cells from 28 donor tissues were isolated using peel-and-digest method and evenly seeded into two wells of an 8-well chamber slide. The cells were left to attach after topical application of Viscoat. At confluence, one well was subjected to end-stage characterization, whereas the other well was passaged into another two wells. The cells at P1 were attached with and without the use of Viscoat. The growth rate was monitored; and at confluence, morphometric analysis, corneal endothelial specific (CD166-Tag1A3 & PRDX6-Tag2A12), mitochondrial and respiration assessment (Tom-20 and Seahorse); function-associated (Na+/K+ATPase & ZO-1); proliferative (Ki-67) marker analysis, and viability (Hoechst, Ethidium Homodimer and Calcein AM-HEC) studies were performed. Cells at P0 (with Viscoat) showed 100% confluence at day 9. Cells at P1 with and without Viscoat showed significant difference of confluence 67.0% v 18.8% respectively (p < 0.05). Confluence rate, cell density, hexagonality, Ki-67 positivity and mitochondrial intensity was significantly higher (p < 0.05), whereas cell-area and polymorphism was significantly lower (p < 0.05) in the cells attached with Viscoat compared with the cells attached without Viscoat. There was no significant difference in oxygen consumption rate between the groups. In conclusion, we observed that acceleration in the attachment of passaged HCEnCs with the assistance of Viscoat, could be beneficial for the propagation of HCEnCs isolated from older donors, to increase their propensity to proliferate, without loss of the expression of vital proteins and heterogeneity in cellular morphology.

Type: Article
Title: Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.exer.2019.107814
Publisher version: https://doi.org/10.1016/j.exer.2019.107814
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Adherence, Cornea, Corneal endothelial cells, Old donor, Passage, Primary cell culture
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
URI: https://discovery.ucl.ac.uk/id/eprint/10086819
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