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Exp Eye Res. 2019 Dec;189:107814. doi: 10.1016/j.exer.2019.107814. Epub 2019 Sep 24.

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

Author information

1
Institute of Ophthalmology, University College London, London, UK; International Center for Ocular Physiopathology, The Veneto Eye Bank Foundation, Venice, Italy. Electronic address: m.parekh@ucl.ac.uk.
2
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore; Duke-NUS Graduate Medical School, Singapore.
3
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore; Duke-NUS Graduate Medical School, Singapore; Singapore National Eye Centre, Singapore; School of Material Science and Engineering and School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore.
4
Institute of Ophthalmology, University College London, London, UK.
5
International Center for Ocular Physiopathology, The Veneto Eye Bank Foundation, Venice, Italy.
6
Institute of Ophthalmology, University College London, London, UK; Moorfields Eye Hospital NHS Foundation Trust, London, UK.

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.

KEYWORDS:

Adherence; Cornea; Corneal endothelial cells; Old donor; Passage; Primary cell culture

PMID:
31560924
DOI:
10.1016/j.exer.2019.107814
[Indexed for MEDLINE]

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