Cultivation and characterisation of human peripheral cornea derived endothelial cells.pdf (93.75 kB)

Cultivation and characterisation of human peripheral cornea-derived endothelial cells [abstract]

Download (93.75 kB)
journal contribution
posted on 06.10.2017, 09:14 by Reka Albert, Lana A. Faraj, Aaron Yeung, Matthew J. Branch, Laura E. Sidney, Sammy Wilson, Owen D. McIntosh, Andrew Hopkinson, Goran Petrovski, Harminder S. Dua
To confirm that human corneal rims left over from DALK/DSEK/PK surgeries could be useful sources for ex vivo endothelial cell expansion. Human corneal rims remaining from DALK/DSEK/PK surgeries were utilized (1:1 sex ratio, age 63+20 years, endothelial cell density >2,500 cells/mm2). The time from death to use varied between 3 days and 1.5 months. Endothelial cells isolated using a two-step, peel-and-digest method, whereby the Descemet’s membrane and endothelial cells were peeled off under a dissecting microscope, followed by digestion in collagenase. The isolated cells were suspended in TrypLE prior to plating onto FNC-coated tissue culture plates. The cells were then cultured in Ham’s F12:M199 (1:1) media supplemented with, ascorbic acid, transferrin, sodium selenite and bFGF. Characterisation of the cultured cells was performed by RT-qPCR and immunofluorescence staining accordingly. The number of isolated endothelial cells was repeatedly low (< 20,000 cells). However, improved techniques allowed to reduce stromal cell contamination. It was observed that endothelial cell proliferation was improved when the culture surface area was reduced. Furthermore, typical endothelial cobble stone morphology was observed when the cell density was high. Cell morphology and growth showed notable difference related to donor age and preservation time. ZO-1, Na/K-ATPase and PITX2 were used to confirm the endothelial phenotype. Preserved human corneal rims can be utilized for ex vivo expansion of corneal endothelial cells but further optimization is needed.

History

School

  • Mechanical, Electrical and Manufacturing Engineering

Published in

Acta Ophthalmologica

Volume

91

Pages

0 - 0

Citation

ALBERT, R. ... et al., 2013. Cultivation and characterisation of human peripheral cornea-derived endothelial cells [abstract]. Acta Ophthalmologica, 91, S252, DOI: 10.1111/j.1755-3768.2013.4766.x.

Publisher

John Wiley & Sons (© Acta Ophthalmologica Scandinavica Foundation)

Version

AM (Accepted Manuscript)

Publisher statement

This work is made available according to the conditions of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Full details of this licence are available at: https://creativecommons.org/licenses/by-nc-nd/4.0/

Publication date

2013

Notes

Presented at: 2015 European Association for Vision and Eye Research Conference (EVER 2013), Nice, France, 18-21 September 2013. This is the peer reviewed version of the following article: ALBERT, R. ... et al., 2013. Cultivation and characterisation of human peripheral cornea-derived endothelial cells [abstract]. Acta Ophthalmologica, 91, S252, which has been published in final form at: https://10.1111/j.1755-3768.2013.4766.x. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

ISSN

1755-375X

Language

en

Exports

Logo branding

Keywords

Exports