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Graefe's Archive for Clinical and Experimental Ophthalmology

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01-03-2018 | Basic Science

Generation and characterisation of decellularised human corneal limbus

Authors: Kristina Spaniol, Joana Witt, Sonja Mertsch, Maria Borrelli, Gerd Geerling, Stefan Schrader

Published in: Graefe's Archive for Clinical and Experimental Ophthalmology | Issue 3/2018

Abstract

Purpose

Limbal epithelial stem cells (LESC) reside in a niche in the corneo-scleral transition zone. Deficiency leads to pain, corneal opacity, and eventually blindness. LESC transplantation of ex-vivo expanded human LESC on a carrier such as human amniotic membrane is a current treatment option. We evaluated decellularised human limbus (DHL) as a potential carrier matrix for the transplantation of LESC.

Methods

Human corneas were obtained from the local eye bank. The limbal tissue was decellularised by sodium desoxychelate and DNase solution and sterilised by γ-irradiation. Native limbus- and DHL-surface structures were assessed by scanning electron microscopy and collagen ultrastructure using transmission electron microscopy. Presence and preservation of limbal basement membrane proteins in native limbus and DHL were analysed immunohistochemically. Absence of DNA after decellularisation was assessed by Feulgen staining and DNA quantification. Presence of immune cells was explored by CD45 staining, and potential cytotoxicity was tested using a cell viability assay.

Results

In the DHL, the DNA content was reduced from 1.5 ± 0.3 μg/mg to 0.15 ± 0.01 μg/mg; the three-dimensional structure and the arrangement of the collagen fibrils were preserved. Main basement membrane proteins such as collagen IV, laminin, and fibronectin were still present after decellularisation and γ-irradiation. CD45-expressing cells were evident neither in the native limbus nor in the DHL. DHL did not convey cytotoxicity.

Conclusions

The extracellular matrix (ECM) of the limbus provides a tissue specific morphology and three-dimensionality consisting of particular ECM proteins. It therefore represents a substantial component of the stem cell niche. The DHL provides a specific limbal niche surrounding, and might serve as an easily producible carrier matrix for LESC transplantation.

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Title: Generation and characterisation of decellularised human corneal limbus
Authors: Kristina Spaniol
Joana Witt
Sonja Mertsch
Maria Borrelli
Gerd Geerling
Stefan Schrader
Publication date: 01-03-2018
Publisher: Springer Berlin Heidelberg
Published in: Graefe's Archive for Clinical and Experimental Ophthalmology / Issue 3/2018
Print ISSN: 0721-832X
Electronic ISSN: 1435-702X
DOI: https://doi.org/10.1007/s00417-018-3904-1

At a glance: The STEP trials

Springer Medicine

Generation and characterisation of decellularised human corneal limbus

Abstract

Purpose

Methods

Results

Conclusions

At a glance: The STEP trials

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusions

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