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Generation and characterisation of decellularised human corneal limbus

  • Kristina Spaniol
  • Joana Witt
  • Sonja Mertsch
  • Maria Borrelli
  • Gerd Geerling
  • Stefan Schrader
Basic Science

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.

Keywords

Epithelial stem cells Decellularisation Tissue engineering Limbus 

Notes

Compliance with ethical standards

Conflict of interest

All authors certify that they have no affiliations with or involvement in any organisation or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licencing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge, or beliefs) in the subject matter or materials discussed in this manuscript.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee (name the institution/committee) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Supplementary material

417_2018_3904_Fig8_ESM.gif (43 kb)
Figure 8

CD45 staining of different tissues. Human conjunctiva (a) and murine lymph node (b) were stained as positive controls and showed CD45 expression of several cells (green, arrow). Neither the native (c) nor the decellularised human limbus (d) expressed CD45, indicating low immunogenicity. The nuclear counterstaining with DAPI (blue) showed that no cell nuclei were evident in the decellularised human limbus, indicating complete cell removal. Scale bar: 100 μm, applies to all images. (GIF 43 kb)

417_2018_3904_MOESM1_ESM.tif (703 kb)
High resolution image (TIFF 703 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.University Eye Hospital DüsseldorfDüsseldorfGermany
  2. 2.DüsseldorfGermany
  3. 3.Laboratory of Experimental OphthalmologyDüsseldorfGermany

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