Generation and characterisation of decellularised human corneal limbus

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

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. 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. 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. 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.