Generation and characterisation of decellularised human corneal limbus
- 276 Downloads
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.
KeywordsEpithelial stem cells Decellularisation Tissue engineering Limbus
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.
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.
- 5.Basu S, Sureka SP, Shanbhag SS, Kethiri AR, Singh V, Sangwan VS (2016) Simple limbal epithelial transplantation: long-term clinical outcomes in 125 cases of unilateral chronic ocular surface burns. Ophthalmology 123:1000–1010. https://doi.org/10.1016/j.ophtha.2015.12.042 CrossRefPubMedGoogle Scholar
- 14.Levis HJ, Massie I, Dziasko MA, Kaasi A, Daniels JT (2013) Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture. Biomaterials 34:8860–8868. https://doi.org/10.1016/j.biomaterials.2013.08.002 CrossRefPubMedGoogle Scholar
- 18.Yurchenco PD (2011) Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb Perspect Biol 3. https://doi.org/10.1101/cshperspect.a004911
- 25.Bray LJ, George KA, Ainscough SL, Hutmacher DW, Chirila TV, Harkin DG (2011) Human corneal epithelial equivalents constructed on Bombyx Mori silk fibroin membranes. Biomaterials 32:5086–5091. https://doi.org/10.1016/j.biomaterials.2011.03.068 CrossRefPubMedGoogle Scholar
- 28.Badylak SF, Taylor D, Uygun K (2011) Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu Rev Biomed Eng 13:27–53. https://doi.org/10.1146/annurev-bioeng-071910-124743 CrossRefPubMedGoogle Scholar
- 30.Huang M et al (2011) Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction. Biomaterials 32:7812–7821. https://doi.org/10.1016/j.biomaterials.2011.07.012 CrossRefPubMedGoogle Scholar
- 36.Crapo PM, Gilbert TW, Badylak SF (2011) An overview of tissue and whole organ decellularization processes. Biomaterials 32:3233–3243. https://doi.org/10.1016/j.biomaterials.2011.01.057 CrossRefPubMedPubMedCentralGoogle Scholar
- 39.Keane TJ, Londono R, Turner NJ, Badylak SF (2012) Consequences of ineffective decellularization of biologic scaffolds on the host response. Biomaterials 33:1771–1781. https://doi.org/10.1016/j.biomaterials.2011.10.054 CrossRefPubMedGoogle Scholar
- 49.Gordon SR (2014) Fibronectin antibody labels corneal stromal collagen fibrils in situ along their length and circumference and demonstrates distinct staining along the cell and stromal interfaces of Descemet’s membrane. Curr Eye Res 39:312–316. https://doi.org/10.3109/02713683.2013.841260 CrossRefPubMedGoogle Scholar