July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Organotypical 3D corneal equivalents from decellularized materials
Author Affiliations & Notes
  • Julia Fernández-Pérez
    Trinity Center for Bioengineering, Trinity College Dublin, Dublin, Ireland
    Department of Mechanical and Manufacturing Engineering, Trinity College Dublin, Dublin, Ireland
  • Mark Ahearne
    Trinity Center for Bioengineering, Trinity College Dublin, Dublin, Ireland
    Department of Mechanical and Manufacturing Engineering, Trinity College Dublin, Dublin, Ireland
  • Footnotes
    Commercial Relationships   Julia Fernández-Pérez, None; Mark Ahearne, None
  • Footnotes
    Support  European Research Council starting grant (EyeRegen 637460)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4124. doi:
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      Julia Fernández-Pérez, Mark Ahearne; Organotypical 3D corneal equivalents from decellularized materials. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4124.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The worldwide shortage of donor corneas available for performing keratoplasty has necessitated the development of tissue engineered alternatives. The aim of this study is to fabricate a three dimensional corneal equivalent using human corneal stromal and epithelial cells and decellularized stromal sheets.

Methods : Porcine corneas were embedded in OCT immediately after collection from an abattoir, sliced into thin sheets and decellularized with a previously established method using SDS, Triton X-100 and nucleases. The sheets were air-dried under sterile conditions and kept at room temperature until used. Human corneal stromal cells were embedded in rat tail collagen to produce a gel that was cast between dried sheets. A 5-sheet construct was obtained with cell seeded gel between each sheet. Constructs were cultured for 3 weeks. Human epithelial cells were seeded on the surface of the construct at high cell density and cultured for another week. Cell viability was assessed by Calcein-AM/Ethidium homodimer staining. Structures of the constructs was visualized by Haematoxylin and Eosin (H&E) staining. Cell phenotype was assessed by immunofluorescent staining.

Results : The obtained decellularized sheets were highly transparent. When stacked into a construct with cell-laden collagen hydrogels, these supported high cell viability after 3 weeks of culture. Stromal cells presented a keratocyte-like morphology. Human corneal epithelial cells attached easily to the sheets, adopted a cobblestone morphology and formed a stratified tight epithelium. Constructs were successfully sutured into an ex vivo porcine model.

Conclusions : Constructs fabricated in this study closely resemble the multi-layered structure of the cornea and have potential as corneal substitutes. Future experiments will explore the addition of endothelial cells as a full thickness corneal implant.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

a) Decellularized porcine corneal sheets; b) Cell viability of constructs after 3 weeks in culture (green = alive, red = dead, scale bar = 200 µm); c) Cross-sectional view of the construct after H&E staining (scale bar = 200 µm); d) Stratified epithelium on construct surface stained with H&E (scale bar = 50 µm).

a) Decellularized porcine corneal sheets; b) Cell viability of constructs after 3 weeks in culture (green = alive, red = dead, scale bar = 200 µm); c) Cross-sectional view of the construct after H&E staining (scale bar = 200 µm); d) Stratified epithelium on construct surface stained with H&E (scale bar = 50 µm).

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