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.