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Louise Morgan, Alex J Shortt, Anna Peters, Julie T Daniels; An ex-vivo tissue model for feasibility testing of corneal RAFT tissue equivalents. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3478.
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© ARVO (1962-2015); The Authors (2016-present)
Human amniotic membrane (HAM) provides a culture substrate and carrier during ex-vivo expansion and transplantation of limbal epithelial stem cells (LESC). We previously described a biomimetic tissue equivalent (TE) called RAFT comprising plastic compressed collagen with comparable handling characteristics and LESC growth supporting properties to HAM. Before proceeding to phase-1 clinical trials of RAFT TE containing living cells it will be necessary to test the safety of this technology in an animal model.<br /> The aim of this study was to develop methods of transplanting RAFT TE transplants. To comply with legal and ethical requirement to replace, reduce and refine the use of animals in medicine and science, we performed this work using an ex-vivo tissue model.
RAFT TE and primary human LESC were prepared as previously described. Whole rabbit eyes from a commercial supplier were used within 72h of enuclation. A central 6mm dia. superficial anterior lamellar keratoplasty was performed and a lamellar pocket created at the edge of the keratectomy. RAFT TE were transplanted to the corneal surface and glued in place using fibrin glue. The RAFT TE edges were tucked into the peripheral pocket. Surgery was performed on 4 separate occasions using a minimum of 3 eyes per experiment. A panel of mouse monoclonal antibodies was assembled to identify donor human and host rabbit cells. Epithelial cells were identified using anti-K3 antibody and stem and immature epithelial cells using anti K15 and p40 (ΔNp63) antibodies. Blood vessels were identified with anti-integrin beta-1 and smooth-muscle actin. Human and rabbit cells were differentiated using anti human nuclear antigen (anti-HNA). H&E and trichrome stains identified the graft, blood vessels and inflammatory cells.
Optimum illumination settings for slit lamp and surgical microscope based photography were defined. A sutureless procedure to transplant RAFT TE was validated. We demonstrated that donor human cells could be distinguished from recipient rabbit cells using anti-HNA antibody. Epithelial, stromal, immune and vascular cells could be reliably identified.
By using an ex-vivo tissue model to develop methods for transplanting RAFT TE the number of live animals to be used in future safety studies has been reduced in compliance with ARVO recommendations, local regulations and ethical obligation regarding the use of animals in research.
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