Limbal epithelial stem cells (LESCs) produce progeny (human limbal epithelial cells, hLEC) responsible for corneal repair and maintenance. The loss, or dysfunction, of LESCs results in LESC deficiency (LESCD) characterised by corneal opacification, neovascularisation and vision loss. Current treatments include LESC transplantation through cultivated limbal epithelial transplantation (CLET). There remains a number of manufacturing and regulatory challenges with CLET, therefore, this research was performed to address these challenges and improve existing protocols. This thesis aims were; to improve the starting number of LESCs by investigating to what extent corneal storage media preserve LESCs. Secondly, to decrease risk of zoonotic agent transmission through animal-derived materials, the potential of human-derived feeders (MRC5 fibroblasts) and serum for hLEC expansion was assessed. The final aim was to examine the feasibility of collagen-based tissue equivalents (TE) containing surrogate niche cells (human dermal fibroblasts, hDFs) as alternative delivery method to current protocols utilising human amniotic membrane, a substrate predisposed to inter-and intra-donor variability. Experiments showed different corneal storage formulations can preserve poorly differentiated cells; however, investigations into other factors (donor age/ hLEC isolation) may further improve the starting number of LESCs. Human derived serum was effective in maintaining hLECs and is an adequate replacement for animal-derived serum in CLET protocols. However, human-derived MRC5 feeders should not replace animal-derived feeders in CLET manufacture due to unfavourable hLEC characteristics observed. Furthermore, future investigations into cytokines expressed by human MRC5 feeders may elucidate factors influencing hLEC behaviour. Finally, changes in hLEC characteristics and increased inflammation-associated mediator expression demonstrated hDFs were not a suitable TE surrogate niche cell; however, the potential role of such mediators in epithelial-stromal interactions should be explored. To conclude; these experiments have improved existing CLET protocols, highlighted lower-risk materials may not always be effective, and that the surrounding culture environment is integral for CLET graft success.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647291 |
| Date | January 2015 |
| Creators | Vernon, A. J. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1463939/ |
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