Clinical Applications of Stem Cells in Ocular Surface Reconstruction.

Rigby, Helen (2005) Clinical Applications of Stem Cells in Ocular Surface Reconstruction. PhD thesis, Lancaster University.

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The central aim of this thesis is to improve current techniques for the ex vivo expansion of epithelial and endothelial cells on amniotic membrane for the treatment of ocular disorders. This thesis is primarily a microscope-based study and documents the findings of several investigations designed to refine tissue engineering of the cornea. Light, scanning and transmission electron microscopy were used to study cultured cell morphology. Observations were quantified and compared with data from control corneas, facilitating statistical analyses. Immunohistochemical techniques were also employed to determine the distribution of extracellular matrix proteins in processed amniotic membrane (AM) carriers. Various components of the culture system (including carrier, media and cell type) were evaluated in a number of separate studies. The results of this thesis show that denuded amniotic membrane encourages better adhesion of in vitro cultured stem cells and as such is a more practical carrier than the cellular variant. A freeze-dried form of amniotic membrane (FD-AM) was produced and found to compare favourably with cryopreserved tissue. Immunohistochemical findings confirmed similar distributions of extracellular matrix proteins in both carriers and FD-AM was used as a culture substrate to create healthy-looking epithelial cell sheets. In the first study of its kind, amniotic membrane was successfully used as a supportive matrix for the culture of quiescent corneal endothelial cells. In addition, evidence from the preservation study suggests that polyphenol antioxidant (extracted from green tea) could be a useful addition to culture media for corneal organ storage as it was found to maintain endothelial morphology and prolong cellular adhesion. In an attempt to remove the risk of allogeneic graft rejection, human oral mucous membranes were evaluated as potential sources of stem cells for autologous corneal grafts. Buccal and gingival epithelia cultured on AM formed well stratified and differentiated cell sheets which closely resembled in vivo corneal epithelium and were found to be useful in the treatment of bilateral limbal stem cell deficiencies. Co-culture with corneal stem cells was found to further induce differentiation of the oral mucosal cells into more comeal-like epithelium. To remove the risks of viral or prion transmission associated with human amniotic membrane, extracellular matrix protein coated gelatin hydrogels were examined as alternative carriers for the cultivation of ex vivo expanded comeal cells and produced some promising results. It was also found that human serum can be effectively used to replace foetal bovine serum in the culture medium, removing a potential risk of zoonose contamination. Morphological and quantitative analyses confirmed that the cells produced using human serum closely resembled those of control cornea. Collectively, the findings described and discussed herein have fostered further understanding of the amniotic membrane carrier and contributed towards significant improvements in the suspension culture system for the ex vivo expansion of stem cells for ocular surface reconstruction. They have also shown that corneal endothelial cells can be cultured successfully on AM and have the potential to treat corneal dystrophies.

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Thesis (PhD)
?? miaapqcellular biology.biochemistry. ??
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02 May 2019 16:29
Last Modified:
16 Jul 2024 05:45