Introduction: Age-related macular degeneration (AMD) results in the deterioration of the retinal pigment epithelial (RPE) layer under the macula. A potential surgical treatment involves the replacement of the diseased RPE cells with healthy cells. Clinically, this requires identification of the appropriate cells to use and the optimal substrate for use as a transplant vehicle. The material under investigation was polyurethane (PU) and ways to manufacture porous PU were explored. Iris pigment epithelium (IPE) is derived from the same embryonic origin as RPE and has been shown to have several of the same functions. IPE can be harvested more easily than RPE. The aim of this study was to compare the behaviour of primary bovine RPE (bRPE), IPE (bIPE) and human RPE (hRPE) cultured on PU membranes with the objective of optimising the cell/substrate combination for transplantation. Morphology and functionality of the cells attaching on the membranes were also evaluated. Methods: The PU used in this study were Z3Ai and Z9Ai (b9'" Biomer Technology Ltd.) membranes with thickness <lOOllm. Several ways of producing porous PU were investigated. One of which was mixing Z3Ai with (dimethyl acetamide) DMAC in a concentration of 5% (vol/vol) and 5% icing sugar (by weight). The mixture was then dried in an oven at 75°C for 24 hours and then rinsed with distilled water. The surface topography was investigated using SEM. Properties of the membranes such as tensile strength, modulus of elasticity and wettability were examined. Freshly harvested bRPE, blPE and hRPE cells were seeded on the b9 films, porous PU membrane and tissue culture polystyrene (TCPS) as control. The monolayer formation of the cells on the surfaces were controlled by optimising the correct harvesting technique, using the correct amount of seeding, controlling the amount of serum and the addition of retinoic acid. The cell morphology was assessed by phase contrast microscopy and stained for F-actin, zonula occludens-I (ZO-i) tight junction marker and DAPI for nuclei distribution. Cytokeratin staining was used to confirm the epithelial phenotype. Phagocytosis assays, selectivity of phagocytosis assay and dextran transport studies were conducted to evaluate the functionalities of the cells on the membranes. An In vitro injury model of the cells attaching on the membranes was also investigated. Results: The porous PU membranes produced were shown to have suitable properties as a replacement of a native Bruch's membrane with Sum thickness and good porosities. All cell types demonstrated good epithelial phenotype, confirmed by cytokeratin expression when attached on the membrane. Nuclei were dispersed evenly, F-actin belt formation with ZO-i at the cell borders were observed. Phagocytosis of photoreceptor outer segments (PaS) demonstrated that the cells attaching on these surfaces were able to phagocytose in a time-dependant manner and show selectivity in phagocytosis when tested with polystyrene beads and blocking with the aVI35-integrin. Dextran transport revealed a reduction in the transport of higher molecular weight dextran through the porous membrane-cells complex. The preliminary In vitro injury model study showed inconsistent cell integration. Conclusion: This project has developed a porous PU with appropriate mechanical properties and porosities. The project has demonstrated that primary BRPE and BIPE were successfully harvested and differentiated into a functional monolayer in vitro. Primary bovine and human RPE and IPE were shown to grow as a differentiated monolayer on non-porous and porous PU substrates. The differentiated monolayer of primary cells on PU substrate displayed functional activities in the ability to phagocytose pas and transporting different sized dextran molecules.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:569211 |
| Date | January 2013 |
| Creators | Alias, Eliesmaziah |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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