Engineering human tissue for the control of cellular adhesion and function for an eventual retinal implant.

摘要

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly individuals. It is believed to be triggered by dysfunction of retinal pigment epithelial (RPE) cells and changes in the underlying Bruch's membrane. One proposed strategy for treating AMD is a dual-layer implant, consisting of an organized monolayer of healthy RPE cells cultured onto a biocompatible substrate. In particular, human anterior lens capsule is explored here as a substitute for Bruch's membrane due to its potential to be an autologous implant. In this thesis, in vitro methods are used to investigate the feasibility of a dual-layer implant.; First, this work focuses on mimicking RPE morphology, since it is believed that cell shape influences function. Native RPE cells, of uniform shape and size, form an organized monolayer. Soft lithography was applied to lens capsule to control cellular adhesion and morphology of an RPE cell line. Poly(vinyl) alcohol was found to successfully confine cells to the defined cytophilic regions of lens capsule of sizes ranging from 15--50 mum in diameter, exhibiting control over the placement of single cells before allowing cells to spread and form a confluent monolayer.; Second, immunofluorescence is utilized to assess the function of the RPE monolayer engineered by soft lithography or by centrifugation. Cells cultured on lens capsule were found to exhibit the characteristic formation of actin belts and tight junctions. RPE cells also have the critical role of renewing photoreceptors by phagocytizing their outer segments. In these studies, cells seeded by centrifugation were found to exhibit phagocytosis and comparisons made with the literature determined that the measured in vitro activity may be sufficient to sustain photoreceptors.; Finally, the permeability of lens capsule is investigated. One important property of Bruch's membrane is to allow flow of nutrients and waste between RPE cells and the choriocapallaris. The permeability of lens capsule, determined by measuring the diffusion of fluorescently-labeled dextran, was compared with reported values for Bruch's membrane. Lens capsule appears to have comparable permeability to amino acids and thus has the potential to act as an appropriate substitute for Bruch's membrane.