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J ZhangJ Marshall, R Chibber, J Hillenkamp, J Marshall; Establishment of an In Vitro Model of Human RPE/Bruch's Membrane . Invest. Ophthalmol. Vis. Sci. 2002;43(13):693.
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Purpose: Interactions between cell and its environment are known to play a major role in several cellular functions and maintenance of a stable differentiated phenotype. An important component of the extracellular environment is the extracellular matrix (ECM). Retinal pigment epithelium (RPE) is a layer of polarized epithelial cells interposed between the nerosensory retina and the Bruch's membrane/choriocapillaris. Bruch's membrane offers structural support and provides important barrier functions and selective filtration of solute molecules between the RPE and choriocapillaris. It is therefore important to provide these cells a similar in vivo environment to promote a development of their in vivo characteristics. The aim of this study was to develop an in vitro reconstituted model of RPE-Bruch's membrane complex, which recaptures the structure and function that is found in the living eye. Methods: Bruch's membrane preparations denuded of cells were prepared by incubating Bruch's/choriod preparations in Ca2+ and Mg2+ free HBSS for 4-6 weeks, and the preparations were washed with fresh HBSS once a week during incubating period. Human RPE cells were established and plated onto denuded human Bruch's membrane. The cells were grown to a confluent state and then maintained for an additional 4-6 weeks. Morphology of the RPE/Bruch's membrane complexes were examined by scanning and transmission electron microscopes. Formation of tight junctions and polarisation of the RPE cells on the complexes were determined by immunofluorescence microscopy. Results: Histological and electron microscopic examination demonstrated that the Bruch's -choroid preparations were free of cells, particularly in choroid, after 4-6 weeks HBSS treatment, indicating a relevant denuded preparation. It was also shown that the human RPE cells grown on these denuded Bruch's membranes form and maintain a health monolayer for entire culture period, and exhibit morphological polarization as seen in vivo. The RPE cells grown under this condition also developed a complete circumferential distribution of tight-junction proteins around each cell at the apical-lateral level. Na/K ATPase and Na/H exchangers were also found to be accumulated and polarized on the apical surface of these cells. Conclusion: For the first time, to our knowledge, we have demonstrated the ability to repopulate a denuded Bruch's membrane complex with a monolayer of RPE cells that exhibit many in vivo cell properties and characteristics. This provides a novel method of culturing cells on a collagen matrix and a useful model for the study of ageing and retina diseases.
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