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Y. Sun, J. Zhang, A. El-Osta, A. Hussain, P. Munro, J. Marshall; Human Retinal Pigment Epithelium and Bruch’S - Choroid Membrane in vitro Organ Culture Working Model. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1757.
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The aim of the study is to establish a human retinal pigment epithelial (RPE) and Bruch’s -Choroids membrane (BCM) organ culture working model that more closely represents the in vivo environment. This will enable us to investigate the cellular and biochemical responses of the RPE-BCM system associated with laser or the other treatments.
Explants of human RPE still attached to BCM and sclera were prepared from donor eyes (12 to 72 hours post-mortem) and clamped in a tailor-made Ussing chamber to isolate the apical sides of the RPE from the choroids. Freshly prepared explants were firstly covered by pure fetal calf serum (FCS) for overnight at 37 degrees C and then transfer to a organ culture system in Dulbecco’s modified Eagle’s medium (DMEM)/F12 nutrient supplemented with 15%FBS and bFGF for various periods up to 8 weeks.The morphology and functional behavior of the system were assessed by using phase-contrast microscopy, transmission electron microscopy and confocal immune-fluorescence. Cell viability and proliferation assays were also used to quantify RPE survival and division at different time points.
The study shows a viable human RPE-BCM organ culture working model, which enables us to maintain RPE cells attached to the Bruch's membrane for up to 4 weeks without significant cell death and detachment. Majority of RPE cells showed Caicein AM positive staining in the examined explants and confirmed by MTT viability assay.For up to four weeks in the organ culture system the RPE cells remained as a monolayer and retained several prominent ultrastructural features, including apical microvillus, intracellular melanosomes and mitochondria. The ability of this system to maintain it native barrier function was also confirmed by labelling the expression of the tight junction protein ZO-1.
These results suggest that the system is suitable for the short-term maintenance of RPE for experimental purposes. The ECM can exert crucial impacts on cell behavior and the ability to maintain RPE cells attached to the Bruch's membrane provides a new in vitro tool for studying the cellular and biochemical activity and its response to external stimuli, including laser treatment.
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