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Audra Shadforth, Damien Harkin, Traian Chirila, Anthony Weiss, Dietmar Hutmacher, Beatrix Feigl; An in vitro 3-D cell culture model for studying pathomechanisms in AMD. Invest. Ophthalmol. Vis. Sci. 2013;54(15):314.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a novel 3-D cell culture model with the view to studying the pathomechanisms underlying the development of age-related macular degeneration (AMD). Our central hypothesis is that the silk structural protein fibroin, used in conjunction with cultured human retinal pigment epithelium cells (RPE) cells and microvascular endothelial cells, can serve as a structural analogue of Bruch’s membrane.
Co-cultures of human RPE cells (ARPE-19 cells grown in Miller’s medium) and microvascular endothelial cells (HMEC-1 cells grown in endothelial culture medium) were established on opposing sides of a 3 micron thick membrane constructed from B. mori silk fibroin. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The phagocytic activity of the RPE cells cultured on silk fibroin was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). In some cultures, membrane defects were created by puncturing with a 27 G needle to mimic breaks in Bruch’s membrane that are established factors of inducing neovascularisation. The architecture of the cultured tissue before and after wounding was examined by confocal microscopy after staining with rhodamine phalloidin and Hoechst 33342 nuclear dye.
The RPE component of the 3D model developed a cobblestoned morphology as well as barrier function and displayed vitronectin-mediated phagocytic activity. Microvascular endothelial cells attached well to the gelatin-coated lower surface of the fibroin membrane and remained physically separated from the overlying RPE layer. The fibroin membranes remained intact throughout the duration of cell culture and were amenable to puncturing without collapse.
The Bruch’s membrane analogue constructed from silk fibroin, vitronectin and gelatin, supports the co-cultivation of RPE cells and microvascular endothelial cells. The resulting RPE layer displays functions similar to that of native RPE and the entire tri-layered structure displays potential to be used as an in vitro model for studying pathomechanisms of AMD including choroidal neovascularization.
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