Past > QUT Faculties & Divisions > Faculty of Health 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 of choroidal neovascularization.ĬopyrightOwnerCopyrightOwner Conclusions Synthetic Bruch’s membranes constructed from silk fibroin, vitronectin and gelatin, support the co-cultivation of RPE cells and microvascular endothelial cells. The fibroin membranes were amenable to puncturing without collapse thus providing the opportunity to study transmembrane migration of the endothelial cells. Microvascular endothelial cells attached well to the gelatin-coated surface of the fibroin membrane and remained physically separated from the overlaying RPE layer. Attachment of ARPE-19 cells to fibroin was unaffected by tropoelastin. Results The RPE layer of the 3D model developed a cobblestoned morphology (validated by staining for ZO-1 and F-actin), displayed barrier function (validated by measurement of TER) and demonstrated cytoplasmic uptake of vitronectin-coated microspheres. The architecture of the synthetic tissue before and after wounding was examined by confocal microscopy after staining for ZO-1 and F-actin. In some cultures, membrane defects were created by puncturing within a 24 G needle. The phagocytic activity of the synthetic RPE was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The effects of tropoelastin on attachment of ARPE-19 cells was also examined. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. Methods 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 synthetic Bruch’s membrane (3 microns thick) constructed from B mori silk fibroin. Our central hypothesis is that the silk structural protein fibroin used in conjunction with cultured human cells can be used to mimic the structural relationships between the RPE and choriocapillaris in health and disease. Purpose 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).
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