Effects of extracellular matrix and Bruch's membrane on retinal outer segment phagocytosis by cultured human retinal pigment epithelium

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Abstract
The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.