The influence of substrate elastic modulus on retinal pigment epithelial cell phagocytosis

Abstract

To better understand if a complex process such as phagocytosis is influenced by substrate stiffness, we investigated the influence of substrate elastic modulus on phagocytosis in the retinal pigment epithelial (RPE) cell line ARPE-19. RPE cells lie on Bruch’s membrane, directly under the retina, and phagocytose the shed photoreceptor outer segments. Bruch’s membrane is known to increase in stiffness by an order of magnitude with age and thus, this study has potential relevance in explaining retinal changes in age-related macular degeneration. ARPE-19 cells were plated on laminin-coated polyacrylamide substrates of varying elastic modulus. After 14 days in culture, a solution of latex fluorescent beads suspended in PBS was placed in each well. After an incubation time of 4 hours, flow cytometry was performed to determine the number of cells that phagocytosed a bead. The number of ARPE-19 cells that phagocytosed a bead decreased continuously as a function of increasing substrate elastic modulus (p=0.0135), and this was found to be a linear relationship (slope=−0.03305 ± 0.01104, R2 =0.4726 per 10,000 cells). Our results suggest that RPE cells display decreased phagocytosis when grown on firmer substrates, and thus, RPE cells in older eyes, in which Bruch’s membrane is stiffer, may demonstrate decreased phagocytosis. Impaired phagocytosis by RPE cells may contribute to impaired metabolism of photoreceptor outer segments and to development of macular degeneration. Material stiffness may be a critical parameter in the development of neural therapies, including retinal prosthetics and stem cell therapies.

The influence of substrate modulus on retinal pigment epithelial cells

“…Therefore, it is possible that the mechanical environment experienced by RPE cells affects their adhesion and initiates or promotes dedifferentiation. This is supported by recent work demonstrating difference in phagocytic ability of RPE cells on different substrate moduli …”

“…This is supported by recent work demonstrating difference in phagocytic ability of RPE cells on different substrate moduli. 31 The cell activity, as measured by a mitochondrial reduction assay, varied on the different moduli. Because it remains difficult to quantify cell number on scaffolds, the cell activity was normalized to the day 1 activity.…”

Collagen vitrigels with low‐fibril density enhance human embryonic stem cell‐derived retinal pigment epithelial cell maturation

“…Although the influence of substrate mechanics on RPEs is relatively unexplored, there are studies that demonstrate that increased elastic modulus in polyacrylamide substrates suppress phagocytosis in ARPE‐19 cells (Boochoon, Manarang, Davis, McDermott, & Foster, ), and stiffer poly(ethylene glycol) diacrylate (PEGDA) hydrogels cause an increase in proinflammatory marker expressions such as IL‐6 (White, DiStefano, & Olabisi, ). These findings provide potential explanations for the decreased function and increased apoptosis of RPE cells in older eyes, which are known to have stiffer Bruch's membranes (Curcio & Johnson, ).…”

Effects of bevacizumab, ranibizumab, and aflibercept on phagocytic properties in human RPE cybrids with AMD versus normal mitochondria

“…Some have proposed that the failure of RPE cells to phagocytose POS disc membranes contributes to accumulation of drusen apically, atrophy of the subretinal retinal pigment layer, and loss of macular photoreceptors causing vision loss. [13][14][15][16] This hypothesis is further supported by Zhao et al, who found in vitro, that RPE cells can migrate towards photoreceptors, resulting in translocation of non-phagocytosized debris from the apical side to the basal side. 17 A separate study by Inana et al (2018) demonstrated that RPE phagocytic function was indeed reduced in AMD.…”