Mechanical Modulation of RPE Cell Phagocytosis through Dynamic Substrate Stiffening

Introduction: : Evolution has produced complex immune systems enabling organisms to distinguish between harmful threats and healthy self-tissues [1-4]. Among the most ancient and conserved components of immunity is phagocytosis—a fundamental process used to eliminate pathogens, facilitate tissue repair, and regulate development [1,3]. This process begins when phagocytes use transmembrane receptors, functioning as molecular “hands,” to recognize biochemical “eat-me” signals on target cells. While the molecular pathways governing phagocytosis are well understood, the role of physical cues from the cellular microenvironment remains less defined. Substrate stiffness, in particular, is a known regulator of cellular behaviors such as proliferation, differentiation, and motility [5]. In disease models, altered stiffness is often used to simulate pathological conditions.

The retina serves as a compelling example of how changes in substrate stiffness can impair phagocytic function. Retinal pigment epithelial (RPE) cells, which rest atop Bruch’s membrane, are essential for retinal homeostasis, in part by phagocytosing shed photoreceptor outer segments (POS) [6,7]. With age, Bruch’s membrane undergoes biomechanical changes marked by increased stiffness, which has been directly associated with diminished RPE phagocytic activity [6]. This impairment contributes to the accumulation of drusen, degeneration of the RPE layer, and subsequent photoreceptor loss, key pathological features of non-exudative age-related macular degeneration (AMD) [5,6]. To study the kinetics of this mechanobiological relationship, we will use magnetorheological elastomers (MREs), iron particle-embedded polymers with stiffness tunable via magnetic fields [8]. MREs will allow us to model Bruch’s membrane stiffening and investigate time- and stiffness-dependent effects on RPE phagocytosis, offering insight into AMD pathogenesis.

Materials and

Methods: : MREs are fabricated by thoroughly mixing Ecoflex Gel (1:1 parts A and B), 50 wt% carbonyl iron powder (lot no. C8M031, Chemical Store), and 25 wt% thinner (lot no. 2105485, Smooth-On, Inc.) [9]. Devices were fabricated, sterilized, and coated with fibronectin following previous literature [9].

RPE cells were seeded onto MRE substrates at 26,000 cells/mm² and cultured for 48 hours under two conditions: with a magnet (increasing stiffness to ~82.49 kPa) or without (baseline stiffness ~ 8.14 kPa) [9]. Silica beads (4 μm in diameter), coated with either 10 mol% phosphatidylserine (PS, “eat me” signal) or phosphatidylcholine (PC, healthy cell mimic controls) were added at 25 beads per cell [10]. Fresh media with lipid-coated beads was added to each well, and magnets were either introduced (stiffening) or removed (softening) based on experimental condition. Phagocytosis was measured from 15 minutes to 4 hours. After fixation, cells were stained with DAPI and CellMask. For each condition, a total of 9 images were captured, 3 images from different locations in each of 3 wells (Fig. 1A).

Gray scale images of nuclei and whole cells were analyzed with CellProfiler using thresholding (Fig. 1A-ii,iii). Ilastik was trained with 15 manually annotated images to identify ingested beads, classifying pixels as “background”, “cell’, or “bead” (Fig.1A-iv). Simple segmented gray-scale images are exported and analyzed in CellProfiler to identify bead locations based on size and pixel intensity (Fig. 1A-iv). Nuclei, cells, and bead locations were used to relate objects and determine bead count per cell (Fig. 1A-iv).

Results, Conclusions, and Discussions:: This study investigates how changes in Bruch’s membrane stiffness affect the phagocytic activity of RPE cells. Using magnetically responsive materials, we simulate both static and dynamic stiffening to model physiological, pathological, and potentially reversible changes in the extracellular environment. Understanding the relationship between mechanical stiffness and selective phagocytic efficiency could provide insight into the mechanobiology of AMD. Our primary focus is on how these mechanical cues influence the efficiency of RPE cells in phagocytosing PS beads, which mimic POS in vivo. Incorporating multiple timepoints allows for a detailed analysis of the temporal regulation of phagocytosis, including the kinetics of bead internalization and the duration of substrate stiffness exposure required to elicit measurable changes in phagocytic activity. To assess phagocytic specificity, PC beads mimicking healthy non-apoptotic cells will serve as a control.

Our preliminary data indicate that RPE cells cultured on stiffer MREs exhibit reduced phagocytosis of PS-coated beads after 4 hours of incubation (Fig. 1B), suggesting that increased substrate stiffness may impair the ability of RPE cells to efficiently internalize apoptotic targets. We also observed a time-dependent increase in bead uptake, with higher numbers of internalized PS and PC-coated beads per cell at longer time points (3 and 4 hours) compared to shorter intervals (15 and 30 minutes). This trend implies that phagocytic activity accumulates over time, potentially due to prolonged cell engagement or delayed internalization kinetics. The observation that PS bead uptake is diminished on stiffer substrates supports the hypothesis that mechanical cues from the microenvironment negatively affect selective phagocytosis and is consistent with prior work in the field [6,11]. These findings suggest that both substrate stiffness and incubation duration are critical factors influencing RPE phagocytic behavior and may interact to affect the efficiency of phagocytosis.

Establishing these relationships will be essential for understanding how age-related stiffening of Bruch’s membrane contributes to phagocytic dysfunction and the progression of retinal diseases such as age-related macular degeneration (AMD). Studying these effects could help the understanding of disease mechanisms and give insight into mechanical thresholds where phagocytosis dysfunction occurs.

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