A novel biosynthetic RPE-BrM (Retinal Pigment Epithelium-Bruch's Membrane) assembly suitable for retinal transplantation therapy

Lotery, Andrew, Ward, Gareth, Alexander, Philip, Johnston, David, Page, Anton, Cree, Angela, Bhaskar, Atul, Mahajan, Sumeet, Grossel, Martin and Ratnayaka, J. Arjuna (2016) A novel biosynthetic RPE-BrM (Retinal Pigment Epithelium-Bruch's Membrane) assembly suitable for retinal transplantation therapy. Investigative Ophthalmology & Visual Science, 57 (12).

Abstract

Purpose : Clinical trial data suggests that treatment of neovascular age-related macular degeneration (AMD) with Vascular Endothelial Growth Factor (VEGF) inhibitors may accelerate RPE atrophy. It is therefore imperative that a novel solution be found to repair/replace the damaged RPE-BrM in disease. We tested the hypothesis whether a novel biosynthetic RPE-BrM assembly is able to meet the manifold demands of the outer retina, with potential to rapidly benefit patients at risk of advancing blindness.

Methods : We used a combination of methyl methacrylate and poly(ethylene glycol) methacrylate P(MMA:PEGM) in a 60:40 ratio to create a synthetic BrM scaffold. Enhanced electrospinning techniques were used to create a nanofibrous web-like structure similar to the inner collagenous layer of native BrM.
Primary RPE cells isolated from murine retinas were seeded on the synthetic BrM scaffold. These RPE-BrM assemblies were maintained in culture for several months up to 1 year, during which time their structure, physiology and functionality were assessed using TEM/SEM and confocal microscopy as well as biochemical studies

Results : Electrospun scaffolds had comparable porosity, diffusional properties and mechanical strength equivalent to human BrM. It also formed an excellent substrate on which primary RPE cells could readily attach and proliferate. Individual RPE cells readily adhered to underlying fibres with vinculin and focal adhesion kinases showing points of contact. Long-term RPE cultures on scaffolds expressed the cell-specific marker RPE65, formed ZO-1 junctional complexes, and showed specialisation of apical membranes with microvilli and expression of Na/K ATPase. RPE monolayers also secreted VEGF preferentially via the basolateral membrane. RPE cells proved fully functional and expressed MerTK and aVb5 on apical surfaces to ingest fed photoreceptor outer segments, which were internalised in a time dependent manner. We then tested the biocompatibility and integrity of the BrM scaffold by subretinally transplanting them in rabbit eyes using a custom-made injector.

Conclusions : We show that a fully-functional RPE-BrM assembly can be made that is suitable for transplantation. This approach has the potential to bring rapid long-lasting benefits to patients with retinal diseases such as AMD or Retinitis Pigmentosa, and could also be adapted for other regenerative treatments.

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Contributors

Author: Andrew Lotery

Author: David Johnston

Author: Angela Cree

Author: Sumeet Mahajan

Author: Martin Grossel

Author: J. Arjuna Ratnayaka

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