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

A novel biosynthetic RPE-BrM (Retinal Pigment Epithelium-Bruch's Membrane) assembly suitable for retinal transplantation therapy
A novel biosynthetic RPE-BrM (Retinal Pigment Epithelium-Bruch's Membrane) assembly suitable for retinal transplantation therapy
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.
0146-0404
Lotery, Andrew
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Ward, Gareth
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Alexander, Philip
ea648f08-18ec-4834-befc-9d2279e7f2bc
Johnston, David
b41163c9-b9d2-425c-af99-2a357204014e
Page, Anton
76ebbfb8-4fe3-495c-afff-1f2f34977fee
Cree, Angela
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Bhaskar, Atul
d4122e7c-5bf3-415f-9846-5b0fed645f3e
Mahajan, Sumeet
b131f40a-479e-4432-b662-19d60d4069e9
Grossel, Martin
403bf3ff-6364-44e9-ab46-52d84c6f0d56
Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Lotery, Andrew
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Ward, Gareth
250b5aa5-a94a-49ec-9569-9250d8e2607b
Alexander, Philip
ea648f08-18ec-4834-befc-9d2279e7f2bc
Johnston, David
b41163c9-b9d2-425c-af99-2a357204014e
Page, Anton
76ebbfb8-4fe3-495c-afff-1f2f34977fee
Cree, Angela
6724b71b-8828-4abb-971f-0856c2af555e
Bhaskar, Atul
d4122e7c-5bf3-415f-9846-5b0fed645f3e
Mahajan, Sumeet
b131f40a-479e-4432-b662-19d60d4069e9
Grossel, Martin
403bf3ff-6364-44e9-ab46-52d84c6f0d56
Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd

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).

Record type: Meeting abstract

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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Published date: 1 September 2016

Identifiers

Local EPrints ID: 423100
URI: http://eprints.soton.ac.uk/id/eprint/423100
ISSN: 0146-0404
PURE UUID: d5f284cd-c817-4b8b-8b15-cc419dbe56d2
ORCID for Andrew Lotery: ORCID iD orcid.org/0000-0001-5541-4305
ORCID for David Johnston: ORCID iD orcid.org/0000-0001-6703-6014
ORCID for Sumeet Mahajan: ORCID iD orcid.org/0000-0001-8923-6666
ORCID for Martin Grossel: ORCID iD orcid.org/0000-0001-7469-6854
ORCID for J. Arjuna Ratnayaka: ORCID iD orcid.org/0000-0002-1027-6938

Catalogue record

Date deposited: 14 Aug 2018 16:30
Last modified: 10 Nov 2021 03:34

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Contributors

Author: Andrew Lotery ORCID iD
Author: Gareth Ward
Author: Philip Alexander
Author: David Johnston ORCID iD
Author: Anton Page
Author: Angela Cree
Author: Atul Bhaskar
Author: Sumeet Mahajan ORCID iD
Author: Martin Grossel ORCID iD

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