'MacuSIM': A microfluidic, in vitro model of the outer retina as an experimental platform for macular disease and therapeutic trials.
Lead Research Organisation:
University of Southampton
Department Name: Clinical and Experimental Sciences
Abstract
Age-related Macular Degeneration (AMD) is a common blinding condition that results in the gradual loss of central vision. This is often associated with difficulties in performing simple daily activities such as reading, driving and recognising faces and is a particular concern for the elderly who are at a greater risk of developing the disease. In fact, one in three people will exhibit some form of AMD by the age of 70. AMD currently affects around 600,000 people in the UK alone and costs the economy £1.6 billion annually. This is predicted to increase as our population ages, where outpatient appointments have risen by 30% over the past 4 years and approximately 200 new AMD cases are reported daily. Although treatments exist for some forms of AMD, these are only applicable to 50% of cases and remain ineffective long-term. Thus, there is no cure for AMD at present, which is largely due to our limited understanding of how the disease develops. This constantly remains under investigation by scientists in order to develop new treatments.
A number of animals are used by AMD researchers to investigate the underlying causes of disease and in the development of new drugs. Of these, rodents are the most commonly used species which have provided important insights into detrimental changes associated with AMD. However, the structure of the rodent eye is markedly different to that of humans and therefore they have been questioned as a relevant model for the study of AMD. In fact, rodents do not have a macula, the specialised part of the eye affected in AMD. Monkeys (non-human primates) offer the closest similarity to the human eye but come with considerable ethical concerns and must be aged for long periods in order to exhibit disease symptoms. There has therefore been a longstanding requirement for a system that is capable of modelling the full disease spectrum of AMD, but that also exhibits fast turnaround times as well as economic viability.
One method of avoiding this difficulty is using cells cultured in a dish, which scientists have used effectively to make discoveries. However, whilst current cell culture models have allowed researchers to investigate individual components of the outer retina and how these are affected in AMD, none have succeeded in modelling the complex relationships and how they may collectively lead to disease. Similarly, no current system models the blood supply to the human retina and how it differs within the macula, the specific region of the eye affected.
In this proposal, we aim to validate and demonstrate the applicability of MacuSIM to AMD research, a novel cell culture model of the outer retina developed by the applicants that is capable of recapitulating the key cell types affected in AMD as well as their reliance on the retinal blood supply. This allows the region of the eye most susceptible to AMD to be investigated outside of animal models for the first time, thus reducing their requirement in AMD research. We will employ cutting edge techniques at the forefront of engineering and physics to manufacture this transformative model and will validate the system for use with various biological applications. Our experiments will then involve demonstrating its relevance to AMD research and benefits over existing methods, to promote its uptake within both the academic and pharmaceutical sectors. Here, we will model increased environmental stresses to the macula and test the ability of compounds to alleviate disease symptoms.
Through this research we hope to provide a more economic and high throughput model that will lead to the faster development of treatments for AMD patients and reduce associated costs to the NHS. In fact, this work will also benefit research into other eye diseases including Best disease, Stargardt disease and retinitis pigmentosa, highlighting the far reaching impacts and benefits of MacuSIM to society.
A number of animals are used by AMD researchers to investigate the underlying causes of disease and in the development of new drugs. Of these, rodents are the most commonly used species which have provided important insights into detrimental changes associated with AMD. However, the structure of the rodent eye is markedly different to that of humans and therefore they have been questioned as a relevant model for the study of AMD. In fact, rodents do not have a macula, the specialised part of the eye affected in AMD. Monkeys (non-human primates) offer the closest similarity to the human eye but come with considerable ethical concerns and must be aged for long periods in order to exhibit disease symptoms. There has therefore been a longstanding requirement for a system that is capable of modelling the full disease spectrum of AMD, but that also exhibits fast turnaround times as well as economic viability.
One method of avoiding this difficulty is using cells cultured in a dish, which scientists have used effectively to make discoveries. However, whilst current cell culture models have allowed researchers to investigate individual components of the outer retina and how these are affected in AMD, none have succeeded in modelling the complex relationships and how they may collectively lead to disease. Similarly, no current system models the blood supply to the human retina and how it differs within the macula, the specific region of the eye affected.
In this proposal, we aim to validate and demonstrate the applicability of MacuSIM to AMD research, a novel cell culture model of the outer retina developed by the applicants that is capable of recapitulating the key cell types affected in AMD as well as their reliance on the retinal blood supply. This allows the region of the eye most susceptible to AMD to be investigated outside of animal models for the first time, thus reducing their requirement in AMD research. We will employ cutting edge techniques at the forefront of engineering and physics to manufacture this transformative model and will validate the system for use with various biological applications. Our experiments will then involve demonstrating its relevance to AMD research and benefits over existing methods, to promote its uptake within both the academic and pharmaceutical sectors. Here, we will model increased environmental stresses to the macula and test the ability of compounds to alleviate disease symptoms.
Through this research we hope to provide a more economic and high throughput model that will lead to the faster development of treatments for AMD patients and reduce associated costs to the NHS. In fact, this work will also benefit research into other eye diseases including Best disease, Stargardt disease and retinitis pigmentosa, highlighting the far reaching impacts and benefits of MacuSIM to society.
Technical Summary
Age-related Macular Degeneration (AMD) is the leading cause of irreversible sight loss in the developed world, accounting for 50% of registered blindness. The disease affects a central region of the retina, termed the macula, and is characterised by atrophy of the Retinal Pigment Epithelium (RPE) and/or choroidal neovascularisation. Disease management currently involves treatment with anti-VEGF inhibitors, although these are only applicable to 50% of cases and remain ineffective long-term. This is due an incomplete understanding of disease aetiology which requires further investigation to devise new treatments. Animal models, including rodents and non-human primates, are employed in AMD research to elucidate factors driving disease onset. However, whilst informative these either lack an anatomical macula (rodents), and/or entail extensive ethics. Associated time frames and costs are also unfavourable compared to in vitro models, which boast additional scientific benefits.
The proposed work aims to reduce and replace the number of animals used in AMD research through the development of MacuSIM, a microfluidic in vitro model that mimics key features of the human macula including the vasculature and environmental stresses specific to macula RPE. We will exploit cutting edge technologies, such as 3D printing and high-precision laser machining to manufacture MacuSIM. Established methods will then be used to validate use for RPE (iPSC-RPE, ARPE-19) and choroidal endothelial (RF/6A) cell culture including immunofluorescence, molecular biology techniques, electron microscopy, ELISA and cell viability assays. This novel approach has the potential to transform research into maculopathies and act as an invaluable drug discovery tool in both academia and industry. Thus, ongoing experiments will demonstrate its benefits over existing methods, where we will recapitulate the oxidative macula environment and test the ability of compounds to alleviate associated pathology.
The proposed work aims to reduce and replace the number of animals used in AMD research through the development of MacuSIM, a microfluidic in vitro model that mimics key features of the human macula including the vasculature and environmental stresses specific to macula RPE. We will exploit cutting edge technologies, such as 3D printing and high-precision laser machining to manufacture MacuSIM. Established methods will then be used to validate use for RPE (iPSC-RPE, ARPE-19) and choroidal endothelial (RF/6A) cell culture including immunofluorescence, molecular biology techniques, electron microscopy, ELISA and cell viability assays. This novel approach has the potential to transform research into maculopathies and act as an invaluable drug discovery tool in both academia and industry. Thus, ongoing experiments will demonstrate its benefits over existing methods, where we will recapitulate the oxidative macula environment and test the ability of compounds to alleviate associated pathology.
Planned Impact
Numerous animal models are employed in Age-related Macular Degeneration (AMD) research to better understand disease pathways and assist the development of new treatments. These include mice, rats, rabbits, pigs and non-human primates. Yet many AMD models do not recapitulate the cardinal features of disease and/or bare limited resemblance to human ocular anatomy. Rodents (mice and rats) are the most widely used species in AMD research, with approximately 3528 reported in the literature per year. However, the rodent retina is markedly different to that of humans and lacks an anatomical macula (the key structure affected in disease). The ability to model this aspect of anatomy is only achievable in non-human primates (NHPs), which develop the major histological and functional changes seen in disease. Still, their use is associated with substantial research costs, extensive ethics, and the time required for AMD symptoms to develop is in excess of 20 years. There has therefore been a longstanding requirement for a model that sufficiently recapitulates the human system, as well as clinical features of AMD, but that is inexpensive and yields rapid results to enhance study efficiency.
'MacuSIM' is a transformative model that fills this void. This novel tool boasts the ability to mimic the human macula in vitro for the first time, and will considerably reduce and replace the use of NHPs and rodents in retinal research. This offers an advanced platform in which to investigate disease associated pathways in the outer retina and conduct high throughput testing of therapeutics. Similarly, it exhibits several economic and experimental benefits over available in vivo models. These include reduced costs, minimal ethics, the timely delivery of results and the ability to interrogate molecular pathways at a unicellular level. We therefore anticipate rapid uptake of 'MacuSIM' by the both the academic and industrial sectors, which will have a widespread impact on animals numbers used in AMD research. For example, our calculations indicate that 'MacuSIM' could replace up to 50% of research involving NHPs, equating to a reduction of between 339 and 573 animals over the next 10 years. This is based on data from the past decade, which predicts the publication of between 16 and 39 papers yearly by 2030 when extrapolated using a linear (R2=0.7353) or second order polynomial (R2=0.7909) relationship respectively. 'MacuSIM' will also replace a sizeable proportion of rodents employed in AMD research through its enhanced similarity to the human system, where reductions of up to 90% are feasible. Whilst at a local level this equates to a reduction of 120 mice per year, the literature suggests the replacement of 227 and 3176 rodents is acheivable annually at national and international levels. In fact, this is likely conservative as we have assumed an average of 36 animals per paper, which is often far exceeded. The impact of 'MacuSIM' on 3Rs metrics will undoubtedly be furthered by its relevance to fields investigating other maculopathies including diabetic retinopathy, Best disease and Stargardt's macular dystrophy.
Throughout the duration of the fellowship, I will promote awareness and uptake of 'MacuSIM' through several avenues to implement such figures into macula research. This will involve presenting research outputs at various conferences/seminars, holding scientific workshops and publishing transparent research articles in academic journals, where we hope to encourage adoption by academic and industrial beneficiaries through the validation and application of 'MacuSIM' . To this end, commercialisation of the system will also be explored to further promote its availability to other researchers and increase 'MacuSIM's' 3Rs impact.
'MacuSIM' is a transformative model that fills this void. This novel tool boasts the ability to mimic the human macula in vitro for the first time, and will considerably reduce and replace the use of NHPs and rodents in retinal research. This offers an advanced platform in which to investigate disease associated pathways in the outer retina and conduct high throughput testing of therapeutics. Similarly, it exhibits several economic and experimental benefits over available in vivo models. These include reduced costs, minimal ethics, the timely delivery of results and the ability to interrogate molecular pathways at a unicellular level. We therefore anticipate rapid uptake of 'MacuSIM' by the both the academic and industrial sectors, which will have a widespread impact on animals numbers used in AMD research. For example, our calculations indicate that 'MacuSIM' could replace up to 50% of research involving NHPs, equating to a reduction of between 339 and 573 animals over the next 10 years. This is based on data from the past decade, which predicts the publication of between 16 and 39 papers yearly by 2030 when extrapolated using a linear (R2=0.7353) or second order polynomial (R2=0.7909) relationship respectively. 'MacuSIM' will also replace a sizeable proportion of rodents employed in AMD research through its enhanced similarity to the human system, where reductions of up to 90% are feasible. Whilst at a local level this equates to a reduction of 120 mice per year, the literature suggests the replacement of 227 and 3176 rodents is acheivable annually at national and international levels. In fact, this is likely conservative as we have assumed an average of 36 animals per paper, which is often far exceeded. The impact of 'MacuSIM' on 3Rs metrics will undoubtedly be furthered by its relevance to fields investigating other maculopathies including diabetic retinopathy, Best disease and Stargardt's macular dystrophy.
Throughout the duration of the fellowship, I will promote awareness and uptake of 'MacuSIM' through several avenues to implement such figures into macula research. This will involve presenting research outputs at various conferences/seminars, holding scientific workshops and publishing transparent research articles in academic journals, where we hope to encourage adoption by academic and industrial beneficiaries through the validation and application of 'MacuSIM' . To this end, commercialisation of the system will also be explored to further promote its availability to other researchers and increase 'MacuSIM's' 3Rs impact.
