Ex vivo human donor retina tissue platform for disease modelling and drug development

 

Summary | Background | Current state of the art
What could your Solution be used for? | Collaboration | 3Rs impact | References

Summary

OcuScience has developed new methods to preserve human retinas from organ donors and make them viable for research for hours after death. We are able to revive and maintain the neural retina from adult human donor eyes in an ex vivo environment and conduct repeated trans-retinal recordings, indicating function which is nearly identical to electroretinography (ERG) seen in living humans. The donor retina can be divided into multiple samples for intra-subject drug testing and screening. Combining OcuScience’s human tissue approach with our proprietary Handheld Multi-species ERG and Ex Vivo ERG Adapter, donor retina tissue which was once discarded can now be used in place of animals for a variety of eye research and drug development applications.


Background

It is known from prior research [1] that retinal tissue has to be recovered and dissected soon after euthanasia to ensure a functional ERG response, a key source of quantifiable data to detect abnormal function of the retina. Working with the Lions Eye Institute for Transplantation and Research in Florida, we have increased the timescale during which recovered tissue can be used and have determined the critical methods needed to capture retina function in ex vivo adult donor human eyes. We now have an established methodology to obtain functional, reliable and repeatable ERGs. In particular, we have a means to sustain the retina in transport with a specialised device  and nutrient solution which assists in preserving the function and tissue integrity; a unique dissection method that reliably yields a region-specific response; and a proven transretinal recording system that is unique in controlling multivariable influences on the data including electromagnetic noise, continuous nutrient perfusion, regulated physiological tissue support (temperature, perfusion rate and pH), and control of input electrode impedance. Each eye yields multiple samples which are processed into Human Retina Biosensors (HRB).

OcuScience has manufactured the versatile Handheld Multi-species ERG (HMsERGTM) for nearly a decade. To support the use of these HRB, we have now combined this with our Ex Vivo ERG Adapter to maintain viability while providing exposure of calibrated light to evoke composite responses from multiple cell types within the neural retina, e.g.. from rod and cone photoreceptors to ganglion cells. Together with simultaneous data acquisition, we have a complete system to test nearly any species of neural retina.  


Current state of the art

The need for and benefit of a human relevant retina model has been well described [2, 3].  Laboratories around the world are investigating the use of stem cells and cell therapy to “build” a fully complex and functional human retina model. OcuScience has taken an alternative approach by developing tools to enable analysis of readily available and functional ex vivo tissue. The inherent advantages of using adult human donor tissue over the current state of research in stem cells and fetal tissue strategies provide a leap forward compared to existing models.

OcuScience has demonstrated proof of concept of drug testing in ex vivo human tissue with several compounds known to affect ERG responses. The system stability and repeatability of the data make it possible to introduce compounds and quantitatively observe the change between baseline and treatment in a dose-dependent manner (see figures A & B below).

Figure A: Ex vivo Flash ERG intensity series of photostimulator evoked responses from a 73 year old male donor retina. This shows photoreceptor and retinal function are nearly identical to human in vivo retinal function morphology. Blue = 1 mcd.s/m-2, Black1 = 10 mcd.s/m-2, Red = 100 mcd.s/m-2, Green = 1,000mcd.s/m-2, Magenta = 3,000 mcd.s/m-2, Black2 = 10,000 mcd.s/m-2

Figure B: Example data showing change in response of HRB in the presence of 2-amino-4- phosphobutyric acid (APB), an on-bipolar pathway blocker, demonstrating data acquisition for drug testing. Baseline (Perfusion Only): Blue = 3,000 mcd.s/m-2, Red = 10,000 mcd.s/m-2. Treated (Perfusion with APB): Green = 3,000 mcd.s/m-2, Black = 10,000mcd.s/m-2.

We observe several significant and novel advantages and potential impacts of our technology over existing testing methods:

  1. The dose response and therapeutic profile may be more predictive compared to animal in vivo and in vitro studies, since the model is utilising human tissue to investigate human disease.
  2. Our system makes it possible to conduct screening of various compounds to measure their physiological effect on human donor retina. By accelerating the identification of target molecule physiological effect on the retina, retinal pigment epithelium (RPE), and similar neurological tissue, we will be able to efficiently identify candidate drugs for many human diseases.
  3. There is a huge opportunity to access a nearly infinite supply of tissue from adult human donor tissue to conduct pharmacogenomic studies to better understand genetic effects on drug response.
  4. We foresee a myriad of adjunct data being collected in conjunction with Ex Vivo ERG. For example, OcuScience already has in house experience with Optical Coherence Tomography (OCT) to combine structural with functional tissue data.

What could your Solution be used for?

Our system is a novel drug development and research tool using a unique adult human donor tissue model and represents a significant improvement in testing pharmaceuticals for toxicology and pharmacological effects [4, 5, 6]. A major advantage of our technology is our ability to test donor tissue from human diseases that are known to affect neurotransmission of photoreceptors, glial cells and ganglion cells such as Age-related Macular Degeneration (AMD), Diabetic Retinopathy and Retinitis Pigmentosa.

The technique could also be used by researchers to monitor the effects of drugs on the central nervous system by observing the effect on retinal nerves. The implications for advancing neurological drug testing using functional donor tissue is a brand new opportunity and we are uniquely positioned to deliver a novel service to pharmaceutical companies using the human retina biosensor.  Our system has the potential to better predict safety and efficacy prior to human clinical trials. By using ex vivo retinal ERG responses, we will now have a closer analogy to a living human and drug effects can be tested in human retina tissue before advancing to human studies.


Need for collaboration

Researchers:
We are looking to work with collaborators who have expertise in various diseases of the retina to provide direction and validation of our human disease models.  We also seek alliances in using “big data” strategies to find candidate drugs and identify pharmacogenetic effects as a novel way to improve safety and efficacy.

OcuScience wants to share our knowledge about ex vivo ERG technology. We provide a variety of ways for researchers to learn how to utilise our technology specifically in their area of expertise.  We also provide the tools to researchers to conduct preliminary ex vivo studies in their own laboratories, prior to testing their compounds in the human retina biosensor.

Clients:
OcuScience is starting a contract research service focused on processing human donor retina for preclinical research and drug testing. We are fully prepared to test client compounds to their specification, conduct ERG functional testing, and structural analysis using OCT. We expect to process 5,000 HRB in the first year and plan to provide our service to any institution or pharmaceutical company to conduct exploratory experiments or test the functional effects of their compounds using our ex vivo HRB. 

Donor Networks:
We would like to collaborate with experts in global organ donor networks. We intend to recover and process tissue worldwide to obtain pharmacogenomic data from various populations and ethnicities around the world, making future drugs safer and more effective.


3Rs impact assessment

Reduction:
Our Solution promises to reduce the use of animals in the discovery of new ophthalmic drugs as well as screen out a significant number of compounds that would fail later in the pipeline, thus avoiding unnecessary animal testing. Although there are estimations of animals used in research, no specific information exists on the number used in ophthalmic and neuroscience in vivo models.  One estimation of the impact of other ex vivo ocular safety testing assays suggest a possible 10% reduction in live animal testing [7]. Given the expanded ability of our HRB, we feel confident that more than 10% of animals used in retinal and CNS safety, toxicity, screening, and dose-response analysis may be spared. As well as reducing animal use in drug discovery/efficacy studies, our biosensor could also be used in basic research, which would not only reduce animal use in this area but would also better inform the development of appropriate drugs for certain conditions.

Replacement:
The HRB could replace live animal testing where the effect of a compound is tested using electrophysiology. Our technology allows tissue structural, immunoassay, and other histological data to be collected alongside functional ERG data, and correlated to this over time. An estimated 3,000 articles are published annually which include data from animal testing of “back of the eye” and CNS effect of compounds. Assuming 50-100 animals could be replaced in each study using our technology, we project 150,000 to 300,000 may be saved annually by adoption of human donor retina testing.


References

For a comprehensive list of publications, please go to http://www.ocuscience.us/pages/publications

  1. Frank RN, Dowling JE (1968). Rhodopsin photoproducts: effects on electroretinogram sensitivity in isolated perfused rat retina. Science 161(3840):487-489.
  2. NC3Rs CRACK IT Challenge: Retinal 3D. http://nc3rs.org.uk/challenge-23-retinal-3d.
  3. National Eye Institute 3-D Retina Organoid Challenge. https://nei.nih.gov/3DROC.
  4. Vinberg F, Kolesnikov AV, Kefalov VJ (2014). Ex vivo ERG analysis of photoreceptors using an in vivo ERG system.. Vision Research 101:108-117.
  5. Kolesnikov AV, Kefalov VJ (2012). Transretinal ERG recordings from mouse retina: rod and cone photoresponses. Journal of Visualized Experiments 14(61) pii:3424 doi:10.3791/3424.
  6. Vinberg F, Kefalov VJ (2015). Simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system. Journal of Visualized Experiments 6(99) e52855 doi:10.3791/52855. 
  7. Hood E (2008). Alternative Test Models: Ocular Safety Assays AcceptedEnvironmental Health Perspectives 116(9):A381.

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