A tissue engineered corneal epithelium replacement for animal testing using human stem cells

Lead Research Organisation: University of Reading
Department Name: Pharmacy


The use of human stem cells in the treatment of serious disease or injury is currently undergoing a phase of rapid development. This intensive research has already produced new treatments for blindness. UK and Japanese scientists are now growing adult stem cells taken from donor corneas (the transparent tissue at the front of the eye) on specialised membranes to create new functional tissue. These regenerated tissues are then transplanted onto the surface of diseased or injured eyes leading to a restoration of the patients' sight. The use of human stem cells need not be limited to clinical theraputic use i.e. aimed at re-establishing vision in humans, they can aslo be used to significantly reduce the number of animals used in medical research. Presently, thousands of rabbits are needed every year to test for the toxic potential of new drugs and cosmetics. The method used in testing on rabbits eyes has changed little over 50 years. Now, by employing the scientific advancements recently made in therapeutic stem cell research to the manufacture of a human tissue based replica in culture, we aim to develop a human stem cell based non-animal alternative to drug and nanomaterial testing. The advantages of this are primarily that the effect of new chemicals/materials can be assessed without any animals being harmed and since the replica tissue is formed from human stem cells the results are also more representative. Furthermore, our non-animal model can assess water insoluble materials such as nanomaterials, something the animal models can not do well. To develop a human stem cell based non-animal alternative to drug testing we will essentially recreate the surface of the human eye in a plastic dish. Using adult stem cells isolated from adult corneal donor tissue and building upon the techniques sucessfully employed for corneal stem cell transplantation, we will identify a suitable protocol and substrate capable of both supporting the formation of a functional corneal epithelium in vitro and being easily sourced. The suitability of the newly developed human stem cell based non-animal alternative to drug testing will be established by vigorous testing of the model's response to known toxic and non-toxic agents. The model's response to toxicity testing will be robustly assessed by microscopy and established toxicity assays.

Technical Summary

We will apply our knowledge of clinical human corneal stem cell transplantation and cornea/amniotic membrane collagen fibre structure to construct a model specifically designed to make possible an accurate non-animal investigation into the effect chemicals and nanoparticles have on the surface of the eye. Corneal stem cells will be isolated from human donor tissue and expanded upon a specifically engineered collagen sheet. The collagen sheet will manufactured in such a way as to closely resemble the native structure underlying corneal epithelial cells within the eye. The bespoke collagen substrates ability to support ex vivo stem cell expansion and epithelial startification will be compared against a previously established and clinically sucessful substrate for in vitro corneal epithelial cultivation, that being amniotic membrane. Validation of the stem cell based model, as a non-animal alternative for oculotoxicity testing, will be accomplished by comparing the dose-dependent toxic effect of a range of chemicals with known toxicity. Furthermore, the toxicity of novel nanoparticles on ocular surface function will also be investigated. Epithelial toxic damage, to our stem cell based model, will be measured directly by microscopy and indirectly by cell toxicity assays.


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Connon CJ (2010) The variation in transparency of amniotic membrane used in ocular surface regeneration. in The British journal of ophthalmology

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Mi S (2012) Tissue engineering a fetal membrane. in Tissue engineering. Part A

Description We have successfully developed a method to create tissue constructs from multiple cell types and a dehydrated collagen gel. Our method retains cells in a physiological and functional state. So far we have used this method to tissue engineer two important tissues (ocular surface and fetal membrane). The method and creation of an artificial ocular surface has been granted patent protection. The patent covers the use of this construct for clinical transplantation to the cornea and ocular toxicity testing as an alternative to animal testing. The method has also enabled the creation of a powerful new corneal model that for the first time can begin to investigate the effect of collagen density and ECM stiffness on both epithelial and stromal stem cell differentiation.

We have created a way to produce collagen substrates with tractable stiffness and importantly de-couple topography from the changes in stiffness (i.e. fibrillar collagen substrates with different mechanical properties but the same surface topography). With this technology we can now design tissue constructs with pre-defined levels of cellular differentiation in the final product. This is important because in situ any tissue will have an established ratio between undifferentiated and differentiated cell types, with our technology we can now facilitate this. We can also use this to model the effect of substrate stiffness on cell behaviour and investigate the importance of a soft tissues mechanical properties to its function.

Finally we have discovered that a dehydrated collagen gel can replace the use of amniotic membrane in ocular surface repair. We have recently discovered that it is the amniotic membranes mechanical properties which give it its therapeutic effect and that by tuning the stiffness of our collagen gels to that of amniotic membrane and similar therapeutic effect should be made. Therefore defined collagen gels with a suitable stiffness could replace the use of poorly defined amniotic membrane for ocular surface repair.

Update for 2020
We have now spun out a company 3D Bio-Tissues Ltd which uses some of the methods developed here to grow transplantable corneas
Exploitation Route Our improved ocular surface model illicts more accurate results when compared to current oculotoxicity tests. Therefore could be used an improved drug toxicity test. An International patent has been awarded for an ocular graft for use as a toxicity test or corneal replacement.
We are now using the collagen constructs as model of tissue compliance for ongoing MRC grant. Without this model we would not have been able to recreate different levels of tissue stiffness.
Also Queen Marys University are now also using this model
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.independent.co.uk/news/science/cornea-3d-printing-eye-transplant-blindness-cure-scientists-newcastle-university-research-a8377206.html
Description Findings formed part of patent application and several publications. Findings have supported a commercial company (TAP) which produces commercial product RAFT based upon our original findings Spun out a company 3dbiotissues.com
First Year Of Impact 2019
Sector Healthcare
Impact Types Economic

Description Smart Materials for Wound Healing: A New Fast Acting in situ Method to Treat Skin and Eye wounds
Amount £122,000 (GBP)
Funding ID BB/J019836/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 01/2014
Description Studentship
Amount £45,000 (GBP)
Organisation Newcastle University 
Sector Academic/University
Country United Kingdom
Start 01/2015 
End 12/2018
Title 3d printing the cornea 
Description We have developed a sophisticated way to 3D print functional cornea 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? Yes  
Impact 2018 CellInk Innovation Award 2018 London Design Awards Winner Presentation of technology to: Royal College of Ophthalmologists Royal Society of Medicine Norwegian Ophthalmic Society New York Optometry Society UK Optometry Society 
URL https://mashable.com/video/newcastle-university-researchers-3d-print-cornea/?europe=true#0fbmIJLyWaq...
Description The present invention relates to the use of a plastically-compacted collagen gel as a substrate for the growth of corneal cells, particularly limbal corneal epithelial stem cells. Cells grown on such a substrate can be cultured to produce artificial ocular epithelia which can be used in ocular toxicity testing or for transplantation. 
IP Reference WO2010133853 
Protection Patent application published
Year Protection Granted 2010
Licensed Commercial In Confidence
Impact Development of model system which underpins MRC grant and research into corneal biology
Description The Company "3D Bio-Tissues" (3DBT) has identified a significant and unique commercial opportunity in the rapidly developing 3D bio-printing market. We will offer superior, transplantable corneal replicates for the treatment of corneal stromal disorders. These tissue engineered replicates will uniquely conform to the hosts' needs in terms of shape and thickness, providing adequate cells and materials for the restoration of the patients' sight. 
Year Established 2018 
Impact Will employ technology developed by Prof Connon on 3D printing the cornea
Description Interviews for national news 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Press release associate with paper describing new way to grow human corneas then led to 2x TV news interviews (BBC and ITV) and BBC Radio NE
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/news/uk-england-tyne-41685982