Construction of an immuno-competent and self reporting human lung model using nanosensor incorporated scaffolds

Lead Research Organisation: University of Nottingham
Department Name: Sch of Molecular Medical Sciences

Abstract

The study of human lung biology has a huge impact on our understanding of the disease process in a number of lung conditions such as asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD), disorders which have significant health and socioeconomic implications worldwide. At the moment, it is difficult to carry out such research on humans, because in many cases it is not safe or procedures are too invasive, and the use of animal models is not always appropriate. For instance, mice do not develop asthma naturally which suggest that the biology of their lungs is different to that of humans. These limitations in the availability of physiologically relevant human lung models are therefore set to continue having a negative knock-on effect on the search for novel targets and molecules for therapeutic interventions. For example, despite enhanced patient care, the morbidity and mortality of asthma has remained high with one asthma related death every 19 minutes and 20 million lost working days per annum in the UK alone. This is partly due to lack of efficient therapeutic strategies and the fact that a large proportion of patients do not respond to treatment. What we want to do in this project is to develop a model of the human lung in the laboratory using cells previously isolated from donated tissue or blood. We will grow these cells on materials that contain sensors that can pick up changes in, for example, oxygen, glucose and acidity. These sensors will allow us to observe how cells respond to stimulation in real time. By growing these cell types on these materials, we can arrange them into layers such that the cells are grown in the laboratory in the same position as in the lung. Such a model would give scientists and pharmaceutical companies a better tool for investigating some aspects of human lung biology, identifying new targets for treatment and testing new drug compounds.

Technical Summary

The study of human lung biology has a huge impact on our understanding of the pathophysiology of lung diseases. Unfortunately, such studies in human have been hampered by technical and ethical issues which have led to the use of animal models that in most cases have little relevance to human disease. These limitations in the availability of physiologically relevant human lung models are thus set to continue having a negative knock-on effect on the search for novel targets for therapy. Thus, there is a real need to develop biotechnologically-advanced human-based lung models which can be used for studying disease pathogenesis, identifying novel therapeutic targets and assessing response to new drug leads. There are at present only a few in vitro models of bronchial mucosa and none that incorporate the immune elements of this tissue, which are essential for sensing cellular and environmental changes, as well as exerting a crucial role in the pathogenesis of lung diseases. This, therefore, questions the physiological relevance of existing models in studying lung disease pathogenesis and intervention. Another equally important shortfall in the existing models is the lack of appropriate means for non-invasive, real-time and integrated monitoring of cellular responses. To circumvent these limitations, we propose to develop an immuno-competent and self-reporting human lung model comprising dendritic cells, epithelial cells and fibroblasts within a perfusable 3D environment on nanosensor-incorporated scaffolds. The presence of the key cell types and the use of nanosensor-incorporated scaffold sheets will enable us to interactively monitor the microenvironment, cell-cell interaction and cell activation without disturbing the 3D architecture of the model. The adequate cellular and structural representation of the lung tissue and being amenable to in situ monitoring make this model an invaluable tool for research in lung biology, disease modeling and drug discovery/delivery.

Planned Impact

This proposal directly addresses the BBSRC's Research and Policy Priority areas of Bionanotechnology, Synthetic Biology and the 3Rs priority for reduction, refinement and replacement of animals, specifically for the purpose of assessment. Those who benefit from this research include scientists with an interest in nanotechnology, lung biology, immunology biomaterials, tissue engineering and developing 3D in vitro models. Another set of main beneficiaries are the many industries involved in the translation and use of these models, policy regulators and the general public at large. The wider beneficiaries will be as follows: The research staff: The research staff employed on this grant will gain interdisciplinary expertise as a result of the project, through collaborative working with researchers from distinctly different fields of science (material science, nanotechnology, tissue engineering, immunology and imaging). It will bring opportunities to publish in new areas, present to a diverse audience, and to strengthen and develop the direction of their careers. Policy regulators: If this technology is successful and enters routine practice, the avoidance of animal use will directly address the 3Rs criteria. Since March 2009 European directives now impose a marketing ban on any cosmetic substances that involve animal usage, and as such a ban on animal use for the purpose of cosmetic testing is effectively now in place. Alternative methods must be used where possible, however the scientific rigour and relevance of many alternative tests is presently lacking. Our technology addresses the need to develop more relevant models while directly addressing new regulatory guidance requiring such change. Industry: This an IPA application and is supported by Kirkstall Ltd., a British company with extensive expertise in developing high throughput screening technologies and multi-chamber bioreactors for growing different tissues. Once the methodology for creating immuno-competent lung epithelia is developed and shown to be effective, it is hoped that it would be translated via industry and become available for routine use. Kirkstall will not only be involved in developing and validating the model, it would also be an ideal vehicle for manufacturing of customised constructs on a commercial basis. This would bring a range of benefits including employment, expertise, and future research and development opportunities. A realistic timescale would be that this could be achieved within 5-7 years. This work would place the UK at the forefront of 3D immuno-competent epithelial models. Currently there are two ECVAM approved epithelial models available for testing. However, both are single cell-type models and as such do not take in to account epithelial-stromal interactions, or immune cell interactions. It is clear from existing research that 3D models populated with these cells give a much more relevant response than single cell type models, so there is an opportunity here for the UK to lead this field, bringing associated economic benefits. General public: The general public as consumers will be exposed directly to a multitude of environmental chemicals. Under European legislation all raw material chemicals must be assessed under REACH legislation. General public concern exists in the use of animal models for such assessment, and so engagement with the public using 3D in vitro models as a basis for accurate and relevant alternatives should be viewed as extremely important. Animals: The major potential beneficiaries of this study will be animals originally utilise

Publications

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Ghaemmaghami AM (2012) Biomimetic tissues on a chip for drug discovery. in Drug discovery today

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Haycock, John W.; Ahluwalia, Arti; Wilkinson, J. Malcolm (2014) Cellular in Vitro Testing: Methods and Protocols

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Morris GE (2014) Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds. in American journal of physiology. Lung cellular and molecular physiology

 
Description The development of a 3D model lung has progressed by optimising the scaffolds that the epithelial cells and fibroblast cells are seeded upon. The scaffolds are electrospun with various fibre diameters to create architecture that resembles human lung tissue.



Furthermore, nanosensors have been incorporated into scaffolds which will allow non-invasive monitoring of analyte concentrations within the 3D structure. Nanosensors that can detect both oxygen and pH at the same time are currently being investigated and if successful will be incorporated into the electrospun scaffolds.



To mimic in-vivo conditions, epithelial cells are cultured at the air-liquid interface (ALI) whilst the fibroblasts remain submerged in media. It has been established that co-culture of the two cell types provides optimal conditions for epithelial cell differentiation as evidenced by monitoring tight junction formation using Trans Epithelial Electrical Resistance (TEER) measurements within commercially available Transwell inserts. To achieve ALI cultures using our novel electrospun scaffolds we have designed and manufactured a device we call "ScaffHolders". It consists of a PTFE platform and a ring which securely hold the scaffolds at the ALI. This allows the TEER measurements and permeation analysis of tight junctions.



To compare static cell culture to flow conditions Quasi Vivo bioreactors are being utilised.

The bioreactors have undergone development work as initially they did not prove leak proof or allowed culturing cells at ALI. The new design is now proving successful and has undergone leak testing using our experimental set-up and the flow rates using a peristaltic pump have been calibrated. A suitable PTFE platform to support and position the scaffold correctly within the ALI bioreactor has been designed and manufactured and is currently being used to support single cell cultures of either epithelial cells or fibroblasts to determine optimal flow rates for their growth.
Exploitation Route Pharmaceutical and Chemical industries within the context of safety testing, disease modelling and testing new therapeutics
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description In this study we developed an immune competent model of human respiratory epithelium. The model is currently being used for testing the effect of different allergens on airways (industrial collaboration). We are also using the model to investigate upper airway infections (academic collaboration)
First Year Of Impact 2014
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description A platform of tissue engineered human lung models to study the pathophysiology of asthma
Amount £500,000 (GBP)
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Private
Country United Kingdom
Start 09/2010 
End 12/2013
 
Description Local Immunomodulation around implants by innovative auxiliary hydrogel-based systems encapsulating autologous and phenotype controlled macrophages.
Amount £5,600,000 (GBP)
Funding ID 602694 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 10/2013 
End 09/2017
 
Description Personalized And/Or Generalized Integrated Biomaterial Risk Assessment
Amount € 7,992,471 (EUR)
Funding ID 760921 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 12/2017 
End 01/2022
 
Description Sensor Integrated Biosensors (SIBs)
Amount £260,091 (GBP)
Funding ID 101101 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 05/2012 
End 04/2014
 
Description Using microscale technologies in tissue engineering of human lung
Amount £4,850 (GBP)
Funding ID BB/I02643X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2011 
End 09/2011
 
Description A microfluidic device for studying innate-adaptive immune cell interaction 
Organisation Brigham and Women's Hospital
Country United States 
Sector Hospitals 
PI Contribution Using a microfluidic device to establish a multi cell culture of immune cells under flow conditions
Start Year 2012
 
Description 'The Politics Show' 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Appearance on the BBC programme 'The Politics Show' for East Midlands today 2011 regarding a feature on alternatives to animal testing.

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Nottingham Student Radio 2012 interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Media (as a channel to the public)
Results and Impact Interview on Nottingham Student Radio 2012 about Fliss Rose's research

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Outreach Activity - Tissue Engineering 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact An interactive outreach activity delivered to A-level students based upon tissue engineering taken into local schools to promote a better understanding of science and to encourage young people embark in a career in science. An interactive presentation including props of actual or imitation scaffolds used for seeding different cell types to produce various tissues to assist the understanding of tissue engineering research.

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Roberts project mentor 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact Roberts project mentor which provides training in A'level student outreach. Assist Outreach participants to produce a suitable presentation/acivity for delivery to 'A' level students

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
URL http://videolectures.net/mlsb2012_ferrari_active
 
Description Royal Society of Chemistry ChemNet Ambassador 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach local
Primary Audience Schools
Results and Impact ChemNet Ambassadors deliver an interactive presentation to local schools to introduce students to chemical sciences and the potential careers that science offers. A training course provided by the Royal Society of Chemistry to support ambassador involvement has been attended. An interactive presentation demonstrating how chemistry is used within other scientific reserach e.g. materials science, nanotechnology and pharmaceuticals.

no actual impacts realised to date
Year(s) Of Engagement Activity 2011