Direct visualisation of epithelial fluid transport at the subcellular scale

Lead Research Organisation: University of Nottingham
Department Name: Faculty of Engineering

Abstract

Epithelial cells are the specialized cells which form the barriers between tissues of the body and the inside or outside worlds. Different organs have different functions and different internal environments, which are set up and maintained by the transporting activities of epithelial cells. Epithelial cells form polarised sheets whose apical and basolateral "ends" face in opposite directions across the barrier. This polarity allows epithelial sheets to pump fluid in a directed fashion to maintain the normal physiology of individual tissues and the entire body. These fluid pumping processes are crucial to normal health and often found to be awry in various disease states. For example, disrupted fluid secretion in cystic fibrosis patients affects the function of their lungs, digestive system, and other organs all through this common pathway. In the eye, disturbed fluid regulation is believed to be responsible for glaucoma, cataract formation, macular degeneration, and retinal detachment. Between them, these diseases cause the majority of blindness worldwide.

The retinal pigment epithelium lies behind the neurons of the retina, and performs a series of functions to make sure vision is preserved by doing the "housekeeping" for the layers of nerve cells. The pigment epithelium is black to avoid reflections within the eyeball, and reaches out tiny fingers to wrap around and protect the vital photoreceptor cells which do the actual "seeing". When the epithelium is disrupted or injured, fluid can accumulate between the neurons and the epithelium like a blister, keeping the epithelium from helping the neurons to work properly. We still don't understand exactly how the epithelium works normally to prevent this from happening, and would like to know how to help the cells repair such damage by boosting their fluid pumping abilities in the right way.

A lot of what goes on when epithelia pump fluid happens between the cells, in a very confined space. Salt is pumped into these tiny spaces and water follows along to create secretion of fluid. Scientists are still not sure exactly how this is done to precisely match the balance of salts and water required for particular functions - from tears and sweat to urine and bile, these secretions can vary widely in their properties. The only trouble is, these tiny spaces which are so important are also too small to look inside very easily.

This project will develop a new tool which will help us understand epithelial fluid transport. It's actually a combination of tools, all being applied together, which will give us information about what the whole tissue is doing - from the large scale right down to the very small. Two new techniques are proposed (one an optical measurement, and one an optical stimulation technique) which will make measurements of salt and water movements in the tiny spaces we believe are crucial to fluid transport. By applying them together with measurements at the large scale, we can then perform experiments to see how the secretion of salt affects secretion of water, and vice versa. It's a bit of a chicken and egg problem, but attacking it by making many measurements at once should give us the answers we are looking for to understand what's going on.

If we understand fluid transport better we can make better choices in treating diseases resulting from fluid transport problems. In particular, this project will give hope to those with retinal disease which might be better treated by strategies we develop during this research project. We hope to make the system available to other researchers looking at fluid transport in other tissues too, so that the benefits of new technology can have the widest impact possible.

Technical Summary

Transport of ions and water by epithelia is essential to homeostasis at the whole body, tissue, and cellular levels. Polarised epithelia use defined sets of ion transporters to accomplish directed movement of fluid transmurally. Most puzzling is the transport of apparently isotonic fluid, in the absence of detectable driving gradients. Several theories explain this by establishment of local ion and/or osmotic gradients within restricted intercellular spaces. These local gradients drive secretion by attracting water and solutes to produce vectorial fluid transport. The tiny geometries believed to be essential to this process have been historically difficult to probe.

Development of a system to address this problem is proposed, enabling direct measurement of water flux correlated with transcellular and paracellular pathways available to transported fluid. Retinal pigment epithelium (RPE), an important fluid transporting tissue, will be cultured on permeable membranes and probed using Raman microspectroscopy to image the movement of D2O ("heavy water") across the barrier. Measurements will be combined with perfusion and electrophysiology to manipulate the biophysics of epithelial transport, and watch the response in terms of water flux at the micron scale across the tissue. Included in the proposal is the deployment of "optogenetics" to introduce light-sensitive transport pathways into the epithelium, which will allow the system to be driven non-invasively with pulses of light to either shunt particular cells by opening cation channels across the membrane (Channelrhodospin-2), or by accumulating Cl- by activating an electrogenic anion pump (Halorhodopsin).

Applying these methods in concert with transmural electrodes to measure or impose voltage and current clamp will allow the process of epithelial fluid transport to be investigated at a level never before seen, and will unmask the fundamental processes by which isotonic fluid transport is obtained.

Planned Impact

Epithelial fluid transport lies at the heart of bodily homeostasis - maintaining fluid and ion balance globally throughout the body and within individual organs or compartments. Some of the seminal discoveries in medicine have related serious disease to defects in epithelial transport, among them cystic fibrosis, kidney disease, gut defects, and ophthalmic diseases. This project will provide a technology base capable of increasing knowledge of these conditions and in many other contexts, by extending observations of fluid dynamics into the subcellular scale; illuminating fundamental biophysical processes which may then be manipulated for clinical benefit. The proposed investigation of retinal pigment epithelium has direct relevance to cutting-edge treatment of retinal diseases such as macular degeneration, retinal detachment, and retinitis pigmentosa. The ARMI retinal modelling collaboration is intended as a tool both for academic research and ultimately as an adjunct to clinical decision making, founding in a rational understanding of fundamental biophysics and cellular physiology. The project will therefore impact the knowledge base which informs the choice of clinical strategies, and potentially lead to the development of novel strategies superior to the current approaches to treating retinal disease.

As well as developing biomedical knowledge, the proposed project will yield a novel multimodal imaging platform which will be applicable to the study of any transporting epithelium or indeed any other tissue. In a broader context, the technology of ratiometric Raman imaging of water flux has potential application in other disciplines such as plant biology, soil science, oceanography, and other areas where water dynamics on a micro scale is important. The instrument proposed could be fitted in principle with any objective lens and used at scales both smaller and larger than that proposed while using fundamentally the same technology. Water flux imaging using D2O as a biocompatible tracer may also be useful in design and optimisation of bioreactors, microfluidic systems, and in evaluation of tissue perfusion in pharmacokinetic or similar studies, both in vitro and in vivo in human and animal subjects.
 
Description We have developed a novel nanomaterial (very thin magnesium fluoride) which is an ideal, nanoporous substrate on which to grow epithelial cells. We have also confirmed both that cells can grow on these nanomaterials as well as their suitability for the latest high-resolution imaging methods using a Raman microspectrometry system also developed in our laboratory. Using these tools we are able to image the movement of water through and across the cells, with the goal of measuring epithelial transport as a platform for drug discovery, physiological research, and personalised medicine. We have further demonstrated that these methods are applicable to the real-time visualisation of fluid transport in living plant tissue, and obtained funding from the Leverhulme Trust to pursue the work in this important area of water resiliance and the assessment of plant physiology and water stress in real time.
We have further discovered a novel biosensing technology based on these nanoporous membrane devices (SERS biosensors), with major potential to revolutionise current approaches to diagnostics and point-of-care monitoring of biomarkers and bioanalytes. This technology has been jointly patented between the Universities of Nottingham and Rochester and is being developed for commercial exploitation by industrial partners in the USA (SiMPore Inc, Rochester, NY) and Europe (ibidi GmbH, Munich, Germany).
With industrial collaborators in New Zealand (Kode Biotech Ltd) we have further developed novel surface chemistries and molecules for the polarisation and differentiation of mature epithelial tissue models on arbitrary materials - both porous and non-porous. This work is being IP protected and will be commercialised by Kode Biotech Ltd via newly-created Kode Diagnostics Ltd, under a joint licensing agreement which is being negotiated between the parties.
A serendipitously discovered family of small molecules has further been shown through our work as being capable of modulating tight junctions in a non-invasive and reversible fashion, which is currently being assessed as a drug delivery enhancement agent for immunotherapies and chemotherapies, aiding their delivery across tissue barriers to their targets within the body. This represents early-stage development which has already attracted the interest of NHS clinicians as an adjunctive prospective therapeutic delivery enhancer of macromolecular therapies across tissue barriers in a reversible fashion.
Exploitation Route Our novel devices and methods to grow and investigate cultures of epithelial cells may become a new standard for physiological studies, providing non-invasive readout of key transport processes against which novel drug targets, compounds, therapies, or personalised approaches may be tested.
Our novel biosensor technology is a revolutionary platform allowing unprecedented sensitivity and functionality in the sensitive and reproducible quantitation of bioanalytes in healthcare as well as industrial contexts.
Our drug delivery enhancement agents offer the potential to deliver next-generation macromolecular therapies across tissue barriers non-invasively, using a fraction (1/1000) of the currently required dose.
Our non-invasive label free method of directly imaging water transport within and across living biological tissues is finding uptake across the field, including the physiological assessment of water transport and uptake in living plants.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Our research has provided a new platform for the study of epithelial transport processes, which may lead to a step change in personalised medicine by providing epithelia on a chipas well as new devices for market which are currently under technology transfer with a USA spinout company from the University of Rochester (Simpore Inc). In addition to these novel sample preparations and cell culture substrates, we have translated the nanoporous membrane technologies co-developed with Rochester into a new class of SERS biosensor which is presently in a rapid development phase for commercialisation between a consortium of academic (Nottingham, Rochester) and industrial (SimPore Inc, Ibidi GmBH) partners. We have transformed this family of novel nanomaterials into a platform technology capable of SERS biosensing, cell culture, and real-time assessment of small and macromolecular transport across and within living systems. In addition we have jointly created (with Kode Biotech Ltd) a pair of novel surface chemistries to encourage the proper polarisation of epithelial barrier tissue models on arbitrary substrates and material porosities. In addition we have used this platform of novel bioreactors to develop and test a novel family of drug delivery enhancement agents capable of booosting drug delivery across these layers in vitro. We have also developed in paralell a set of epithelial tissues from the gut, eye and lung which are capable of optical modulation of their fundamental physiology, with the goal of controlling and manipulating physiology in these important model systems non-invasively using photonic means. In the future, these new epithelial microdevices will offer a platform technology for the assessment of physiology, drug delivery and toxicity in vitro, and replace animal experiments by providing an alternative human-relevant means of assessing impact on physiology and health in vitro. A new, multi-lateral collaboration between academic (Nottingham, Rochester, RIT) and industrial (Kode Biotech, ibidi, SiMPore) partners, whose scope and scale will be further cemented and broadened by the upcoming Munch symposium in September 2018 and, hopefully, one additional international symposium at Nottingham in summer 2019 - approval for this further activity is pending from BBSRC, via a requested no-cost extension to the award.
First Year Of Impact 2016
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description HERMES Travel Fellowship
Amount £3,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 06/2014
 
Description Hermes Innovation Fellowship
Amount £27,500 (GBP)
Funding ID Commercialisation of a nanoporous, re-writable, SERS biosensor 
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 07/2016
 
Description International Collaborators Fund
Amount £6,500 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 07/2017
 
Description NSF PFI:ATT
Amount $199,000 (USD)
Organisation National Science Foundation (NSF) 
Sector Public
Country United States
Start 06/2016 
End 06/2018
 
Description Proximity to Discovery
Amount £16,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 06/2016 
End 02/2017
 
Description Research Project Grants
Amount £283,147 (GBP)
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 11/2016 
End 11/2018
 
Description UNICAS Interdisciplinary research fund
Amount £15,000 (GBP)
Organisation University of Nottingham 
Department University of Nottingham Interdisciplinary Centre for Analytical Science (UNICAS)
Sector Academic/University
Country United Kingdom
Start 04/2013 
End 08/2013
 
Description University of Nottingham Regenerative Medicine and Stem Cells Research Priority Area
Amount £15,000 (GBP)
Funding ID A2QRVX 
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 07/2018
 
Description University of Nottingham Research Board Discipline Bridging Award
Amount £47,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 02/2014 
End 02/2015
 
Description Whitaker Foundation Travel Fellowship
Amount $15,000 (USD)
Organisation Whitaker Foundation 
Sector Charity/Non Profit
Country United States
Start 07/2013 
End 10/2013
 
Description Whittaker Foundation International Scholars Program
Amount $34,500 (USD)
Organisation Whitaker Foundation 
Sector Charity/Non Profit
Country United States
Start 08/2015 
End 07/2016
 
Title Hydrodynamic Raman imaging 
Description We have established a method for the assessment of fluid movement within and across cultures of biological cells using non-invasive Raman Microspectroscopy, employing a naturally-occurring "heavy" isotope of water (deuterium oxide). By this method, direct physiological measurements can be carried out of key epithelial transport processes in preparations of living cells - epithelia on a chip. 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact This technique is early in its development, but has resulted in the creation of a novel nanoporous nanomaterial to support living cell cultures to ensure compatibility with this novel method. 
 
Title Imaging hydrodynamics within plant tissues 
Description We have developed our novel Raman microspectrometer into an instrument capable of revealing in real time the hydrodynamics within living plant roots as they take up deuterated water. We are able to visualise its movement within and across tissue barriers and to begin to unpick the subcellular basis of epithelial water transport in this novel system. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2016 
Provided To Others? Yes  
Impact We are collaborating with the Centre for Plant Integrative Biology at Nottingham to explore hydrodynamics in roots of Arabidopsis plants explsoed to heavy water tracer agents. This is providing the basis for further funding from the Leverhulme Trust which will launch our novel method towards a field-deployable real time analytic instrument capable of monitoring the hydrodynamics within living plants. 
 
Title Novel surface chemistries for polarising epithelial tissues on arbitrary substrates 
Description In collaboration with Kode Biotech Ltd we have co-developed a set of novel molecules for the surface chemistry modification of arbitrary substrate, such that epithelial tissues can be adhered and polarised on any material - porous or non-porous - in contrast to decades of previous work. These molecules are presently under IP negotiation for protection in multiple jurisdictions globally. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Epithelial tissues are classically viewed as incapable of proper differentiation, polarisation, or maturation towards normal physiological function on non-porous substrates. Requiring porous substrates has limited the use of epithelial tissues in epithelial microdevices especially in an imaging context. Opening the door for proper polarisation on arbitrary porous and non-porous substrates has major potential in the design of tissue-on-chip and lab-on-chip devices for the biosensing and bioimaging readout of biomarkers, physiology, fluid and molecular transport, and barrier function. This ability will be of wide interest to industry and academia, and is currently under IP review with a view to protecting the IP in several jurisdictions. 
 
Title Optogenetic epithelial cell lines 
Description We have successfully achieved the expression in mammalian epithelia (gut Caco-2, Lung Calu-3, Retinal Pigment Epithelium ARPE-19) of optogenetic constructs (ChR2, HR) in order to manipulate physiology and behaviour in real time using non-invasive photonics. Current work is focussing on the modulation of cell volume, fluid transport, and intercellular communication to achieve coordinated activity and physiological control. 
Type Of Material Cell line 
Provided To Others? No  
Impact This is a recent advance, with the potential to create novel drivers and detectors of fluid and molecular transport within epithelial microdevices, organs on a chip, lab on a chip - driving and manipulating processes using native cellular physiology modulated non-invasively by photonic means. 
 
Title Picosecond ultrasound in living cells 
Description We have jointly developed a novel laser ultrasound microscopy platform capable of imaging mechanical contrast in living cells. We are presently applying this to image hydrodynamics in real time within living epithelial tissues using deuterated water as a density-based contrast agent. 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact We are applying our novel method to image diverse cell types including cardiac myocytes (in collaboration with Oxford University), brain parasites (UoN Vet School) and cultured models of human and mammalian epithelial tissues (lung, retina, gut). 
 
Title PreFACE - Predictive Focus Automatic Correction Engine 
Description PreFACE is a novel image processing method capable of extracting, in real time, from a single image, accurate defocus correction signals for the instantaneous closed-loop control of focus in high-content imaging. 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact Development of commercial product (Cairn Research Ltd), development of high-content screening platform (Ibidi GmbH), Development of open-source PreFACE engine for the open-source microscopy community (µManager). 
 
Title PreFACE - Predictive Focus Automatic Correction Engine 
Description This novel technology, arising from an industrially-focussed MEng project in conjunction with ibidi GmbH, addresses one of the major outstanding problems in biomedical science - that of maintaining the focus of a microscopy system over long time periods in the face of thermal and mechanical disturbance. This is especially important in the context of big pharma assays for behaviour and morphology, in addition to research and academic studies. The method is unique, patentable, and is capable of precisely determining both the magnitude and direction of defocus of an imaging system from a single image or series of images. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact ibidi GmbH has incorporated PreFACE into their new developmental imaging system product. Cairn Research Ltd, Faversham, Kent is in licensing discussions with the University of Nottingham tech transfer office about funding IP capture and developing the system to a full commercial product to install within the microscopy platforms of 3 of the 4 major global manufacturers - Nikon, Olympus, Leica. At this stage the IP is not yet protected, and the technology has been made available to the industrial partners for developmental and commercialisation purposes. 
 
Title Raman microspectroscopy in living cells 
Description We have designed and constructed a state-of-the-art tool for the label-free chemical imaging of biological samples - a Raman microspectrope - which uses biocompatible near infrared lasers to probe the molecular composition of samples at the submicron scale. We are using this advanced too to demonstrate its applicability to samples from frozen tissue sections, live cells in culture, and dynamic tissue engineered physiological microsystems. 
Type Of Material Improvements to research infrastructure 
Year Produced 2014 
Provided To Others? Yes  
Impact Our new bespoke instrument is among the most powerful in the world in terms of its applicability to living cells, and there are many researchers at the University of Nottingham who are obtaining pilot data using our equipment, with the goal of joint funding bids in the near future. We have further obtained several small interdisciplinary and discipline-bridging grants to pump-prime efforts in parasite biology, corneal wounding, and age-related macular degeneration. 
 
Description Consortium for the real-time control of microscope focus in high-content screening 
Organisation Ibidi
Country Germany 
Sector Private 
PI Contribution Resulting from a MEng industrially-focussed research project, a student-led technology development for the real-time extraction of defocus error signal from microscope images was invented (PreFACE - the Predictive Focus Automatic Correction Engine). This innovation is currently being evaluated for patent and being developed under license by Cairn Research Ltd (Faversham, Kent) into a commercial product capable of being retrofitted to 3 of the major 4 microscope manufacturers globally (Nikon, Olympus, Leica).
Collaborator Contribution Ibidi GmbH are early-adopter users of the technology, incorporated within their lead industrial prototype high-content imaging product line. Cairn Research Ltd are key commercial implementors - funding IP protection and development under license from the University of Nottingham.
Impact Patent under consideration, commercial PreFACE product under industrial development, open-source PreFACE engine in development for the open-source microscopy community.
Start Year 2017
 
Description Epithelia-on-a-chip Consortium 
Organisation Rochester Institute of Technology
Country United States 
Sector Academic/University 
PI Contribution We have developed a bespoke Raman microspectroscope which we are using to study the transport of fluid by layers of epithelial cells. We have provided cell cultures and assays, hosted a US graduate student in our lab, implemented epithelial cultures on novel nanoporous nanomembrane substrates, and begun to encapsulate cell cultures in microfluidic devices for physiological measurements. We have further advised the US group on approaches to live cell imaging and maintenance in vitro which has led to a step-change in operational workflow in the Nanomembranens Research Group (NRG) at Rochester.
Collaborator Contribution The NRG have produced and characterised novel nanoporous nanomembrane materials for our project, including a world-first nanoporous magnesium fluoride nanomembrane which is compatible with Raman microspectroscopy in a regime appropriate for live cell work. the UoR group has contributed access to expertise and fabrication facilities unavailable in this country, and provided a leg up into the world of nanomaterials and bioengineering microdevices which has provided a step change in our own experimental approach. This partnership has been extended to a wider constortium involving Rochester Institute of Technology (RIT) as well as joint spinout company (SimPore Inc). In-kind contributions include bespoke wafer design and manufacture and multiple years of technical support as well as supply of novel nanostructured materials optimised for Raman microspectroscopy on living cells.
Impact Two travel grants have been funded, allowing reciprocal research visits by Dr Webb (UoN) and Prof McGrath (UoR) to each others laboratories. In addition the travel funding provided for a 3 month secondment of Greg Madeijski (UoR) to Nottingham, and a week-long knowledge transfer interaction with Dr Pascut (UoN) embedded within the NRG for a short period. We are preparing major joint funding bids to fund both the interaction and the epithelia-on-chip concept, and Dr Webb is now co-supervising the PhD of Mr Madeijski along with Prof McGrath. TTO outputs are currently at the disclosure stage at both Rochester and Nottingham, under mutual NDA/CDA. In 2015-16 we have hosted Mr Madejski at Nottingham under a Whittaker Foundation international scholars programme award, which has led to the serendipitous discover of a novel biosensor based on nanoporous nanomembrane technology - this is described elsewhere.
Start Year 2013
 
Description Epithelia-on-a-chip Consortium 
Organisation SimPore Inc
Country United States 
Sector Private 
PI Contribution We have developed a bespoke Raman microspectroscope which we are using to study the transport of fluid by layers of epithelial cells. We have provided cell cultures and assays, hosted a US graduate student in our lab, implemented epithelial cultures on novel nanoporous nanomembrane substrates, and begun to encapsulate cell cultures in microfluidic devices for physiological measurements. We have further advised the US group on approaches to live cell imaging and maintenance in vitro which has led to a step-change in operational workflow in the Nanomembranens Research Group (NRG) at Rochester.
Collaborator Contribution The NRG have produced and characterised novel nanoporous nanomembrane materials for our project, including a world-first nanoporous magnesium fluoride nanomembrane which is compatible with Raman microspectroscopy in a regime appropriate for live cell work. the UoR group has contributed access to expertise and fabrication facilities unavailable in this country, and provided a leg up into the world of nanomaterials and bioengineering microdevices which has provided a step change in our own experimental approach. This partnership has been extended to a wider constortium involving Rochester Institute of Technology (RIT) as well as joint spinout company (SimPore Inc). In-kind contributions include bespoke wafer design and manufacture and multiple years of technical support as well as supply of novel nanostructured materials optimised for Raman microspectroscopy on living cells.
Impact Two travel grants have been funded, allowing reciprocal research visits by Dr Webb (UoN) and Prof McGrath (UoR) to each others laboratories. In addition the travel funding provided for a 3 month secondment of Greg Madeijski (UoR) to Nottingham, and a week-long knowledge transfer interaction with Dr Pascut (UoN) embedded within the NRG for a short period. We are preparing major joint funding bids to fund both the interaction and the epithelia-on-chip concept, and Dr Webb is now co-supervising the PhD of Mr Madeijski along with Prof McGrath. TTO outputs are currently at the disclosure stage at both Rochester and Nottingham, under mutual NDA/CDA. In 2015-16 we have hosted Mr Madejski at Nottingham under a Whittaker Foundation international scholars programme award, which has led to the serendipitous discover of a novel biosensor based on nanoporous nanomembrane technology - this is described elsewhere.
Start Year 2013
 
Description Epithelia-on-a-chip Consortium 
Organisation University of Rochester
Country United States 
Sector Academic/University 
PI Contribution We have developed a bespoke Raman microspectroscope which we are using to study the transport of fluid by layers of epithelial cells. We have provided cell cultures and assays, hosted a US graduate student in our lab, implemented epithelial cultures on novel nanoporous nanomembrane substrates, and begun to encapsulate cell cultures in microfluidic devices for physiological measurements. We have further advised the US group on approaches to live cell imaging and maintenance in vitro which has led to a step-change in operational workflow in the Nanomembranens Research Group (NRG) at Rochester.
Collaborator Contribution The NRG have produced and characterised novel nanoporous nanomembrane materials for our project, including a world-first nanoporous magnesium fluoride nanomembrane which is compatible with Raman microspectroscopy in a regime appropriate for live cell work. the UoR group has contributed access to expertise and fabrication facilities unavailable in this country, and provided a leg up into the world of nanomaterials and bioengineering microdevices which has provided a step change in our own experimental approach. This partnership has been extended to a wider constortium involving Rochester Institute of Technology (RIT) as well as joint spinout company (SimPore Inc). In-kind contributions include bespoke wafer design and manufacture and multiple years of technical support as well as supply of novel nanostructured materials optimised for Raman microspectroscopy on living cells.
Impact Two travel grants have been funded, allowing reciprocal research visits by Dr Webb (UoN) and Prof McGrath (UoR) to each others laboratories. In addition the travel funding provided for a 3 month secondment of Greg Madeijski (UoR) to Nottingham, and a week-long knowledge transfer interaction with Dr Pascut (UoN) embedded within the NRG for a short period. We are preparing major joint funding bids to fund both the interaction and the epithelia-on-chip concept, and Dr Webb is now co-supervising the PhD of Mr Madeijski along with Prof McGrath. TTO outputs are currently at the disclosure stage at both Rochester and Nottingham, under mutual NDA/CDA. In 2015-16 we have hosted Mr Madejski at Nottingham under a Whittaker Foundation international scholars programme award, which has led to the serendipitous discover of a novel biosensor based on nanoporous nanomembrane technology - this is described elsewhere.
Start Year 2013
 
Description Laser ultrasound dynamic imaging for the study of epithelial transport and cell volume regulation 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have partnered with a collaborating group in Advanced Optics, led by Prof Matt Clark, and jointly developed a laser ultrasound instrument capable of revealing mechanical contrast from living biological cells at below the optical diffraction limit. Through a joint PhD supervision (Fernando Perez-Cota, viva passed in Feb 2016) we have demonstrated and applied this method to an array of biological cell types to reveal morphology and subcellular detail. We have recently extended this approach into dynamic imaging of hydrodynamics by exploiting the availability of innocuous heavy isotopes of water (D2O) in order to reveal fluid fluxes into and across cells by monitoring changes in their accoustic properties in the presence of these denser solutions.
Collaborator Contribution The Clark lab (SIOS) are experts in ultrasonic methods for non-destructive testing of materials. Together we have applied these methods at an unprecedentedly small scale, in order to target living biological cells. This involved the design and construction of a novel nanotransducer to launch ultrasonic waves under picosecond ultrasound interrogation, with the dual purpose of providing optical shielding from the exciting laser wavelengths which are injurious to living tissue. An entire bespoke microscope system has been created and implemented with phase contrast optics in order to perform correlative imaging with the novel method. Future work will focus on using picosecond ultrasound as a complementary method for revealing hydrodynamics to our main Raman microspectroscopy approach, which suffers certain crucial limitations in imaging at the required scales to probe dynamics across and between mammalian cells. This approach may result in a generalised method for contrast improvement or enhancement in cellular ultrasound imaging by manipulating density contrast within biomaterials - both living and fixed. As a dynamic method for assessing transmembrane or transcellular transport of fluid in living systems the method is under development, crossing engineering hurdles to make our imaging system compatible with life processes over extended periods.
Impact The first ultrasonically-created images in living cells have been published in Nature Scientific Reports. The recent discovery that heavy water can reveal mechanical contrast and thus allow hydrodynamics to be directly visualised has yielded a novel method which will be an important correlative approach with which we can independently investigate epithelial hydrodynamics using this technologically-distinct and novel approach.
Start Year 2014
 
Description Novel commercial bioreactors for dynamic cellular assays 
Organisation Ibidi
Country Germany 
Sector Private 
PI Contribution We have developed novel microporous cell culture substrates in collaboration with the University of Rochester, NY. These devices are inherently designed to be compatible with the commercial bioreactor microfluidic platform currently marketed by ibidi GmBH, sold under cross-selling agreements with our other industrial collaborator SimPore Inc (Rochester, NY).
Collaborator Contribution We have partnered with ibidi GmBH (Munich) - a leading market player in biomedical imaging consumables market, with a presence in 47 global markets. In collaboration with our other industrial partner, SimPore Inc (Rochester, NY) we are developing prototype consumable devices based on our novel Raman-silent microporous MgF2 membrane materials - developed jointly with the University of Rochester. This foundation has led to fruitful interactions including student placements with the company in Munich. This interaction has led to the in-kind supply of microfluidic constructs and components for the creation of prototype devices for testing in our lab.
Impact Student research visit (August 2016) leading to development of prototype illumination device for development imaging system. Prototype bioreactor consumables for consideration in the Raman-silent cellular imaging market (epithelia on a chip). We have an agreement in principle for the creation of full commercial prototypes for market testing once the IP issues have been agreed between parties (in process).
Start Year 2016
 
Description Novel surface chemistries for polarisation of epithelial tissues on arbitrary surfaces 
Organisation Kode Biotech Ltd
Country New Zealand 
Sector Private 
PI Contribution Kode Biotech Ltd has contributed know-how and synthetic and analytical capacity to create a set of novel surface chemistry to our specifications, designed to coat in a single step any given surface and expose appropriate surface chemistries to actively drive the polarisation and maturation of epithelial tissues in vitro. Our Nottingham lab has contributed the cellular biology and functional imaging and analysis tools to assess the maturity and polarisation of these epithelial tissue mimics on a range of materials in vitro. Dr Webb was appointed as a Research Fellow of the Centre for Kode Technologies at the Auckland University of Technology, New Zealand in July 2017.
Collaborator Contribution We have co-developed a set of proprietary surface modification agents based on the Kode FSL platform which, when applied to arbitrary surfaces, bind and expose surface chemistries appropriate for the attachment and proper differentiation, polarisation and maturation of in vitro models of epithelial tissues. Importantly these molecules are capable of this remarkable feat even on non-porous surfaces, in contrast to decades of previous work. The IP and scientific implications of these observations are under rapid development via the Tech Transfer apparatus at Nottingham and the company is aggressively leading their commercialisation.
Impact A pair of manuscripts are under preparation, and an additional manuscript in Scientific Reports was published in Feb 2018. Two potential IP filings are in process. An invited talk has been delivered by Prof Steven Henry to Nottingham in Nov 2016 during a sponsored research visit to the Optics and Photonics Research Group. An invited talk was delivered by Prof Steven Henry to our inaugural Epithelia-on-a-chip consortium meeting in August 2016 at the University of Rochester, NY.
Start Year 2015
 
Description Real-time functional volumetry via ratiometric fluorescence imaging in living systems 
Organisation University of Auckland
Country New Zealand 
Sector Academic/University 
PI Contribution The Webb lab collaborates extensively with the Molecular Vision Laboratory, University of Auckland Department of Physiology, in the field of live-cell imaging and dynamic physiology of volume regulation and fluid transport in epithelia of the eye. This includes joint PhD supervision and the Honorary appointment of Dr Webb to the University of Auckland Department of Physiology through 2019. The collaboration continues to yield regular joint publications, research visits, and joint funding applications.
Collaborator Contribution The Molecular Vision Laboratory are world experts in the mapping and functional imaging of epithelial transport systems in living and fixed tissues of the eye (ocular lens, retina). In addition to joint development of imaging technologies and assays for the real-time capture of dynamic events from living cells and tissues, the collaboration has resulted in 2 joint PhD students and a path to regular student and researcher exchanges under the Universitas21 framework (commencing Q4/18)
Impact Petrova R.S., Webb K.F., Vaghefi E., Walker K., Schey K.L., Donaldson P.J. Dynamic functional contribution of the water channel AQP5 to the water permeability of peripheral lens fiber cells. Am J Physiol Cell Physiol 314: C000-C000, 2018. doi:10.1152/ajpcell.00214.2017.
Start Year 2014
 
Description Real-time hydrodynamic imaging in living systems 
Organisation University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution With colleagues at Würzburg, the Webb lab have developed a pair of related fluorescent nanoparticle biosensors capable of reading out in real time the diffusion of water within living systems at sub-micron resolution. The technologies have been synthesised at Nottingham in interdisciplinary collaboration with the Pharmacy school, and tested and calibrated at Würzburg.
Collaborator Contribution Würzburg is a world centre of excellence in the subcellular dynamics of small-molecule signalling systems. Our novel nanosensors, jointly developed, are the perfect control system to establish the fundamental limits of diffusion of small molecules within living systems, thereby capturing the fundamental biophysics and constraints dictating the kinetics and distribution of water within living system, cells, and subcellular structures.
Impact A pair of distinct nanoparticle biosensors (130nm polymer particles, 10nm semiconductor particles) have been developed and are currently under consideration for patent at the University of Nottingham
Start Year 2016
 
Description SERS biosensing 
Organisation SimPore Inc
Country United States 
Sector Private 
PI Contribution We have serendipitously discovered that a novel Surface Enhanced Raman Scattering (SERS) biosensor can be fabricated at scale using semiconductor processing methods. This novel technology has been patented by a team of inventors spanning Nottingham (Webb, Pascut) and Rochester (Madejski, McGrath), with a prior date of 1st October 2015. This method of sensor fabrication is a game-changer for the scalable manufacture of porous biosensors with novel functionalities, and will enable the design and optimisation of devices for diagnostics and point-of-care therapeutic information-gathering based on the highly sensitive detection of particular bioanalytes. The Nottingham contribution has been to complete the final manufacturing steps, and to assess the biosensor performance using our state-of-the-art bespoke Raman microspectrometer. The Nottingham TTO liased with Rochester to lead the patent, which was filed in the USA for strategic reasons.
Collaborator Contribution The team at Rochester provided expert manufacturing design and silicon fabrication which provided the underpinning technology for the novel biosensor. Drawing on established background IP held by SimPore (as a spin-out company from Rochester) to add significant value to existing technology as well as enabling the creation of an entirely new class of scalably-manufactured biosensors. Mr Madejski, as co-inventor, has translated his tenure at Nottingham into a significant, serendipitous benefit with major potential to revolutionise disease diagnosis and biomarker detection in a range of contexts.
Impact This collaboration is multidisciplinary, spanning semiconductor manufacture, bioengineering, optical engineering, and biochemistry/electrochemistry. The outputs so far include a patent-protected IP platform, as well as the creation of commercial-quality prototypes of a novel biosensor platform which are presently undergoing market feedback in collaboration with commercial partners (and logical licensees) SimPore Inc.
Start Year 2015
 
Description SERS biosensing 
Organisation University of Rochester
Country United States 
Sector Academic/University 
PI Contribution We have serendipitously discovered that a novel Surface Enhanced Raman Scattering (SERS) biosensor can be fabricated at scale using semiconductor processing methods. This novel technology has been patented by a team of inventors spanning Nottingham (Webb, Pascut) and Rochester (Madejski, McGrath), with a prior date of 1st October 2015. This method of sensor fabrication is a game-changer for the scalable manufacture of porous biosensors with novel functionalities, and will enable the design and optimisation of devices for diagnostics and point-of-care therapeutic information-gathering based on the highly sensitive detection of particular bioanalytes. The Nottingham contribution has been to complete the final manufacturing steps, and to assess the biosensor performance using our state-of-the-art bespoke Raman microspectrometer. The Nottingham TTO liased with Rochester to lead the patent, which was filed in the USA for strategic reasons.
Collaborator Contribution The team at Rochester provided expert manufacturing design and silicon fabrication which provided the underpinning technology for the novel biosensor. Drawing on established background IP held by SimPore (as a spin-out company from Rochester) to add significant value to existing technology as well as enabling the creation of an entirely new class of scalably-manufactured biosensors. Mr Madejski, as co-inventor, has translated his tenure at Nottingham into a significant, serendipitous benefit with major potential to revolutionise disease diagnosis and biomarker detection in a range of contexts.
Impact This collaboration is multidisciplinary, spanning semiconductor manufacture, bioengineering, optical engineering, and biochemistry/electrochemistry. The outputs so far include a patent-protected IP platform, as well as the creation of commercial-quality prototypes of a novel biosensor platform which are presently undergoing market feedback in collaboration with commercial partners (and logical licensees) SimPore Inc.
Start Year 2015
 
Title Nanostructured materials 
Description A novel nanoporous membrane and method of fabrication is taught, which confers an extremely sensitive surface enhanced Raman scattering (SERS)-based sensing substrate capable of large enhancements of Raman signatures of analytes bound to its surface. The material has the ability to perform flow-through sensing and is erasible using standard electrochemistry by virtue of its electrical contiguity. The material is fabricated using silicon wafer processing, lending itself to scalable and reproducible manufacture and solving a major barrier to the high-throughput fabrication of SERS sensors in the field. Licensing arrangements are under negotiation with SimPore Inc Rochester, NY and other parties via the University of Nottingham and University of Rochester technology transfer offices. 
IP Reference PCT 62/235 929 
Protection Patent application published
Year Protection Granted 2017
Licensed Commercial In Confidence
Impact We have solved a major problem in the field - that of the scalable manufactur and reproducible enhancement factor associated with SERS-based measurements - allowing calibration, repeated usage, and erasible operating modes for SERS-based sensing platforms.
 
Title PreFACE - Predictive Focus Automatic Correction Engine 
Description Presently under consideration for patent, funded by Cairn Research Ltd (Faversham, Kent), PreFACE is capable of extracting a focus error correction signal in real time from a single image with sub-micron accuracy for the closed-loop control of microscope focus during high-content screening and time-lapse assays. Patent relates to both software algorithm as well as hardware implementation. IP capture is being funded by industrial partner (Cairn) and commercialisation and licensing discussions are advanced between Cairn Research and the University of Nottingham tech transfer office. 
IP Reference  
Protection Copyrighted (e.g. software)
Year Protection Granted
Licensed Commercial In Confidence
Impact PreFACE is being instrumented within the new high-content screening platform developed by ibidi GmbH (Munich) and will be released to the open-source microscopy community as a µManager plugin during Q4/18, following patent filing. A retrofittable product is under commercial development by Cairn Research Ltd for global sale in support of 3 out of 4 major microscopy manufacturers worldwide (Nikon, Olympus, Leica).
 
Title PreFACE - Predictive Focus Automatic Correction Engine 
Description PreFACE is a novel image processing method capable of correcting the focus of a microscope imaging system indefinately in real time under closed-loop control. The underlying algorithm will be released to the open-source microscopy community as a µManager plugin in Q4/18 following patent filing, and will be sold in support of stand-alone imaging platforms (ibidi GmbH) and OEM components fitted to Nikon/Olympus/Leica microscope platforms. 
Type Of Technology Systems, Materials & Instrumental Engineering 
Year Produced 2017 
Impact PreFACE will change the way that high-content and time-lapse imaging is performed across the world - presently a trade secret being developed for patent, the technology provides unprecedented functionality at extremely low cost, solving one of the crucial outstanding problems in the field - that of maintaining microscope focus for long periods under changing environmental conditions. 
 
Description Inaugural Symposium and Workshop - Epithelial Microdevices 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact an inaugural 3-day Symposium and workshop was held at the University of Rochester, NY on the theme of "Epithelial Microdevices".
Participants were drawn from the UK, USA, and New Zealand and from several US states. Approximately 15 academics, 10 postgraduates and a similar number of undergraduates were brought together to focus on current themes in epithelial microdevices, with particular emphasis on the possiblities enabled by novel microporous and nanoporous membrane technologies and the ability to arbitrarily polarise epithelial tissues within devices and on surfaces.
Year(s) Of Engagement Activity 2016