Creativity@home-Novel Tools for Single Cell Manipulation

Lead Research Organisation: Imperial College London
Department Name: Dept of Chemistry

Abstract

Cells which are amongst the smallest units of life are made up of smaller components including lipids and proteins that combine together to perform functions that are vital to the life of the cell. Lipids form the backbone of membranes that help to compartmentalize them whereas proteins are the worker molecules in cells, making, building and moving things throughout. Interestingly, these molecules do not work in isolation, instead they often come together to perform higher order functions and their levels throughout a cell vary both as a function of time and space. They fluctuate as a function of time in response to stimuli, some external and some internal to the cell. Unfortunately, the state of the art in terms of our ability to measure the inner workings of these systems is such that it is still not possible to measure the protein levels in or accurately deliver or remove material to or from a single cell (either to the surface of to a sub-cellular location). This proposal puts forwards a series of feasibility studies to trial a series of new technologies that have the ability to address these technological bottlenecks thereby unlocking the inner workings of cells.

Planned Impact

This project will be of benefit to both the academic and industrial communities and is aligned with two of the EPSRC's strategic areas (Nanoscience through engineering to application and Towards Next Generation Healthcare). These feasibility studies aim to build upon two-state-of the-art technologies, namely SDMs and MAC chips that are wholly unique to the UK. If successful, this programme will make a significant and substantial contribution to innovation in UK industry, with a 2-5 year timeframe for academic exploitation and 5-10 year timeframe for commercial realisation. However, the individual elements, e.g. single cell delivery protocols and single cell protein readout modules will be exploitable on shorter time scales of ca. 1-2 years. The development of new tools for the manipulation and analysis of single cells will benefit both sectors and include technical improvements in microfluidics, holographic trapping, protein arrays, and liquid handling platform and proteomic analysis. In addition, the tools and techniques under development here have the potential to provide enhanced capabilities for end-users in basic biology, biomarker discovery, diagnostics and drug discovery. In particular the ability to undertake quantitative spatial and temporal proteomics will provide these sectors with new insights to the spatio-temporal dependencies of cellular signalling pathways. These applications can be directly exploited by the pharmaceutical industry, medical sector, biotechnology SMEs and instrumentation manufacturers. The focus of the programme is concerned with providing tools for the better study of single (including rare) cells. There are obvious benefits to the general population of research that has the potential to impact on our understanding of the relationship between biological heterogeneity and signalling pathway regulation that may result in disease states. This offers the potential to drive novel therapeutic interventions developed in response to single cell behaviours. The ability to deliver material to single cells will impact upon the design of drug delivery systems with tailored drug release kinetics, single cell transfection agents and single cell delivery and transplantation protocols. As the developments of this project go beyond current state-of-the art and provide a range of new platforms for single cell analysis it is inevitable that we will generate intellectual property with significant commercial value. The result will be improved healthcare as well as enhanced economic competitiveness of the United Kingdom by generating wealth and potential future employment. Our strategy for realising the commercial potential of these technology elements is outlined in the accompanying Pathways to Impact document.

Publications

10 25 50
 
Description The aim of this project was to investigate how optically trapped bio-emulsion droplets (Smart Droplet Microtools: SDMs) in microfluidic devices can be used to undertake a variety of novel single cell analysis functions using the biological process of phagocytosis as a soft focus for these feasibility studies. The aim was not to determine how well each function worked but whether they would work at all. With respect to objective (a) we were able to trigger binding of the macrophages (RAW 264.7 macrophages) to a variety of delivery systems, ranging from collagen-coated polystyrene beads through to latex spheres coated with rabbit IgG and finally a novel type of SDM based upon a solid-core of silicon dioxide coated with a lipid bilayer. The latter SDMs offer superior trapping capabilities with respect to previous generations of SDMs and have opened up the possibility of using SDMs to transfer material from one membrane to another (see below). In addition, not only were we able to demonstrate that we could trigger phagocytosis with SDMs but that we could also do so simultaneously at multiple sites on the surface of a cell with spatial and temporal specificity using holographic optical traps. Due to the adhesion forces involved in the uptake of the SDMs we were not able to sample the macrophages (either surface or sub-cellular locations, objective e) but we were able to demonstrate that the new solid core SDMs could be used to remove material with spatial specificity from human biliary epithelial (BE) colon cancer cells (objective b). In order to demonstrate that we could use microfluidic antibody capture chips to measure proteins level we had to show for the first time that we could both uptake material from a surface and then release it onto another using SDMs as the cargo transporters. We were successful in doing so, isolating green fluorescent protein (EGFP) labeled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells, before transferring them (and their associated lipids) to an S-layer supported lipid bilayer system. Not only did this demonstrate we could unload an SDM cargo, it also demonstrated that the SDM platform could be used to pattern model membrane surfaces with spatial control, which is currently a bottleneck in membrane engineering. In terms of delivery to single cells we demonstrated that the macrophages could uptake SDMs at user defined sites on the cell surface and extended this capability even further by undertaking single cell transfection. These results have underpinned: (i) a successful EPSRC Programme grant application (EP/J017566/1) (ii) 2 publications (Soft Matter, SMALL, under preparation) and (iii) numerous (iii) presentations to industry with all the PDRAs on the project benefitting from multi-disciplinary research training. These developments have led to new tools and technologies for single cell analysis and the construction of patterned membrane surfaces.
Exploitation Route There are two main ways in which this research is being exploited. Firstly there is its application for the study of biology in order to further our understanding of a range of key biological functions. The fields of proteomics and genomics have developed in ways which emphasise the rate and volume of data acquisition and analysis. They have by necessity worked on large populations of cells reporting on population averages rather than their distributions and have been unable to analyse cells which are only produced in small numbers. In addition genome sequence information provides powerful insights into cellular complexity but limited information pertaining to how individual parts of a cell are integrated in time and space to form dynamic cellular processes. These feasibility studies have demonstrated that SDMs can address many of these issues as they can be used to perform "nanosurgery" on single cells whilst retaining cellular viability, both removing material, delivering material between membrane surfaces and delivering material to sub-cellular locations to trigger e.g. single cell transfection. These are key elements required to underpin spatial and time-resolved proteomic measurements.
By developing techniques to study and manipulate cells at the single cell level it is possible to better understand and manipulate the molecular networks that make cells function. The ability to deliver (and measure) molecular doses of biological reagents to a specific subcellular location, is a largely unmet technological challenge hampering our understanding of cell biology. Our SDM studies have led to a platform that offers unprecedented control with respect to cell manipulation and targeted intervention and we therefore anticipate that it will find widespread applications in bioengineering and healthcare. We have already demonstrated the potential with respect to single cell transfection but anticipate downstream applications in fields such as theranostics, drug delivery systems with tailored drug release kinetics, single cell transplantation and micromechanical systems. This technology has been integrated into a facility at Imperial College London where a consortium of around 50 research collaborators are making use of the facility for subsequent biological projects.

Secondly there is the application of SDMs in the emerging field of molecular membrane engineering as it will facilitate the construction of artificial membranes with user defined levels of patterning which will revolutionize the fabrication of smart, soft materials based on the lipid bilayer as the basic structural motif. These developments are being exploited through a recently awarded programme grant (EP/J017566/1) with applications in nanomedicine, liquid crystal templating and artificial cells.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description By demonstrating the ability to remove and delivery material to single cell and with sub-cellular specificity we have unlocked a technology that has the capability to address a number of key bottlenecks in the pharmaceutical industry. We are exploring industrial applications of this technology with respect to being to deliver known amounts of drug molecules to a given cell so as increase the accuracy of drug screens. In addition, we are using this technology to support the construction of artificial protocells for smart delivery purposes. This approach has most recently been extended to underpin new approaches for manufacturing artificial tissues which may have clinical applications.
First Year Of Impact 2016
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Marie Curie Initial Training Network: NextGenAgriChem
Amount £3,260,000 (GBP)
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 03/2014 
End 03/2018
 
Description Collaboration with Syngenta 
Organisation Syngenta International AG
Department Syngenta Ltd (Bracknell)
Country United Kingdom 
Sector Private 
PI Contribution co-exploitation of MAC chip and SDM technologies for study of single cell plant proteomics
Collaborator Contribution Compounds, training, secondments for students on Marie Curie ITN programme
Impact Collaboration just started, exploring potential of the technology: multi-disciplinary activity.
Start Year 2013
 
Title Coupling of SDMs and holographic traps to undertake multiple biopsies of single cells 
Description By coupling holographic trapping platforms with solid core SDMs we are now able to undertake multiple simultaneous biopsies of single cells with controlled temporal and spatial resolution 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2011 
Impact This technology has opened up the possibility of undertaking biopsies at the cellular level, patterning of lipid membrane to generate new materials, the ability to transplant materials between cells and artificial membranes and the possibility of being able to undertake spatially and temporally specific measurement of the proteome of single cells. 
 
Title Lipid membrane patterning using smart droplet microtools 
Description We have demonstrated that we are are to pattern lipid membranes by demonstating that we can both uptake material from one membrane surface (protein and lipid) and then release it onto another using SDMS (silicon dioxide SDMs coated with lipid bilayers) as the cargo transporters. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2013 
Impact New technology for transplanting materials between artificial cells. 
 
Title Solid core smart droplet microtools (silicon dioxide core with lipid bilayer coating) 
Description We have developed a new generation of micron sized solid core smart droplet microtools (silicon dioxide core with lipid bilayer coating). These offer superior trapping capabilities with respect to early generations of SDMs consisting of oil cores. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2011 
Impact This technology has opened up the ability to transplant materials between cells and artificial membranes. 
 
Description International Microfluidics Congress 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International conference: disseminate findings of research and look for new end users of the technology
Year(s) Of Engagement Activity 2015
 
Description Presentation at Chemistry Taster day to secondary school students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Presentation at Chemistry Taster day to secondary school students.

Student engagement
Year(s) Of Engagement Activity 2012
 
Description Presentation at SET for Britain 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presentation at SET for Britain, 12th March 2012.

Exposure of research to broad cross sector of society and outreach training for personnel
Year(s) Of Engagement Activity 2012
 
Description Presentation to the general public (Friends of Imperial College) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation aimed at addressing public concerning associated with manufacturing artificial cells and manipulating biological systems.
Year(s) Of Engagement Activity 2015
 
Description Single Cell Analysis Conference, May 2014, Boston, MA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Demonstration of new capabilities of MAC chip and SDM technologies

Increased awareness of MAC chip and SDM platforms within the industrial sector.
Year(s) Of Engagement Activity 2014
 
Description Single Cell Analysis Congress, November 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Presentation on MAC chip and SDM technologies and their applications for the clinic and frontiers such as time-resolved single cell analysis.

Awareness of SDM technology to new end users.
Year(s) Of Engagement Activity 2013
 
Description Technology showcase presentation to industry (AstraZeneca) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Workshop and associated talk aimed at translating EPSRC breakthrough to industry.
Year(s) Of Engagement Activity 2015