A novel experimental tool to investigate the mechanics of cell monolayers at tissue, cellular, and subcellular scales

Lead Research Organisation: University College London
Department Name: London Centre for Nanotechnology

Abstract

Many of the cavities and free surfaces of the human body (e.g. gut, lungs, blood vessels) are lined by a layer of cells one-cell thick (a monolayer). Exposure to mechanical stresses is a normal part of physiology for such monolayers: lung alveoli deform during respiration, intestinal epithelia resist peristaltic movements in the gut, and endothelia are exposed to pulsatile fluid shear stresses in blood flow. The mechanical function of monolayers is particularly apparent in disease when mutations or pathogens affecting the cell skeleton (cytoskeleton) or intercellular junctions result in increased fragility of tissues (e.g. epidermis bullosa, staphylococcus blistering). Despite clear physiological relevance, little is presently known about the mechanics of cell monolayers.

Cells within these monolayers are tightly connected to one another by intercellular junctions: tight junctions form barriers restricting the passage of solutes whilst adherens junctions and desmosomes integrate the cytoskeletons of constituent cells into a mechanical continuum. To date, research in cell mechanics has primarily focused on isolated cells and much is now known about their mechanical properties as well as the underlying biology in normal physiology and disease. Comparatively little is known about the mechanics of monolayers and how it relates to the mechanical properties of the tissue's cellular constituents and their cytoskeleton. This is primarily due to the lack of specific experimental techniques to assess the intrinsic mechanical properties of tissues while monitoring cellular and subcellular traits.

We aim to develop a novel tool to stretch cultured cell monolayers that are mechanically isolated from any substrate. During tissue deformation, the applied mechanical tension will be directly measured and monolayers will simultaneously be imaged at subcellular, cellular and tissue length scales, such that the architecture of the sub-cellular components, the shape of the cells and their eventual reorganisation can be accurately monitored as a function of the imposed force.

The studies enabled by this novel instrument will allow us to understand how the structure of individual cells and their arrangement relative to one another participate in setting the mechanical properties of whole tissues. As a consequence, we will be able to understand how pathological changes in the proteins that form part of the cytoskeleton or the intercellular junctions can have catastrophic consequences for tissue mechanics.

Technical Summary

Exposure to mechanical stresses is a normal part of physiology for monolayers and their mechanical function is particularly apparent in disease when mutations or pathogens affecting the cytoskeleton, adherens junctions, or desmosomes result in increased fragility of tissues. Despite clear physiological relevance, little is known about the mechanics of monolayers and how these relate to the mechanical properties of the individual cells constituting the tissue. The challenge is to design an instrument that enables precise control over the mechanical environment of tissues on time scales from 0.1s to hours while allowing for simultaneous high resolution imaging of subcellular structures.

We propose to develop a versatile new experimental tool for measuring the mechanical properties of cell monolayers and applying well-characterised steady forces onto cell monolayers to study their responses to mechanical stresses at all the relevant time- and length-scales.

The tool will require precise control of the deformation and monitoring of the applied tension, with a compact design suitable for live microscopy imaging and environmental control. This will be achieved by using a closed-loop piezo drive to control deformation and a force sensor with an accuracy of 10uN to monitor tension. Control software will allow testing of time-dependent properties by imposing constant stress conditions or cycles of deformation. A laser ablation system will be built to create controlled cuts in the stretched tissue to characterise their resistance to fracture and measure their macroscopic adhesion energy. Image acquisition and image analysis will characterise cellular dynamics within the monolayer. High magnification microscopy combined with GFP-tagging will provide data regarding the subcellular organisation of the material. This new tool will pave the way for interdisciplinary investigations of monolayer mechanics and mechanotransduction at the molelular, cellular, and tissue levels.

Planned Impact

Impact will be ensured through accomplishing the following set of specific objectives.

Academic impact

Academic advancement and innovation:
To ensure the novel instrument has the highest possible research impact, we will present our technological developments and preliminary results generated at high profile conferences that cover relevant topics including tissue engineering, biophysics, developmental biology and cell biology in 2013 and 2014. We expect the instrument to attract interest from many fields in the global scientific community and that this will lead to the development of new international research collaborations over the next couple of years. Where possible we will disseminate our findings in general audience and/or open access journals. Furthermore, we expect that the imminent publication of our preliminary results in PNAS will bring attention of a wide audience onto the new instrument.

Training and professional development:
Both GC and AK are actively involved in interdisciplinary training activities at UCL and Cambridge University. GC runs the "quantitative biology" module for the UCL Systems Biology training program, participates in teaching in the CoMPLEX DTP, and is a member of the new interdisciplinary BBSRC DTP. AK is an important contributor to the development of the Bioengineering curriculum in Cambridge, and teaches a number of relevant subjects ranging from material sciences to physiology. The project described here will be used to introduce students from different backgrounds to interdisciplinary research in the life sciences. Elements of the work will be used as exemplar projects for students in the CoMPLEX and BBSRC DTPs. The mechanical aspects of the project will also form the basis of a couple of 4th year engineering projects in Cambridge and are likely to attract students with a Mechanical/Bio Engineering background.

Throughout the course of the project, the post-doc involved, already largely independent and technically capable to develop this project, will receive further cross-disciplinary mentoring and benefit from regular interactions both in Cambridge and London. In addition, he will be involved in mentoring students and develop his own mentoring and leadership skills. This will aid his progression towards an independent group leader position.

Societal and economic impact

Commercialisation and exploitation:
We expect our instrument to evolve towards a generic characterisation method within a couple of years and we will strive to use standardized components to enable interested laboratories to rapidly and easily build their own instrument using off-the-shelf components. We envisage that it could be utilized to study the effect of pathologic genetic mutations on tissue mechanical properties and test how drug treatments affect macroscopic tissue properties. Should there be interest from the pharmaceutical community, we will study the possibility of designing a new prototype in a format suited to high throughput screening.

Both the UCL and Cambridge University have efficient mechanisms to assist academics in the development of commercial applications of their research outputs and in the management of intellectual property rights (see for instance Cambridge Enterprise or UCL Business).

Increasing public engagement and understanding:
Previously members of the team have been involved in interactions with the wider community through public discussions and school visits. Through this type of outreach we expect this work to reach a wide audience, giving the public a better understanding of multidisciplinary research and an appreciation of the remarkable natural world in which we live. We expect to participate in one public engagement event during the course of this short project. We will use these opportunities to stress the important role played by basic science and engineering research in driving societal advances.
 
Description The goal of this BBSRC tools and resources grant was to develop a versatile new experimental tool for measuring the mechanical properties of cell monolayers and applying well-characterised steady forces onto cell monolayers to study their responses to mechanical stresses at all the relevant scales. The instrument was designed to work on millimeter size suspended epithelia. Our objectives were to design an instrument that could:
(a) stretch epithelia on time-scales ranging from the fraction of a second to tens of hours.
(b) accurately record stress on the tissue in the 10-105Pa range with an acquisition rate >10Hz.
(c) image the evolution of cellular and subcellular morphology within the tissue during stretching.
(d) study tissue fracture with a high degree of control.

Objective a was achieved by integrating a fast and precise motorised micromanipulator into the experimental setup. This micromanipulator is piloted via Labview such that movement and feedback routines can be implemented to suit the experiments to be conducted. With this setup, strain rates >75%/s can be achieved signifying that a monolayer can be extended by 30% length in less than 0.5s. Driving at such fast rates allows the study of the rheology of monolayers. For long duration experiments designed to examine cellular responses to application of extrinsic mechanical stresses, the micromanipulator is utilised to apply the initial extension with high precision. The micromanipulator prong is then fixed at the final position using glue and detached from the micromanipulator arm. This allows multiple experiments to be carried out in parallel.

Objective b was achieved by integrating an SI-KG7B force transducer into the setup. This allows measurement of forces up to 10mN with a 1µN precision at rates of >1kHz, far exceeding our original goals. Data acquisition is integrated into Labview, allowing force feedback routines to be implemented.

Objective c was achieved by purchasing a long working distance high NA 30x objective and integrating the experimental platform into an environmentally controlled chamber. The new objective allows imaging of large fields of view with high signal and allows zooming on scanning laser confocals.

Objective d required the integration of a laser ablation system to the setup to create controlled multi-cellular cracks in the tissue. The laser ablation system was built to interface directly to the FV-1000 laser lines and controlled through Olympus software (similar to the one described in 11). It allows the generation of subcellular cracks by cutting single junctions as well as multicellular cracks ranging from 1 to 10 cells in length. The fracture can be detected by optical microscopy using the objective and chamber described in objective c. Combined with molecular perturbations, these measurements will be applied to understanding the magnitude and biological origin of intercellular adhesion.
Exploitation Route The tool developed for this grant is now currently enabling research on:
-a) the biological origin of the elastic and viscous properties of monolayers
-b) the intercellular adhesion energy in monolayers
-c) the role of oriented mitoses and cell rearrangement in dissipating applied stresses (with Prof Buzz Baum, LMCB, UCL)
-d) the mechanical properties of Drosophila wing disk (With Dr Yanlan Mao, LMCB, UCL).
Sectors Pharmaceuticals and Medical Biotechnology

URL http://www.nature.com/nprot/journal/v8/n12/full/nprot.2013.151.html
 
Description We are not aware of others using the method at this stage. The work has however been published in Nature protocols and other laboratories, in and outside of academia, are now free to reproduce the setup. It is therefore likely that the tool will be picked up in future, if it has not been done already.
 
Description EMBO Long term fellowship
Amount £75,000 (GBP)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 09/2016 
End 09/2018
 
Description ERC consolidator grant
Amount € 2,000,000 (EUR)
Funding ID 647186 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 09/2015 
End 08/2020
 
Description In vitro mechanical testing and in silico modelling to investigate fragile tissues
Amount £30,000 (GBP)
Funding ID M368 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 03/2017
 
Description Multi disciplinary award
Amount £440,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 09/2020
 
Description Royal Society international network grant
Amount £12,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 02/2020
 
Description The mechanics of epithelial tissues
Amount £689,610 (GBP)
Funding ID BB/M003280/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 11/2017
 
Title A novel method for examining the mechanical properties of living cell sheets 
Description Cell monolayers line most of the surfaces and cavities in the human body. During development and normal physiology, monolayers sustain, detect and generate mechanical stresses, yet little is known about their mechanical properties. We describe a cell culture and mechanical testing protocol for generating freely suspended cell monolayers and examining their mechanical and biological response to uniaxial stretch. Cells are cultured on temporary collagen scaffolds polymerized between two parallel glass capillaries. Once cells form a monolayer covering the collagen and the capillaries, the scaffold is removed with collagenase, leaving the monolayer suspended between the test rods. The suspended monolayers are subjected to stretching by prying the capillaries apart with a micromanipulator. The applied force can be measured for the characterization of monolayer mechanics. Monolayers can be imaged with standard optical microscopy to examine changes in cell morphology and subcellular organization concomitant with stretch. The entire preparation and testing protocol requires 3-4 d. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This new method has enabled the investigation of cell monolayers using techniques and theories from Engineering and Physical Sciences. This is allowing to understand how monolayers react in response to application of force and what cellular structure allow the resistance and generation of mechanical forces in tissues. 
URL http://www.nature.com/nprot/journal/v8/n12/full/nprot.2013.151.html
 
Description Deciphering the role of signalling in cell sheet morphogenesis 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution The goal of this collaboration is to understand how signalling controls cell mechanical properties and in turn the mechanical properties of cell sheets. When signalling is locally activated, this will create local differences in mechanics that will lead to morphogenesis. My team is generating the constructs and carrying out the experiments to control signalling using optogenetics. We are also carrying out experiments in which we measure mechanical properties of cell sheets before and after activation of signalling either locally or globally.
Collaborator Contribution Our collaborator is a group of theoretical physicists. They are using our experiments to model cell sheet morphogenesis based on changes that occur at the single cell level.
Impact This is a multidisciplinary collaboration involving Physics and Biology. This collaboration has given rise to a grant application to control signalling at the single cell level.
Start Year 2016
 
Description Mechanisms of oriented cell division in stretched monolayers 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution This research utilises our experimental system to examine how tissue stretch orients cell division along the the axis of tension.
Start Year 2013
 
Description Optogenetics for investigating cell mechanics 
Organisation University of Grenoble
Department Laboratory for Interdisciplinary Physics
Country France 
Sector Academic/University 
PI Contribution My team is providing expertise in designing and actuating optogenetic probes for controlling cell signalling.
Collaborator Contribution My partners are providing expertise in measuring the forces exerted by cells on the substrate.
Impact N/A. Interdisciplinary collaboration.
Start Year 2018
 
Description Regulation of planar polarity by mechanical stresses 
Organisation University of Rennes 1
Country France 
Sector Academic/University 
PI Contribution I am providing expertise on applying mechanical forces to biological tissues. I am providing expertise on image analysis to examine the extent of recruitment of proteins in response to mechanical stresses.
Collaborator Contribution My partners are providing expertise in Developmental Biology.
Impact No impact yet. Multi-disciplinary collaboration.
Start Year 2018
 
Description Understanding the mechanical control of YAP activation 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution The goal of this project is to understand how YAP signalling is controlled by mechanical cues. My team will carry out the experiments aiming to test under what mechanical conditions YAP relocalises to the nucleus.
Collaborator Contribution Our collaborators will generate the reagents necessary for visualising the different components of YAP in single cells. They will also engineer all of the mutants necessary to understand YAP signalling in response to mechanical cues.
Impact Multi disciplinary collaboration: Physics and Biology.
Start Year 2017
 
Title Rheos 
Description The software library provides a computational framework to model the rheological response of materials presenting a power law behaviour. Such behaviour has been observed across a broad range of biological materials. 
Type Of Technology Software 
Year Produced 2018 
Open Source License? Yes  
Impact It has provided us with a reliable mechanism to extract material parameters in monolayers and embryo tissues. This has allowed the prediction of complex behaviours in monolayers, as recently reported in https://www.biorxiv.org/content/10.1101/543330v1. 
URL https://github.com/JuliaRheology/RHEOS.jl
 
Description Interview with TV 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I participated in an interview for NHK, a major TV network in Japan. I described how charities in the UK fund research.
Year(s) Of Engagement Activity 2017
 
Description Interview with newspaper 
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 Public/other audiences
Results and Impact I provided opinions on the risk of Brexit to Science research in the UK.
Year(s) Of Engagement Activity 2018