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

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.

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.

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.