Award Title: Experiencing the micro-world - a cell's perspective
Funder: Medical Research Council

Abstract: In the body, most cells grow in close contact with other neighbouring cells and with a local matrix of proteins and sugars that combine to provide an instructive microenvironment. Until recently, most research labs (in both academic and industrial settings) have used 2D cultures of cells on plastic to study cell behaviour, a significant departure from what is actually happening in vivo that can limit the applicability of their research. However, there has been a recent and dramatic shift away from traditional 2D culture to the use of complex, 3D cultures, that more effectively mimic the micro-environment experienced by cells in vivo. This development impacts directly on fields such as regenerative medicine, drug discovery and cancer research, with significant opportunities for improved in vitro modelling of cell behaviour. Despite these improvements in culture techniques, the interaction of the cells with their local microenvironment - a key target in therapies for cancer, wound healing, and fibrosis etc. - remains a 'black box' with technologies unable investigate these environments at the cell level. This proposal will 'open that box', developing the technology and methodology urgently required to fully explore 3D cell cultures on length scales comparable, or smaller than, single cells. 

The currently accepted protocol to characterise natural and synthetic matrices, uses a bulk rheometer to produce a single, averaged value of the viscosity and elasticity of the material, destroying the sample in the process. Information about the matrix local to the cells growing inside the samples is lost. Our vision is to image and characterise 3D cell culture environments in all three spatial dimensions, over an extended time course, and on a single multifunctional instrument so that the information can be integrated and mapped. To achieve this we will develop a minimally-invasive technique to measure the 3D micro-rheology of the extracellular matrix using nano- (smaller than the cells) and micro-sized (can be the same size at the cells) beads as local probes. These probes will be held at a fixed position within the matrix using an optical trap and their Brownian motion in all three spatial dimensions tracked using multiplane imaging. The micro-rheology (viscosity and elasticity) of the extracellular matrix local to the probe is extracted from temporal analysis of the Brownian motion. To achieve deep 4D (x,y,z, time) images of live 3D cell cultures, we will combine light sheet microscopy with adaptive optics (a technique for correcting for sample aberrations that reduce image quality deep into complex samples). The final multifunctional platform will be the exciting culmination of these 4 microscopy techniques - optical trapping, multiplane imaging, light sheet microscopy and adaptive optics - capable of imaging and micro-mechanically sensing the 3D environment close to cells. 

The output from this work will be the innovation required to allow scientists to study how cells interact with their local microenvironment, combining technologies in a way that's not been possible previously, to observe both the cells, and the forces they exert and are responding to, as they grow and move in 3D space over time. The ability to study cell behaviour in this way is of importance for developing therapies for diseases where cells respond abnormally to signals from their local matrix, such as cancer, providing targets for new drug design. We will include a demonstration of how this can work in our study using both traditional anti-cancer drugs and more innovative therapies such as functionalised nanoparticles. We anticipate that the technology will be useful to both academics and industry (particularly drug discovery in the pharmaceutical industry) and we will work closely with these groups throughout the course of this project to ensure that, once proven, this technology can work for them.


Research Activity Codes: 1.4 METHODOLOGIES AND MEASUREMENTS
Health Categories: GENERIC HEALTH RELEVANCE