Funded Project Summary:
Dr Iestyn Pope’s team at Cardiff University was recently awarded £53,950 to take the following project forward:
Incorporating deuterium into 3D organoids to identify cell types and track cellular metabolic rates using CARS microscopy
Organoids are 3D microtissues that reproduce more accurately how a tissue behaves compared to a 2D culture on plastic. This is because it allows the cells to interact with each other and to organise their own microenvironment. Recently, we and others have shown that patient-derived cancer organoids retain the cellular diversity that matches that of the tumours from which they are derived. Thus 3D cell culture has the potential to revolutionise technical approaches in personalised medicine and drug discovery.
Typically, identification of different cell types within cancer organoids, and many of the experiments undertaken on them are performed by first tagging the cells or molecules of interest with a fluorescent marker, which can then be visualised on a microscope. However, using this technique there is always the potential that the addition of the fluorescent marker alters the behaviour of the cell or molecule you are interested in following.
Over recent years, Coherent anti-Stokes Raman Scattering (CARS) microscopy has emerged as a powerful label-free way of rapidly imaging live cells and tissues with high 3D spatial resolution and quantitative chemical information. Rather than looking at a fluorescent marker, CARS directly probes the bonds of the molecules that make up the cells and tissues of interest. One such bond that is frequently probed is the bond between carbon and hydrogen (CH2) since it is present in large numbers within biological components. Recent work has demonstrated how the addition of deuterated water (also known as heavy water, since the hydrogen atoms that make up the water contain an extra neutron compared to regular hydrogen) can be used to generate carbon–deuterium (C–D) bonds in molecules within cells. Owing to the heavier isotopic mass, deuterated bonds can be easily distinguished from other cellular signatures when imaged with CARS microscopy.
We hypothesise that this will allow us to perform two crucial experiments on 3D cell models:
1) As different types of cell make different biological molecules, we should be able to tell the cells apart simply by the way in which they use the deuterium from the heavy water.
2) Different types of cells metabolise material in different ways, and this heterogeneity could potentially affect their susceptibility to therapeutics. By studying the turnover of deuterium we should be able to understand the cells metabolism in more detail without using fluorescent markers.
In this study we intend to use CARS microscopy to image organoids that have been cultured in media containing deuterated water. The aim being to identify the different cell types that make up the organoid structures without the need for fluorescent markers. Similarly by feeding the cells with deuterated compounds we will be able to observe the uptake or metabolic rate of the individual cells and what effects different drugs have on this rate. This will allow us to study these complex systems with minimal interference or modification, allowing long term studies of the effect of therapeutic drugs on cell development and metabolism – a current unmet need within therapeutic design.