Southampton Imaging: 3D imaging at millimetre to nanometre scales for regenerative medicine using multiple complimentary modalities

Regenerative medicine aims to make tissues and organs to repair damaged and diseased tissues and restore the body to its original health. It uses stems cells and precursor cells that, under exactly the right conditions, will change into the specialized cells needed to repair the tissue. Scaffolds upon which the cells can grow and be guided are used to help organize the cells into the right structures. We are now in a unique position to create new soft and hard tissues (e.g. liver, neural, cartilage, bone) to help aid treatment for all. Important in this goal of moving to clinical application will be to ensure the new formed tissue is completely safe.

One of the most important factors that determines the effectiveness and normal functioning of a tissue is the way it is structured or arranged (the architecture of the tissue- like a bridge). This tissue architecture is important at many different scales, from individual cell components to the large scale organization of structures such as bones or blood vessels and nerves supplying the organs. Therefore to understand the appropriate structures required and check that the tissue constructs made are performing correctly we need to be able to 'see' these by using microscopic imaging at different magnifications. Because the cells and tissues are three-dimensional (3D) structures, we need to see how they fit together in 3D to understand their architecture - in the same way we can understand how the elements of a building fit together and function effectively in 3D by walking around it.

This application is for three imaging systems specially designed to create 3D images of tissues and scaffolds at 3 different scales. The highest magnification is provided by a scanning electron microscope combined with a microtome- this gradually removes very thin (<50nm) slices from the sample imaging the surface that is revealed after each slice. This creates a stack of images representing the structure of the cells down to the components making up the cells and the fine detail of scaffolds- with this technique the membranes within the cells can clearly be seen. The next level of magnification uses a light microscope that shines a very thin sheet of light (4-10 micro meters). This allows us to look at much larger blocks of tissue (up to a cm cube - a sugar cube) without cutting it and again create a 3D image stack to represent this. Individual cells are easily seen and they can be labelled so we can identify cell types and track them over time. However there are some samples that light will not penetrate or that are too large - for these samples the third instrument, a high resolution microCT (computed tomography) imaging device is used. This uses X-rays to image through large samples without damaging them and the design of this new instrument can allows us to distinguish individual cells and some of their features in a way that is not currently possible.

These systems will add to the wide range of existing imaging facilities in Southampton that are supported by 12 expert imaging staff. The existing expertise in sample preparation and biological image interpretation is essential for these cutting edge imaging techniques to be used effectively. An additional problem in 3D imaging at all scales is the very large digital image sets that are produced- each taking large amounts of storage; 50-1000Gb (equivalent of 10-200 DVDs each!). Southampton University is a world leader in advanced computing and image processing - we already have state of the art computing hardware and software. In this project we will work with our collaborators in the University to further develop these specifically for the processing and analysis of images of regenerative medicine samples.

We expect regenerative medicine to transform human health over the next 10-30 years and in order to fulfill this promise as quickly and safely as possible it is essential we can image the generated structures and tissues.

Increased aging populations pose new challenges and emphasize the need for approaches to augment and repair tissue lost through aging, trauma or disease. Regenerative medicine promises to deliver specifiable replacement tissues. As living tissue is inherently 3D, comprehensive quantitation of cell interaction, differentiation and tissue architecture necessitates the use of 3D analytical platforms with appropriate resolution, structural differentiation and volumetric analysis capabilities, along with time resolved, ex vivo and in vivo capabilities. Southampton Imaging offers an open access resource to imaging these tissue constructs pre- and post-implantation, vital to understand and validate the tissue structure and function.

To deliver appropriate tissue resolution we request a Gatan 3View system to produce nanometer scale 3D image stacks by electron microscopy, able to visualise cell organelles and local cell-scaffold interactions. This will be complemented by a LaVision light sheet microscope, capable of imaging blocks of up to 1 cm cube substantially extending the depth and volume that can be imaged. This would for example include representative vascular networks that can be specifically labelled by immuno fluorescence or affinity methods. The platform allows non-destructive imaging of smaller live samples and critically reduces the light flux permitting long-term imaging of live tissues and cell migration in 3D tissues. Together with an Xradia ultra-high resolution microCT with phase contrast imaging, we will be able to image large volumes of tissue constructs including bio-compatible matrices of all types as well as hard and soft tissue elements. The requested equipment will integrate with extensive imaging infrastructure (including 12 dedicated supported staff), computational and image processing capacity (including UK's most powerful supercomputer) providing a unique Image resource for the Regenerative Medicine community.

A multidisciplinary open-access 3D imaging service for regenerative medicine will substantially impact on the ability to provide and apply best cutting-edge practice in 3D imaging. We anticipate that project outputs will have significant bearing on the quality of life through improved regenerative medicine capability. By imaging the whole 3D structure of substantive tissue or implant volumes, it will enhance the development and implementation of effective biomaterial and implants. Quantitation of cell interactions, differentiation and tissue architecture necessitates the use of these 3D analytical platforms (X-ray, light and electron) with appropriate resolution including time resolved, ex vivo and in vivo analysis.

The extent and degree of detailed analysis possible with these technologies will also be improved, deepening understanding and reducing the time required to develop clinically effective tissue regenerative solutions. Critically, by building on extensive existing imaging facilities and expertise in the biological, imaging, engineering and computing domains the time from identifying a regenerative medicine imaging requirement to its application will be reduced. Shared experience and solutions in Southampton will allow data handling and analysis problems to be effectively provided, increasing the value of the data and reducing the time taken to appropriately analyse the images.

Our strategy is to share our data and the approaches developed and used for analysis with the wider scientific community to provide immediate access, enhancing collaboration and research.

We will also disseminate results at various stem cell, regenerative medicine, tissue engineering and microfluidics meetings. The applicants engage with the regenerative research community and have collectively presented keynote and plenary lectures at over 30 meetings around the world in the last 2 years. The enhanced research capability proposed holds significant translational therapeutic potential. As part of our established Excellence with Impact programme, we will build on links with Business Fellows to promote innovative collaborations such as that already sought by Xradia in anticipation of the project (see letter attached).

The research team will work as part of an active multidisciplinary team of biologists, tissue engineers, bio-engineers and, where appropriate, with clinicians and industry. This equipment grant will contribute substantially to the training and development of the Regenerative Medicine community through accredited postgraduate courses and the ability to train researchers at the interdisciplinary boundaries of regenerative medicine and imaging.

In order to maximize broader impact to the general public, we will organize a number of educational outreach programmes (Oreffo as part of the Acellular Hub will bid for outreach funds available in the hub) building on our links to a new school education programme, the LifeLab Southampton. This invites local school pupils, their parents and teachers to experience laboratory-based experiments learn how lifestyle affects lifelong health. We will continue our active involvement with BBSRC & EPSRC public outreach. All applicants have enjoyed significant media coverage for their work with presentations on Sky News, BBC TV stations, Radio Stations and the national and international press and science press (The Engineer, Materials Today and Nature Middle East amongst others). We will continue to actively engage with the press to showcase Southampton Imaging.